WO1993025577A2 - Active component of parathyroid hypertensive factor - Google Patents
Active component of parathyroid hypertensive factor Download PDFInfo
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- WO1993025577A2 WO1993025577A2 PCT/US1993/005626 US9305626W WO9325577A2 WO 1993025577 A2 WO1993025577 A2 WO 1993025577A2 US 9305626 W US9305626 W US 9305626W WO 9325577 A2 WO9325577 A2 WO 9325577A2
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- hypertensive factor
- parathyroid hypertensive
- parathyroid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the identification, characterization and synthesis of an active component of parathyroid hypertensive factor which comprises a polypeptide linked to a phosphoglyceride (sometimes referred to as phospholipid) .
- the component exhibits activities substantially similar to parathyroid hypertensive factor and therefore is indicated in the control of extracellular calcium uptake, and is related to hypertension and some other diseases in mammals.
- Methods for screening patients for the presence of the circulating factor using antibodies against the component are described.
- Antagonists of the parathyroid hypertensive factor component have been developed and the use of such antagonists in methods of treating patients with diseases which involve intracellular calcium elevation are also described.
- Hypertension is generally defined as the elevation of the systolic and/or diastolic arterial blood pressure above a nominal value of 140/90 mm Hg.
- Diseases associated with hypertension include arteriosclerosis, hypertensive renal failure, stroke, congestive heart failure and myocardial infarction.
- arteriosclerosis hypertensive renal failure
- stroke stroke
- congestive heart failure myocardial infarction
- myocardial infarction Although numerous methods of treatment have been found to be effective in the reduction of arterial blood pressure, the etiology of essential hypertension remains essentially unknown.
- a genetic predisposition to hypertension is generally accepted, but the number of different drugs which have been found effective in the treatment of hypertension, and the fact that these drugs seem to operate by eliciting different pharmacological responses, suggests that there may be different primary causes for essential hypertension.
- SHR spontaneously hypertensive rats
- WKY Wistar- Kyoto
- PTH Parathyroid hormone
- Ir-PTH immunoreactive parathyroid hormone
- This vasodilating action of PTH also has been related to a specific region of the molecule separate from the region mediating hypercalcemic effects [See: Pang et al., Endocrinology, 112. 284 (1983)].
- PTH has also been shown to inhibit calcium entry into vascular smooth muscle [See: Pang et al.. Life Sci.. 42. 1395 (1988)] through L-type calcium channels [Wang et al., FEBS, Vol. 282, No. 2, pp.331-334 (1991) ] .
- This paradox is further heightened by the fact that hypertensive patients with increased PTH levels exhibit decreased serum ionized calcium levels [See: Resnick et al., New Engl. J. Med.. 309. 888 (1983); Hvarfner et al., Acta Med. Scand.. 219. 461 (1986)]. It would be expected that the serum ionized calcium levels would be elevated if PTH were primarily elevated.
- dialyzed plasma from hypertensive human subjects was infused into normotensive SD rats and shown to produce hypertension. Plasma from these subjects also increased calcium uptake in rat tail arteries i ⁇ vitro. Dialyzed plasma from normotensive patients produced no significant increase in blood pressure.
- the factor has been shown to regulate extracellular calcium uptake, and can be inhibited by increases in dietary calcium levels.
- the factor has been isolated and a method for screening for the factor using antibodies raised against the factor is described.
- the factor has a molecular weight of approximately 2,700 daltons and has the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle. From bioassay data, the factor in humans and rats has been found to be substantially similar.
- the present invention relates to the identification of an active component of parathyroid hypertensive factor which comprises a polypeptide linked to a phosphoglyceride and has the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle.
- Antibodies raised against the component are used in an immunological assay to test for the presence of parathyroid hypertensive factor or portions thereof, the presence being indicative of hypertension or another disease related to an increase in calcium levels.
- Antagonists of the parathyroid hypertensive factor component have been developed and the use of such antagonists in methods of treating patients with diseases which involve intracellular calcium elevation are also described.
- Figure 1 shows the effect of semi-purified PHF on norepinephrine (NE) induced tension development in rat tail artery.
- Semi-pure PHF potentiates the NE effect.
- Figure 2 shows the effect of LPI on tension development induced by NE in rat tail artery. LPI potentiates NE-induced increase tension as does PHF.
- Figure 4 shows LPC significantly augments the increase in intracellular calcium induced by KCl in vascular smooth muscle cells.
- Figure 5 shows SHR parathyroid gland culture media will stimulate phosphodiesterase activity.
- Figure 6 shows WKY parathyroid gland culture media does not stimulate phosphodiesterase activity.
- Figure 7 shows LPS stimulates phosphodiesterase activity.
- Figure 8 shows the peak effect of FMOC-synthesis
- FIG 9 shows FMOC-synthesis PHF component reveals a typical PHF-like increase in blood pressure that is prevented by digestion with phospholipase D.
- Figure 10 shows that LPS causes a delayed, sustained increase in blood pressure in normotensive rats.
- Figure 11 shows that LPI causes a delayed, sustained increase in blood pressure in normotensive rats.
- Figure 12 shows that FMOC-synthesis PHF component potentiates norepinephrine-induced increase in tension in rat tail artery strips.
- Figure 13 shows the effect of FMOC-PHF (0.01 ⁇ g/mi bath medium) on the [Ca 2+ ]j increment induced by 15 mM KCl in VMSC isolated from rat tail artery.
- the upper panel shows a summary of five individual experiments.
- the [Ca 2+ ]j increment in control group is 22.52 ⁇ 4.82 (nM) . ⁇ Significantly different from the control, p ⁇ 0.05.
- the lower panel shows the original tracing of one typical experiment.
- the effect of KCl alone (a) , in the presence of FMOC-PHF (b) , and after washes (c) .
- the cells were incubated with FMOC-PHF for 45 mins. before challenge with KCl.
- Figure 14 shows the time-dependent effect of pure PHF (0.1 m£ SHR plasma equivalent/m£ bath medium) on L-type Ca 2+ channel activity in a VMSC isolated from rat tail artery.
- the left side of this figure shows the original records at the three test pulses.
- the right side shows control current (I) voltage (V) relationships (•) and that obtained at 35 mins. ( ⁇ ) or 50 mins. (T) after application of pure PHF (0.1 m£ SHR plasma equivalent/m£ bath medium) .
- the holding potential was -40 mV.
- Figure 15 shows the effect of IBMX (5 x 10 5 M) and nifedipine (10 "6 ) on the L-type Ca 2+ current activated by pure PHF (0.1 m£ SHR plasma equivalent/m£ bath medium) in a VMSC isolated from rat tail artery.
- the time-dependent peak current change was monitored continuously for 105 mins. after application of pure PHF, IBMX and nifedipine at the indicated time.
- Figure 16 shows the effects of FMOC-PHF on calcium channel currents and that FMOC-PHF increased current (downward direction) .
- Figure 17 shows the effect of PHF on calcium channel currents and reversal by IBMX.
- the present inventors have identified an active component of parathyroid hypertensive factor which exhibits properties similar to parathyroid hypertensive factor.
- the component comprises a polypeptide linked to a phosphoglyceride and which has the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle. That is, when the component is administered to, for instance, a rat, one begins to see a rise in blood pressure at about 20-30 minutes after administration, which peaks out at about one hour. At the same point in time, one also sees an increase in extracellular calcium uptake by vascular smooth muscle cells - that is, uptake peaks out at around one hour.
- the polypeptide of the component preferably has from 5-20 amino acid residues and more preferably from 5- 10 amino acid residues.
- the amino acids comprising the polypeptide of the component can be any of those chosen from the group of basic amino acids consisting of Alanine (Ala) , Arginine (Arg) , Asparagine (Asn) , Aspartic acid (Asp) , Cysteine (Cys) , Glutamine (Gin) , Gluta ic acid (Glu) , Glycine (Gly) , Histidine (His) , Isoleucine (lie) , Leucine (Leu) , Lysine (Lys) , Methionine (Met) , Phenylalanine (Phe) , Proline (Pro) , Serine (Ser) , Threonine (Thr) , Tryptophan (Trp) , Tyrosine (Tyr) and Valine (Val) with the component retaining substantially similar properties to par
- Non-basic amino acids may replace the basic amino acids in the component.
- Such non-basic amino acids would include for example ornithine, sarcosine, norleucine, N-methyl-phenylalanine and the like.
- the structure of the polypeptide represents Tyr-Ser-Val-Ser-His-Phe-Arg. [SEQ ID N0:1].
- the phosphoglyceride of the component may be linked in any position as long as the component retains properties substantially similar to parathyroid hypertensive factor, including the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle. It is preferred that the linkage be at one of the Serine residues and more preferably, at the Serine residue in position 4 (as depicted in the sequence above) .
- the phosphoglyceride is a lysophosphatidic acid or a phosphatidic acid.
- the parathyroid hypertensive factor component has a molecular weight between about 1,000 and about 2,700 daltons as measured by mass spectrometry. Preferably the molecular weight is in the range of about 1,000 daltons to about 2,000 daltons and more preferably is about 1,350 daltons. It is noted, however, that there may be additional non-interfering amino acids on either side of the component polypeptide which would make the molecular weight of the component molecule much higher than 2,700.
- the parathyroid hypertensive factor component may be bound to a biologically active compound or molecule and this can serve as targeting agent for the transport of biologically active compounds to specified sites within cells, tissues, organs or the like.
- biologically active compounds examples include hormones, renal drugs, diuretics, neurotrans itters and similar compounds.
- the biologically active compounds would, for example, be targeted to cells such as vascular smooth muscle, renal and cardiac cells and the like. Methods of binding the biologically active compound or molecule to the component are well known in the art.
- the parathyroid hypertensive factor component may be bound to a detectable marker or label which would serve to identify the presence of the component in a biologically sample.
- markers and labels are fluorescein, HRP, biotin and the like. Methods of binding the markers and labels to the component are well known in this art.
- the parathyroid hypertensive factor component has the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle, has a structure which is different from parathyroid hypertensive factor and has the following structure identified as:
- R is hydrogen, a alkyl, a alkenyl or at least one fatty acid group
- R 2 is as previously defined for R,
- each of X and Y are oxygen or sulfur
- each of A and B are from 0 to 20 additional amino acids with the proviso that at least one of A and B does not have an amino acid sequence corresponding to that of PHF which has been isolated from a hypertensive individual
- n 1 to 5.
- the variable X and Y of the parathyroid hypertensive component structure are oxygen.
- fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA (eicosopentanoic acid) and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- the fatty acid R 2 is preferably selected from the group of fatty acids consisting lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- the fatty acid groups comprising R are preferably from 1 to 10 in number. More preferably, the fatty acid groups are from 1 to 5 in number and most preferred is 1 fatty acid group.
- the fatty acid groups comprising R 2 are preferably from 1 to 10 in number. More preferably, the fatty acid groups are from 1 to 5 in number and most preferred is 1 fatty acid group.
- parathyroid hypertensive factor component having the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle, in a manner similar to naturally occurring PHF which has been purified from a hypertensive subject, and having the following structure identified as:
- Rj is hydrogen, a Cj-C 22 alkyl, C,-C 22 alkenyl or at least one fatty acid group and R 2 is as previously defined for R j .
- the fatty acid R is preferably selected from the group of fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- the fatty acid R 2 is preferably selected from the group of fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- the fatty acid groups forming R j or R 2 may range in number from 1 to 10. Preferably the numbers of fatty acid groups range from 1 to 5. Most preferred is 1 fatty acid group.
- the present invention is also directed to a process for producing a parathyroid hypertensive factor component having the property of delayed onset of an increase in blood pressure of a normotensive rat when administered thereto, the increase in blood pressure temporally correlating with an increase in extracellular calcium uptake by vascular smooth muscle, in a manner similar to naturally occurring PHF which has been purified from a hypertensive subject, which has a structure different from parathyroid hypertensive factor and which has the following structure identified as:
- R is hydrogen, a C,-C 22 alkyl, a C,-C 22 alkenyl or at least one fatty acid group
- the phospholipid is either lysophosphatidic acid or phosphatidic acid.
- the polypeptide is produced by chemical synthesis.
- the polypeptide may be synthesized in a conventional peptide synthesizer well known in this art.
- a representative example of such a synthesizer is the Applied Biosystems peptide synthesizer Model 430A.
- the procedures for synthesizing peptides using an automated peptide synthesizer are conventional in this art.
- Recovery of the component is accomplished by standard purification and separation techniques well known in this art. Representative examples of such techniques include ion exchange and/or reversed-phase HPL chromatography, and TLC.
- the present invention also is directed to a process for chemically synthesizing the parathyroid hypertensive factor component.
- R is hydrogen, a Cj-C 22 alkyl, a C,-C 22 alkenyl, or at least one fatty acid group
- R 2 is as previously defined for R,
- each of X and Y are oxygen or sulfur
- each of A and B are from 0 to 20 additional amino acids
- n l to 5, comprising the steps of: (a) chemically synthesizing a polypeptide having the structure A-Tyr-Ser-Val-OH, wherein A is as previously defined and purifying said polypeptide; (b) preparing an ester of the polypeptide; (c) adding L- ⁇ -lysophosphatidylyserine (LPS) to the ester to produce a product having the structure
- A is as previously defined and LP is L- ⁇ - lysophosphatidyl, and purifying the product; (d) obtaining a product having the structure
- step (e) by acidifying said product of step (c) and extracting the acidified product and recovering the same by evaporation; (e) preparing an ester of the product of step (d) ; (f) chemically synthesizing a polypeptide having the structure NH 2 -His-Phe-Arg-B-OH and purifying the polypeptide; (g) preparing an ester of the polypeptide of step (f) which has the structure NH 2 -His-Phe-Arg-B-0 " ; and (h) combining the ester of step (e) and the ester of step (g) to produce the parathyroid hypertensive factor component.
- the ester of the polypeptide of step (b) is an N-hydroxysuccinimide ester and is prepared by addition of the polypeptide to a mixture containing dicyclohexylcarbodii ide and dioxane.
- the LPS added to the ester in step (c) is first dissolved in a bicarbonate buffer to form a solution having a pH of 7.
- acidification preferably occurs by addition of sodium citrate to pH of 2.8.
- the ester of step (e) is preferably an N- hydroxysuccinimide ester and is prepared by addition to the product of step (d) of a mixture containing dicyclohexylcarbodiimide, dioxane and tetrahydrofuran.
- the ester of step (g) is preferably first dissolved in a bicarbonate buffer to form a solution having a pH of 7 before the solution is combined with the ester of step (e) .
- Chemical synthesis is carried out in conventional peptide synthesizers well known in this art. For example, synthesis may be carried out in peptide synthesizer model 430A manufactured by Applied Biosystems. Procedures for producing the peptides well known in this art.
- polyclonal and monoclonal antibodies may be raised against the component and used in any assay for PHF. Procedures for producing polyclonal and monoclonal antibodies are well known in this art.
- mice can be immunized by implantation of amino phenolthiol ether discs to which the component has been affixed, according to the procedure of Viamontes et al., J. Immunol. Meth. 94. 13-17 (1986).
- the mice are then boosted with antigen in Freund's incomplete adjuvant at two-week intervals and the antibody titer assayed by enzyme-linked immunosorbent assay (ELISA) using the parathyroid hypertensive factor component of the present invention as the antigen.
- ELISA enzyme-linked immunosorbent assay
- Monoclonal antibodies may be prepared from the spleens of polyclonal antibody producing mice.
- the required hybridoma and MCA may be obtained using the method of Langone and Van Vunakis, "Methods in Enzymology", 121. 1-947 (1986), using such modifications as are known to skilled practitioners in this art.
- the method of detection using polyclonal and/or monoclonal antibodies is not specifically limited and includes radioimmunoassay, enzyme immunoassay, enzyme- linked immunosorbent assays, and assay systems based on the formation of an immunoprecipitate.
- a method for detecting the presence of parathyroid hypertensive factor or a portion thereof would comprise the steps of raising polyclonal antibodies in an animal (mammal or bird) by injecting a solution containing the parathyroid hypertensive factor component of the present invention into the animal; collecting serum containing polyclonal antibodies raised against the parathyroid hypertensive factor component; screening a sample which may contain parathyroid hypertensive factor or an antigenic portion thereof by an immunoassay method using the polyclonal antibodies; and detecting the presence of the PHF or its antigenic portions in the sample.
- the immunoassay method for detecting the presence of parathyroid hypertensive factor or a portion thereof is preferably an enzyme linked immunoassay, an enzyme linked immunosorbent assay or an immunoprecipitation assay.
- the sample may be a biological sample. Examples of the sample would include serum, plasma, urine, tissue, cells and the like.
- the component have the following structure identified as:
- Rj is hydrogen, a C,-C 22 alkyl, a C,-C 22 alkenyl or is at least one fatty acid group
- R 2 is as previously defined for j
- each of X and Y are oxygen or sulfur
- each of A and B are from 0 to 20 additional amino acids with the proviso that at least one of A and B does not have an amino acid sequence corresponding to that of PHF which has been isolated from a hypertensive individual
- n 1 to 5, and more preferably:
- Rj is hydrogen, alkyl, C,-C 22 alkenyl or at least one fatty acid group and R 2 is as previously defined for R,.
- the fatty acid R, and R 2 are preferably selected from the group of fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- a method for the identification of parathyroid hypertensive factor or a portion thereof in a patient would comprise the steps of raising antibodies to the parathyroid hypertensive factor component of the present invention by injecting said component into an animal (mammal or bird) ; isolating antibody-secreting B-lymphocytes from the immunized animal; fusing the antibody-secreting B-lymphocytes with myeloma cells to form hybridomas; selecting and cloning the hybridomas which secrete parathyroid hypertensive factor component antibody; propagating the antibody- secreting hybridomas; isolating a monoclonal antibody from the hybridomas; and screening a sample which may contain parathyroid hypertensive factor or its portions by an immunoassay method using the monoclonal antibodies.
- the immunoassay method may be an enzyme linked immunoassay, an enzyme linked immunosorbent assay, or an immunoprecipitation assay.
- the sample is preferably a biological sample.
- the sample could include serum, plasma, urine, tissue, cells and the like.
- the patient may be a mammal or a bird and is preferably human.
- the component have the following structure identified as:
- R is hydrogen, C j -C 22 alkyl, a C,-C 22 alkenyl or at least one fatty acid group
- R 2 is as previously defined for R,
- each of X and Y are oxygen or sulfur
- each of A and B are from 0 to 20 additional amino acids with the proviso that at least one of A and B does not have an amino acid sequence corresponding to that of PHF which has been isolated from a hypertensive individual
- n 1 to 5 and more preferably:
- R is hydrogen, C,-C 22 alkyl, C,-C 22 alkenyl or at least one fatty acid group and R 2 is a previously defined for R j .
- the fatty acid Rj and R 2 are preferably selected from the group of fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- a particularly desirable embodiment of such an assay system would be in the form of a diagnostic kit which could be used in a physician's office or a clinic, in the absence of sophisticated analytical instruments.
- Such methodology has been used for the detection of hormones and other immunoreactive substances in body fluids.
- One example is a commercially available kit for the detection of early pregnancy. PHF or its fractions could, therefore, be qualitatively or quantitatively detected by the use of such methods.
- the present invention also provides for a kit for the detection of parathyroid hypertensive factor or an immunologically reactive portion thereof in an animal, comprising, in a single package an antibody to the parathyroid hypertensive factor component bound to a solid phase; a secondary antibody to anti-parathyroid hypertensive factor component antibody labeled with an enzyme; a substrate for said enzyme label on said secondary antibody; and standard solutions of parathyroid hypertensive factor or a component or portion thereof.
- a further kit for the detection of parathyroid hypertensive factor or a portion thereof in an animal comprises, in a single package, an antibody to the parathyroid hypertensive factor component; a solid phase upon which said antibody attaches; and a standard solution of parathyroid hypertensive factor or a component thereof.
- the preferred component has the following structure:
- R j is hydrogen, a C,-C 22 alkyl, a C,-C 22 alkenyl or at least one fatty acid group
- R 2 is as previously defined for R j
- each of X and Y are oxygen or sulfur
- each of A and B are from 0 to 20 additional amino acids with the proviso that at least one of A and B does not have an amino acid sequence corresponding to that of PHF which has been isolated from a hypertensive individual
- n 1 to 5 and more preferably:
- R is hydrogen, alkyl, C,-C 22 alkenyl or at least one fatty acid group and R 2 is previously defined for R,.
- the fatty acid R, and R 2 are preferably selected from the group of fatty acids consisting of lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, 7-linolenic acid, arachidonic acid, EPA and Nervonic acid. More preferable are oleic acid and stearic acid, and most preferred is oleic acid.
- the present invention is also directed to antagonists of the parathyroid hypertensive factor. While not being bound to any theory, it is believed that the parathyroid hypertensive factor consist of two parts: the activation part (phospholipid part) ; and the recognition or binding part (peptide part) . Again, it is believed that the phospholipid plays a role in allowing transport of the peptide through cell membrane to reach a receptor site where binding of the peptide occurs. By altering the linkage of the phospholipid at the binding site of serine in position 4, a parathyroid hypertensive factor antagonist can be produced. It is believed that the antagonists bind to the parathyroid hypertensive factor site but do not activate parathyroid hypertensive factor activity and thus can further prevent the action of authentic parathyroid hypertensive factor. Antagonist of the present invention preferably have the following structure
- Xaa is any amino acid other than Serine and PL is a phospholipid.
- the phospholipid is either lysophosphatidic acid or phosphatidic acid. More preferably, Xaa is selected from the group of amino acids consisting of lysine, tyrosine and leucine.
- the phospholipid Since the peptide and the phospholipids must maintain a certain tertiary structure for biological activity, modification of the peptide sequence may produce such tertiary changes that the binding part may not be effected but the phospholipid may be spatially moved and the analogues may become antagonists. Furthermore, if the phospholipid is as important as the activation part of the molecule, its composition may affect its ability to produce the biological response. By changing the composition of the fatty acids and the addition of the fatty acids in the second carbon of the glycerol backbone, antagonists may be produced.
- antagonists to the parathyroid hypertensive factor may be designed by (1) having the phospholipid linked to various positions on the peptide, (2) altering the peptide sequence, (3) changing the phospholipid composition and (4) others that may allow the molecule to bind but not to activate the biological pathway leading to the hypertensive response.
- the antagonists of the present invention can be used in the treatment of hypertension and other diseases related to intracellular calcium elevation (e.g., non- insulin-dependent diabetes mellitus; atherosclerosis; congestive heart failure; cancer, including breast, colon, kidney and leukemia; inflammatory bowel disease and asthma) .
- diseases related to intracellular calcium elevation e.g., non- insulin-dependent diabetes mellitus; atherosclerosis; congestive heart failure; cancer, including breast, colon, kidney and leukemia; inflammatory bowel disease and asthma.
- the antagonists of the present invention may be administered to a warm-blooded mammal in need thereof, particularly a human, by parenteral, topical, oral, rectal administration or by inhalation.
- the antagonists may be conventionally formulated in parenteral or oral dosage form compounding about 1 to about 200 mg per unit of dosage with a conventional vehicle, excipient, binder, preservative, stabilizer, color, agent or the like as called for by accepted pharmaceutical practice.
- parenteral administration a 1 to 10 m£ intravenous, intramuscular or subcutaneous injection would be given one to four times daily.
- the injection would contain an antagonist of the present invention in an aqueous isotonic sterile solution or suspension optionally with a preservative such as phenol or a solubilizing agent such as ethylenediaminetetraacetic acid (EDTA) .
- a preservative such as phenol or a solubilizing agent such as ethylenediaminetetraacetic acid (EDTA)
- EDTA ethylenediaminetetraacetic acid
- acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
- sterile, fixed oils are conventionally employed as a solvent or suspending medium. Synthetic monoglycerides, diglycerides, fatty acids (such as oleic acid) find use as fixed oils in the preparation of injectables.
- the antagonists of the present invention can be prepared in the form of suppositories by mixing with a suitable non-irritating excipient such as cocoa butter or polyethylene glyc ⁇ ls.
- a suitable non-irritating excipient such as cocoa butter or polyethylene glyc ⁇ ls.
- the antagonists of the present invention can be prepared in the form of ointments, jellies, solutions, suspensions or dermal adhesive patches.
- antagonists of the present invention may be administered by a Spinhaler turbo-inhaler device obtained from Fisons Corporation of Bedford, Massachusetts, at a rate of about 0.1 to 50 mg per capsule, 1 to 8 capsules being administered daily for an average human.
- a liquid aerosol the compounds of the present invention are administered at the rate of about 100 to 1000 micrograms per "puff" or activated release of a standard volume of propellant.
- the liquid aerosol would be given at the rate of 1 to 8 "puffs" per day with variation in dosages due to the severity of the conditions being treated, the weight of the patient and the particle size distribution of the aerosol.
- a fluorinated hydrocarbon or isobutane find use as propellants for liquid aerosols.
- Daily doses are in the range of about 0.01 to about 200 mg per kg of body weight, depending on the activity of the specific compound, the age, weight, sex and conditions of the subject to be treated, the type and severity of the disease, the frequency and route of administration. As would be well known, the amount of active ingredient that may be combined with the carried materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration.
- Some forms of cancer are characterized by an increase in intracellular free calcium [see: Okazaki et al.. Cane. Res.. 46 (12 Pt 1), 6059-6063 (1986); Lipton and Morris, Cane. Chemother. Pharmacol.. 18(1), 17-20 (1986); Chien and Warren, Cane. Res.. 46(11), 5706-5714); Shirakawa et al., Cane. Res.. 46(2), 658-661 (1986); and Meyer, J. Hypertens.. 5 (suppl. 4), S3-S4 (1987)].
- Parathyroid activation also has been associated with certain forms of cancer [see: Palmer et al., Am. J. Epidemiol..
- the plasma was then homogenized with 20 volumes of chloroform:methanol (2:1 v/v) and allowed to sit overnight at 4 degrees C.
- the organic phase was collected and washed with 5 volumes of 0.1 M NaCl.
- the extract was then concentrated by rotary evaporation at 40-50°C and freeze dried.
- a BCA assay was performed to determine the amount of protein in the sample.
- SD rats were anesthetized using Na pentobarbital, (50 mg/kg, i.p.) and catheters were inserted in the jugular vein for injection of plasma and drugs and in the carotid artery for measurement of b.p.
- the chloroform methanol extract of SHR plasma was redissolved in saline and administered by bolus injection.
- the blood pressure increased slowly, in a manner similar to PHF.
- the maximum increase in blood pressure of 16.4 ⁇ 3.5 mmHg occurred at 45 to 50 min. (n 5) .
- the test indicates that PHF is extracted by chloroform:methanol.
- Thyroparathyroid glands were excised from SHR rats and cultured for up to 7 days in Hank's medium (Gibco) , with the media changed daily. The production of PHF may be stimulated by reduction of calcium from the medium. Pooled media, after dialysis and filtration analogous to the treatment of plasma samples, was lyophilized. Gland culture medium, dialyzed, filtered and lyophilized as described in the previous example, was first extracted with 20 volumes of ether, followed by 10 volumes of chloroform methanol (4:1). The two extracts and the remaining aqueous phase were lyophilized and reconstituted in saline for a blood pressure bioassay as described in Example 1 and the following results obtained:
- a plasma sample from SHR rats (equivalent to 20 m£ original plasma) was dialyzed, filtered and lyophilized according to Example 1. The sample was dissolved in 10 m£ 0.1 N HC1 and then incubated at room temperature for 3 hr. The sample was then neutralized with 0.1 N NaOH. A control was prepared in a similar manner except that no plasma sample was included in the incubation.
- the sample is then dialyzed against ice-cold water and the pH adjusted to 7.0 with HCl and diluted to the original volume of plasma.
- a control was prepared in a similar manner except that the plasma sample was omitted.
- a buffer comprising 50 mM glycine as a buffer, having a pH of 8.0 and containing 1 mM ZnCl 2 is prepared.
- a plasma sample from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in the buffer (6 mg/m£) .
- the phospholipase C enzyme from Bacillus cereus (commercially available through Boehringer Mannheim) is dissolved in the buffer solution (4000 units/m£) and 1 m£ sample plus 0.1 m£ enzyme is incubated at 37°C for 4 hrs. The sample is then heated to 96°C for 5 min.
- Control 1 PLC and buffer
- Control 2 Buffer
- Control 3 plasma sample and buffer.
- the 9.8 mmHg result indicates positive parathyroid hypertensive factor activity.
- PHF activity was inactivated by phospholipase C (PLC) .
- a buffer comprising 50 mM Tris as a buffer, having a pH of 7.4 and containing 20 mM CaCl 2 , and 100 mM NaCl is prepared.
- a plasma sample from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in the buffer (6 mg/m£) .
- the phospholipase A 2 enzyme from Na a moeambique moeambique (commercially available through Sigma Chemical Co.) is dissolved in the buffer (1500 units/m£) and 1 m£ sample plus 0.1 m£ enzyme is incubated at 37°C for 2 hrs. The sample is then heated to 96°C for 5 min.
- Control 1 plasma sample and buffer
- Control 2 Enzyme and buffer.
- a bioassay was conducted as in Example 1. The results of the assay are as follows:
- the 8.8 mmHg result indicates positive parathyroid hypertensive factor activity. PHF activity was not inactivated by phospholipase A.
- the procedure is conducted in accordance with the protocol established in Bergmeyer et al. (1983) in Methods of Enzymatic Analysis (Bergmeyer ed.) 3rd ed, vol. 2, pp. 288-291.
- the phospholipase D enzyme is obtained from cabbage and is commercially available through Boehringer Mannheim.
- Triton X-100 7.8 containing 0.2% Triton X-100 is prepared.
- a plasma sample from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in 2 m£ of the buffer. Phospholipase D in an amount of lmg/m£ is added to the solution which is then incubated at 37°C for 20 minutes.
- Controls were prepared as follows:
- Control 3 Plasma sample and buffer
- Control 1 Control 2 -4.6 mmHg 11.5 mmHg 2.7 mmHg 0.6 mmHg
- the assay is conducted according to the procedures described in Methods in Enzymology . Vol. 182, pp. 602-613 (1990) and J. Biol. Chem.. Vol. 240, p. 1619 (1965) .
- TPCK N- tosyl-phenylalanine chloromethyl ketone
- a plasma sample (equivalent to 20 m£ of original plasma) from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in 2 m£ of a buffer made up of 50 mM NH 4 HC0 3 , pH 7.5. Trypsin-TPCK in an amount of 36 ⁇ g is added and the solution is incubated at 37°C for 18 hr. The sample is then heated to 96°C for 5 min and the pH adjusted to 7.0.
- Control 1 Buffer only Control 2: Buffer and Trypsin-TPCK Control 3: Buffer and plasma sample
- a buffer containing 0.1 M ammonium bicarbonate (pH 8.5) is prepared.
- a plasma sample from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in 2 m£ of buffer.
- the sample is incubated at 37°C for 4 hrs and the reaction halted by acidification to a pH of 4 by addition of an appropriate amount of acetic acid.
- the sample is then heated to 96°C for 5 min.
- a control was prepared using the above procedures by omission of enzyme from the sample.
- a buffer containing 50 mM NH 4 HC0 3 , 0.4 M NaCl, 2 mM MgCl 2 is prepared.
- a plasma sample from SHR rats was dialyzed, filtered and lyophilized according to Example 1 and dissolved in 2 m£ of buffer. Chymotrypsin A 4 is added
- Controls were prepared using the above described procedures by omission of enzyme from the sample.
- the assay indicates that PHF activity is sensitive to inactivation by chymotrypsin, and that PHF contains a peptide-like structure.
- control peptide used is NSP (non-specific peptide) having the structure G-L-N-R-K-Y-L-V) .
- Incubation was at 37°C for a period of l hour.
- the reaction was terminated at the appropriate time by heating the sample to 95°C for 5 min.
- the samples were bioassayed according to the procedure of Example 1 .
- the blood pressure bioassay results are shown in Table 1.
- PHF like (agonist) activity Table 1
- lysophosphatidic acid with oleic or palmitic acid gave the best biological activity.
- phosphatidic acid with dioleic acid gave some activity.
- the other analog peptides did not give biological activity.
- N-Boc-L- valine and N- ⁇ -Boc-N g -p-tosyl-L-arginine-PAM resins 1% divinylbenzene, 0.5 mmol/gm substitution
- BOC tert- butyloxycarbonyl
- FMOC 9-fluoronylmethyloxycarbonyl
- L- ⁇ -lysophophatidylserine (1- stearoyl-sn-glycero-3-phospho-O-serine) , sodium salt was purchased from Avanti Polar Lipids (Alabaster, AL) .
- Peptide synthesis was carried out on an Applied Biosystems peptide synthesizer Model 430A.
- the analytical HPLC instrument consisted of a Hewlett Packard 1090M liquid chromatograph (Avondale, PA) equipped with 250 m£ autosampler and diode-array detection system coupled to HP 9000 series 300 computer, HP 9153C disc drive, HP 2225A Think Jet printer and HP 7440A Color Pro Plotter.
- Peptides were synthesized using the general procedure for solid-phase peptide synthesis as described here. All amino groups with the exception of FMOC-Tyr, were protected at the ⁇ -amino position with the BOC-group and the following side-chain protecting groups were used: benzyl ether (Ser) , tert-butyl (Tyr) , toluenesulfonyl (Arg and His) . All amino acids were double coupled as preformed symmetrical anhydrides (with the exception of FMOC-Tyr which was coupled as an HOBT active ester) , first in DMF and then dichloromethane. Difficult couplings were monitored using ninhydrin and, in the event that coupling efficiency was less than 99% the amino acid was coupled manually a third time as an active ester, followed by acetylation.
- Boc-groups were removed at each cycle with an 80 sec. reaction with 33% TFA/dichloromethane (v/v) , followed by a second reaction with 50% TFA/dichloromethane (v/v) for 18.5 min. Neutralization were carried out using 2 x 1 min washes with 20% DIEA/DMF (v/v) . Following couplings, when necessary, any unreacted free amino groups present were acetylated with 25% acetic anhydride/dichloromethane (v/v) for 10 min.
- the peptides were cleaved from the resin support by treatment with hydrogen fluoride (10 m£/g resin) containing 10% anisole and 2% 1,2-ethanedithiole for 1 hr at -4°C. Crude peptides were purified initially on the
- PI was prepared by standard BOC chemistry methods as described above. PI was purified by RP-HPLC.
- N-hydroxysuccinimide ester was prepared using DCC in dioxane saturated solution.
- the sodium salt of LPS was dissolved in pH 7 bicarbonate buffer and added to the ester.
- the product was then purified by RP-HPLC.
- the free acid version of the above structure was prepared by acidifying by addition of sodium citrate to pH 2.8 and extracting using ethylacetate.
- P1-LPS-P2 FMOC-Tyr-Ser-Val-Ser-His-Phe-Arg-O"
- Na + K + ATPase activity was measured as the ouabain-sensitive 86 Rb uptake in rat tail artery using the method of Deth et al. Briefly, male Sprague-Dawley rats weighing 300 to 400 g were sacrificed by pentobarbital overdose, and the tail arteries removed and cut into strips.
- the strips were rinsed in ice-cold Krebs solution (composition: NaCl, 117 mM; KCl, 5 mM; NaHC0 3 , 27 mM, NaH 2 P0 4 , 1 mM, MgS0 4 , 1.2 mM, CaCl 2 , 1.25 mM, glucose 11.0 mM) and incubated in oxygenated (95% 0 2 , 5% C0 2 ) Krebs buffer at 37°C for 2 hours. For the final 30 minutes, Krebs solution with no KCl and 122 mM NaCl was used to sodium-load the tissues.
- Krebs solution with no KCl and 122 mM NaCl was used to sodium-load the tissues.
- Lysophosphatidyl serine (30 micromolar) shows a 46% increase in 86 Rb uptake (p ⁇ 0.01).
- Lysophosphatidyl inositol 600 micromolar shows a 31% increase in 86 Rb uptake (p ⁇ 0.05).
- the method was essentially similar to the one described by Pang, et al. (1980) .
- Sprague-Dawley rats of either sex were anesthetized with pentobarbital (50 mg/kg) and the tail artery or aorta were isolated and removed and then placed in KHS buffer (115 mM NaCl, 5 mM KCl, 2.1 mM CaCl 2 , 1.2 mM MgS0 4 , 1.2 mM NaH 2 P0 4 , 25 mM NaHC0 3 , and 11 mM glucose) oxygenated with 95% 0 2 , 5% C0 2 .
- KHS buffer 115 mM NaCl, 5 mM KCl, 2.1 mM CaCl 2 , 1.2 mM MgS0 4 , 1.2 mM NaH 2 P0 4 , 25 mM NaHC0 3 , and 11 mM glucose
- the vessels were cut helically and strips of approximately 1.5 cm were secured in a Sawyer-Bartlestone chamber containing KHS.
- the force development of the helical strips was measured with a Grass FT .03 force displacement transducer and recorded on a Grass Model 79D polygraph.
- Isolated tail artery helical strips were equilibrated for 1 hour prior to addition of any vasoconstricting agents.
- the strips were preloaded with a resting tension of 0.7g and incubated at 37°C (Pang et al., 1985). In all cases, the responsiveness of strips was first tested by adding 60 mM KCl and only responsive tissue was used in experiments. There were two series of experiments.
- HBSS enzyme solution I (0.2 mM Ca) which was composed of collagenase/dispase (1.5mg/m£, Boehringer Mannheim GmbH) , elastase (0.5 mg/m£, sigma type Il-a) , trypsin inhibitor
- the arteries were then transferred into 5 m£ serum free Dulbecco's modified Eagle's medium (DMEM) and triturated using a fire polished pasteur pipette until the medium turned cloudy.
- DMEM Dulbecco's modified Eagle's medium
- the cell suspension was placed in 35 mm culture dishes in DMEM. After 4-6 hours, 10% fetal calf serum was added to the medium.
- fetal calf serum was added to the medium.
- approximately 1 x 10 6 cells were seeded on a microscope coverslip (25 mm circle) , which was placed in a 35 mm culture dish in FCS-free DMEM. After 4-6 hours, 10% FCS was added to the medium. The cells were cultured in a Co 2 incubator at 37°C.
- vascular smooth muscle cells Primary cultured vascular smooth muscle cells were incubated for 45 minutes in DMEM containing 5 ⁇ M fura-2 acetoxymethyl ester (Molecular Probes, INC.) at room temperature. During the incubation, cells were kept in a dark compartment. The cells were gently washed five times with 5K buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 1 mM CaCl 2 , 0.5 mM NaH 2 P0 4 , 10 mM Hepes, pH 7.4), and kept in the same buffer.
- 5K buffer 145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 1 mM CaCl 2 , 0.5 mM NaH 2 P0 4 , 10 mM Hepes, pH 7.4
- the monolayer cells in 1 mM Ca 2+ 5K buffer were challenged by testing substances and the change of R was recorded for a certain period time.
- the experiment was carried out in 1 mM Ca 2+ 5K buffer. The cells were then washed gently with 5K buffer and kept in same buffer for 5 mins. Testing substances were then applied for 45 mins. and R was recorded again, the cells were stimulated with NE again and the R was recorded. After washing the cells with 5K buffer for 3 times, NE was applied again and the change of R was recorded.
- Figures 3 and 4 show the increase in intracellular calcium induced by PHF and Figure 4 shows an increase induced by lysophosphatidyl choline.
- Figure 13 shows the effect of FMOC-PHF an intracellular calcium increase induced by KCl, and that FMOC-PHF potentiated in the effect of KCl.
- the measurement of cGMP-PDE activity is based on the conversion of [ 3 H]cGMP into [ 3 H]GMP and its subsequent hydrolysis by snake venom into [H]Guanosine (Wallace, et al., 1983; Wells, et al., 1975).
- the testing substance was added to a mixture containing 60 mM TES, 3 mM MgCl 2 , 0.8 mg/m£ BSA, 25 ⁇ M EGTA, 50uM CaCl 2 , 1 mM DTT, 0.5 ⁇ M cGMP and [ 3 H]-cGMP ("10-15 x 10 3 cpm) at a final pH of 7.5 for a total volume of 200 ⁇ £.
- the reaction was started by the addition of a fixed quantity of CaM-deficient PDE (Sigma Chemical Co., St. Louis, MO). Incubation carried out for 15 mins at 30°C, was terminated by boiling for 5 mins. The second reaction was initiated by the addition of 20 ⁇ £ (10 mg/m£) of snake venom (Crotalus atrox) . After 10 mins., the incubation was stopped with 700 ⁇ £ of a second termination medium containing 0.1 mM guanosine. The total incubation mixture was then applied onto a column of QAE-Sephadex A-25 (formate form) to separate labeled nucleotides from the labeled nucleosides formed.
- CaM-deficient PDE Sigma Chemical Co., St. Louis, MO
- Incubation carried out for 15 mins at 30°C, was terminated by boiling for 5 mins.
- the second reaction was initiated by the addition of 20 ⁇ £ (10 mg/m£) of snake venom (Crotalus
- the resin was eluted with 4 m£ of 20 mM ammonium formate (pH 6.5), and the radioactivity of the eluate containing [ 3 H]guanosine was determined in a liquid scintillation counter.
- a standard curve of PDE stimulation by CaM was obtained by the addition of different concentrations of purified bovine heart CaM (Sigma) (0.1-200 ng/tube) .
- the testing substance was also assayed in the presence of 10 ng/tube CaM.
- the maximum stimulation of PDE was obtained in the presence of 100 ng/tube CaM. Results are demonstrated in Figures 5, 6 and 7.
- Example 4 The same peptides as in Example 4 are used in this example. Each of the peptides (0.5 mg) and a lipid mixture of Table 2 were incubated with phospholipase D (1 mg) in 50 mM Tris buffer, pH 7.8 in a final volume of 1 m£. Simultaneously, the appropriate controls were also incubated. Incubation was at 37°C for 1 hour. The reaction was terminated at by heating the samples to 95°C for 5 minutes. The samples were bioassayed for their ability to antagonize the elevation in blood pressure of normotensive (SD) rats induced by dialyzed, filtered and lyophilized (DF) SHR plasma.
- SD normotensive
- DF lyophilized
- Ca 2+ channel activity was determined in single VSMC isolated from rat tail artery by using the whole cell version of the patch clamp technique (Hamill et al., 1981) .
- Whole cell Ca 2+ current measurements were carried out by using an Axopatch-IB patch clamp amplifier (Axon Instruments) .
- Patch micropipettes were pulled from borosilicate thin wall glass capillary tubes (OD 1.2 mm, ID 0.9 mm, FHS, Brunswick, Maine, USA) with a two stage micropipette puller (Narishige, PP83, Japan). The micropipettes were fire polished using a microforge (Leitz, Wetzlar, Germany). The tip diameter was approximately 1 ⁇ m with a resistance of 2-8 Mega Ohms.
- the cell attached to the bottom of a 3 cm Petri dish and was bathed in the medium containing 110 mM Tris, 5 mM CsCl, 20 mM HEPES, 30 mM glucose, 20 mM BaCl 2 , and 0.5 ⁇ M TTX.
- the currents were monitored using a digital oscilloscope (Nicolet Instrument Co., Madison, Wisconsin, USA) and filtered at 3 KHz with a low pass filter (Axon Instruments, Foster City, CA, USA) .
- PClamp software version 5.5
- a labmaster interface Analogmaster interface
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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AU46332/93A AU674018B2 (en) | 1992-06-12 | 1993-06-14 | Active component of parathyroid hypertensive factor |
EP93916506A EP0644895A1 (en) | 1992-06-12 | 1993-06-14 | Active component of parathyroid hypertensive factor |
JP6501751A JPH07508001A (en) | 1992-06-12 | 1993-06-14 | Parathyroid-derived hypertensive factor active component |
US08/387,820 US5739274A (en) | 1992-06-12 | 1993-06-14 | Active component of parathyroid hypertensive factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US89765992A | 1992-06-12 | 1992-06-12 | |
US07/897,659 | 1992-06-12 |
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WO1993025577A2 true WO1993025577A2 (en) | 1993-12-23 |
WO1993025577A3 WO1993025577A3 (en) | 1994-02-17 |
Family
ID=25408212
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PCT/US1993/005626 WO1993025577A2 (en) | 1992-06-12 | 1993-06-14 | Active component of parathyroid hypertensive factor |
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US (1) | US5739274A (en) |
EP (1) | EP0644895A1 (en) |
JP (1) | JPH07508001A (en) |
AU (1) | AU674018B2 (en) |
CA (1) | CA2137829A1 (en) |
WO (1) | WO1993025577A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0734723A1 (en) * | 1993-12-29 | 1996-10-02 | Idemitsu Materials Co., Ltd. | Therapeutic composition for hyperparathyroidism of patient subjected to artificial dialysis |
WO1997049807A2 (en) * | 1996-06-21 | 1997-12-31 | Johanne Tremblay | A NOVEL HYPERTENSION RELATED CALCIUM REGULATED GENE (HCaRG) |
EP1012163A1 (en) * | 1997-07-11 | 2000-06-28 | CV Technologies Inc. | A preparation derived from shark cartilage for treatment of diseases related to excessive phf or excessive intracellular calcium |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US7186797B2 (en) * | 2001-08-10 | 2007-03-06 | Epix Pharmaceuticals, Inc. | Polypeptide conjugates with extended circulating half-lives |
TWI322815B (en) * | 2003-06-26 | 2010-04-01 | Cms Peptides Patent Holding Company Ltd | Biologically active peptide comprising tyrosyl-seryl-valine (ysv) |
US20050261475A1 (en) * | 2004-02-13 | 2005-11-24 | Harvard Medical School | Solid-phase capture-release-tag methods for phosphoproteomic analyses |
BRPI0922708A2 (en) * | 2008-12-05 | 2018-11-06 | Univ California | minihepidine peptides and methods of use thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4385050A (en) * | 1981-06-26 | 1983-05-24 | Armour Pharmaceutical Company | Antihypertensive polypeptides |
WO1989003836A1 (en) * | 1987-10-29 | 1989-05-05 | Board Of Regents Of The University Of Washington | Factors associated with essential hypertension |
WO1990011074A1 (en) * | 1989-03-22 | 1990-10-04 | Pang Peter K T | Parathyroid hypertensive factor |
Family Cites Families (1)
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US5554728A (en) * | 1991-07-23 | 1996-09-10 | Nexstar Pharmaceuticals, Inc. | Lipid conjugates of therapeutic peptides and protease inhibitors |
-
1993
- 1993-06-14 AU AU46332/93A patent/AU674018B2/en not_active Ceased
- 1993-06-14 US US08/387,820 patent/US5739274A/en not_active Expired - Lifetime
- 1993-06-14 WO PCT/US1993/005626 patent/WO1993025577A2/en not_active Application Discontinuation
- 1993-06-14 CA CA002137829A patent/CA2137829A1/en not_active Abandoned
- 1993-06-14 EP EP93916506A patent/EP0644895A1/en not_active Ceased
- 1993-06-14 JP JP6501751A patent/JPH07508001A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4385050A (en) * | 1981-06-26 | 1983-05-24 | Armour Pharmaceutical Company | Antihypertensive polypeptides |
WO1989003836A1 (en) * | 1987-10-29 | 1989-05-05 | Board Of Regents Of The University Of Washington | Factors associated with essential hypertension |
WO1990011074A1 (en) * | 1989-03-22 | 1990-10-04 | Pang Peter K T | Parathyroid hypertensive factor |
Non-Patent Citations (1)
Title |
---|
JOURNAL OF HYPERTENSION vol. 11, no. 3 , March 1993 pages 245 - 251 C.G. BENISHIN ET AL. 'Production of polyclonal antisera to parathyroid hypertensive factor from sponaneously hypertensive rats' * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0734723A1 (en) * | 1993-12-29 | 1996-10-02 | Idemitsu Materials Co., Ltd. | Therapeutic composition for hyperparathyroidism of patient subjected to artificial dialysis |
EP0734723A4 (en) * | 1993-12-29 | 2001-04-11 | Kowa Tekuno Sachi Co Ltd | Therapeutic composition for hyperparathyroidism of patient subjected to artificial dialysis |
WO1997049807A2 (en) * | 1996-06-21 | 1997-12-31 | Johanne Tremblay | A NOVEL HYPERTENSION RELATED CALCIUM REGULATED GENE (HCaRG) |
WO1997049807A3 (en) * | 1996-06-21 | 1998-02-26 | Johanne Tremblay | A novel hypertension related calcium regulated gene (hcarg) |
EP1012163A1 (en) * | 1997-07-11 | 2000-06-28 | CV Technologies Inc. | A preparation derived from shark cartilage for treatment of diseases related to excessive phf or excessive intracellular calcium |
EP1012163A4 (en) * | 1997-07-11 | 2003-01-29 | Cv Technologies Inc | A preparation derived from shark cartilage for treatment of diseases related to excessive phf or excessive intracellular calcium |
Also Published As
Publication number | Publication date |
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EP0644895A1 (en) | 1995-03-29 |
AU674018B2 (en) | 1996-12-05 |
US5739274A (en) | 1998-04-14 |
CA2137829A1 (en) | 1993-12-23 |
WO1993025577A3 (en) | 1994-02-17 |
AU4633293A (en) | 1994-01-04 |
JPH07508001A (en) | 1995-09-07 |
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