JPH03501850A - Factors associated with essential hypertension - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Factors associated with essential hypertension field of invention The present invention describes novel factors associated with essential hypertension and similar factors to such factors. Involved in discovery. Specifically, the present invention provides a method for determining hypertensive factors (hyp!r) in patient samples. Qualitative and quantitative biochemical analysis to detect It concerns the method. The present invention also applies such hypertension factors and similar factors. Treatment methods, such as regulating hypertension, vasoconstriction, and increased sodium excretion. Ru.

Furthermore, the present invention also relates to a treatment method using a new blocking agent against a new factor. Ru.

Background of the invention 6. Essential hypertension is the number one cause of morbidity and mortality in the United States. NO Although millions of people are affected by it, its etiology is still unknown. A few years Blast before! in is a hypothetical natriuretic hormone or factor. proposed a theoretical system linking the etiology of systemic hypertension (Blmusjein et al. Annuals of lnj! rna! M! dicine, 98: (No. Part 2') 785-792 (In2)). First, the large number of patients with essential hypertension Some people have a genetic defect in their renal sodium transport system, which causes them to have more sodium than people with normal blood pressure. Less excretion. As a result, hypertensive patients retain more water in their bodies than healthy individuals. It's easy. As sodium accumulation increases, the extracellular fluid volume (ECV) gradually expands. However, when the extracellular fluid volume expands, circulating factors act to increase sodium retention. and increase sodium excretion (excreting sodium with urine).

The hypothesized factor is Na, an enzyme involved in the reabsorption of sodium from urine by renal tubules.

It performs this function by inhibiting K-ATPase.

Causes of sodium loss (increased sodium excretion) and water loss (diuresis) from the kidneys This factor is called natriuretic hormone, based on the claim that There is.

In reality, a new equilibrium state is created so that sodium and water content appear to be "normal". Even when the condition is established, inhibitory factors circulate in the blood at high concentrations. Unfortunately, Na. The action of K-ATPass is not limited to kidney cells, but also smooth muscles, such as blood vessel walls, It is also widely distributed in the heart and nervous tissues. Therefore, this circulating factor is present in these tissues. It also causes vasoconstriction in the blood vessels, resulting in high blood pressure.

Circulating Na, which occurs with volume expansion and/or hypertension, -ATPgse inhibition Many reports have been published regarding controlling factors. Purified enzyme in vitro Based on reduced substrate consumption due to Many researchers have used enzyme inhibition assays to compare the plasma of hypertensive patients with the blood of healthy individuals. They point out a significant difference in plasma. Plasma to separate the 10 inhibitory fractions in serum Electrophoresis was used. Increase in circulating inhibitory factors increases intracellular sodium in red blood cells and It was also associated with an increase in intracellular calcium in platelets. It's not my first attempt However, C1oi1 etc. are heat-resistant, low molecular weight, anionic Na - When a substance with an ATPase inhibitory effect is isolated and injected into the cerebral ventricle, Blood pressure increased (J, F, C1oix, etc. Advances in Neph rology, 14: 161-171 (1985)'), Ouabain Suppression of Na-ATPase by Na-ATPase occurs at hormone-like concentrations; Some researchers have turned to substances such as endotonic glycosides.

For example, there are many unexplained modes that occur during digoxin radioimmunoassay. It has been reported frequently. Digoxin-like immunity from plasma of newborns, patients with renal disease and pre-childhood syndrome Reactive Substances (“DLIS”) have been discovered. Some of these reports are uncompensated This can be supported by matrix effects, drug and antibody cross-reactivity. DLIS (s) was extracted from plasma, urine, brain, liver and adrenal tissue. acids, bases, heat and An attempt was made to elucidate the properties of DLIS(s) by medium extraction, and the results were As a result, it cross-reacts with anti-digoxin antibodies and has an inhibitory effect on Na-ATPatc. 1 to 5 types of low molecular weight (200 to 700 daltons) and markedly polar substances quality was achieved. However, little is known about its physical properties, and isolation protocols are extremely diverse. Such a hypothetical natriuretic hormone There are no publications regarding the full picture of enzyme inhibition or enzyme binding by Mon, and research is limited. It has just begun (Am, J, Physiol, etc. of Hat+p!rt, , 24716: F919-F924 (1984)).

It has been reported that false positive reactions occur in DLIS tests in the plasma of dialysis patients. (Grauc et al., Ann, Intern, Med, 99:604 -608 (1983)). digoxin immunity in plasma obtained from these patients. This finding was considered important at the time in view of the interfering effect of DLIS on epidemic measurements. deer However, it has also been pointed out that plasma DLIS levels are high in obese dogs (Gr. Na+urc such as ube+ 305: 6646 (198B)).

Various biochemistry for measuring so-called natriuretic hormones or factors A measurement method has been proposed.

U.S. Patent No. 4.665.019 to Ham17n et al. - A method for measuring plasma levels of inhibitors of ATPgse is shown. Hamlyn The method is based on the inhibition of Na, K”ATPgse by protein-depleted plasma. It does not specify a specific natriuresis factor. Nard i etc. have a molecular weight of 10,000 to 17,000 daltons and are associated with hypertension. Hypertension or hypertension in mammals by detecting at least one type of protein. claim to have developed a method to diagnose the presence of a predisposition to hypertension (Nudi et al. No. 4,321,120). More recently, anti-digoxin antibodies have been used. Radioimmunoassay (R IA) method has been reported but does not identify the factor (Morise et al. ndocrinol, 32(3): 405-11 (1985)).

Recently, Kuske et al. I tried to elucidate the conflicting situation (conflicting 5NuNion)'' (K lin L+ehentihe+, 65: 553-59 (19?)') . Regarding “conflict states”, Kuske et al. (Na=+K”)-ATPage inhibitors have been reported to be quite diverse. (Delardcner et al., Phyaol, Rev. 65: 658 -759 (1985)).

These factors obtained from different sources are associated with digoxin antibodies and serum (Cra BOS etc. FE BS Led, 175: 223-228 (year of publication unknown) , and Kelly et al.], Biol, Chem, 260:11396-114 05 (1985)'), and in partially purified fractions of serum, urine, and tissue fractions ( Ann, Rev. PhySiol, 46:343-35. 8 (1984); the above literature of Crabos et al.; De Wardcner Physiol, Ppv, 65: 658-754 (1985), and Kelly et al. (cited above) Immunity against (Na-+K”)-ATPage Conflicting results regarding reactivity were observed. Low molecular weight substances are extremely polar (De Wardener's Ann, Cl1n, Biochem, 19:137-140 (1982)) or peptidic (Buc Karew's above literature; Grubar et al.'s Proe, SOC, E+ker B iol, Med, 159:436-467 (197B); Klingmu l l e r etc. K11n Wochensiher, 60: 1249 -1253 (1982); Rengl Phyaol of Krame+ etc. , 8:80-89 (1983); Morgan et al., J. Biol, Ch. em, , 260: 13595-13600 (1985)), and are steroidal (Biochc+n such as C1oix, Biophys.

Res, Commun, 131: 1234-1240 (1985)) This was suggested. Furthermore, unsaturated fatty acids (Biochc of Bidud et al. m.

Bioph7s, Act, 769: 245-252 (1984); Tamur2, etc.], Bio, Chew, 260: 9672-96 77 (1985)) and Dehydroni Special Table Sen 3-501850 (4) Piandrosterone sulfate (Rcs of Vxsdev et al., C.

mmun, Chem, Pgjhol, Pha+maco1. , 49:38 Fu 3 99 (1985)), digitalis-like properties were observed. conflict condition Despite Kuske's efforts to elucidate the Purification and identification of the modified hormone was not possible.

Much has thus been made regarding the nature of the hypothetical natriuretic hormone or factor. However, prior to the present invention, the isolation of factors closely related to hypertension in a broad sense, No theory has been successfully refined and identified. In addition, the present inventors found that innate hypertension One of a group of hypertensive factors that allows chemical synthesis of derivatives or analogs of the factor. The general chemical structure was elucidated. Identification and differentiation of such hypertension factors and their derivatives Separation and synthesis enable the development of a range of different therapeutic and diagnostic applications. .

Summary of the invention The present invention discloses that specific factors in animal tissues or body fluids are associated with essential hypertension. It is based on observation. Such endogenous hypertension factors (herein referred to as “HF ”) was isolated, purified, and its chemical structure was elucidated.

Therefore, the physical and biological properties of endogenous HF and its synthetic analogs HF can be characterized by its general chemical structure. HF is a polar lipid, e.g. For example, the molecular weight is about 521 to 541, and the ultraviolet spectrum has a maximum at about 186 nm. Physically characterized as a polar lipid such as an unsaturated phospholipid or sulfolipid can be attached. Biologically, HF is Na, -ATPaae, Ca, Mg-AT suppressing at least one of P age and calmodulin-activated Ca-ATPxse. and can form a complex with anti-digoxin antibodies.

As is clear from the chemical structure, HF and its analogues are represented by the following formula: Contains compounds: CH, -0-RI CH-R2 R1 is C10 to C26 unsaturated alkyl having at least three double bonds or is Asil, R2 is -OH, -H, -CH, or CIO to C26 unsaturated alkyl ester , R3 is S or Pl R4 represents C1 to C6 saturated alkyl, and R5 represents NH or alkylamine. vinegar.

Furthermore, the present inventors found that two molecules of the same molecular weight, each associated with essential hypertension, discovered that HF exists in the form of a mixture of the two compounds HF-1 and HF-2. . Surprisingly, we found that HF-1 and HF-2 are associated with essential hypertension. It was also discovered that the two main physiological effects are different from each other. in isolated and purified form Administration of HF-1 mainly induces an increase in sodium excretion, whereas administration of HF-2 mainly induces an increase in sodium excretion. This mainly causes vasoconstriction. Therefore, in the following explanation, the term HF is Natural mixture of HF-1 and HF-2 or predetermined separated HF-1 and separated HF-2 shall mean a mixture.

The present invention relates to the administration of HF, HF-1, HF-2, or similar substances to animals. Also provides a method for regulating hypertension, increased natriuresis, and vasoconstriction by . The present invention also provides specific monoclonal and polypeptides for HF, HF-1 or HF-2. Clonal antibodies and fusion cell lines, i.e. hybrids producing monoclonal antibodies It also provides a method for making porcelain.

The present invention also detects the presence of HF, HF-1 or HF-2 in patient samples. In vitro (in vitro) biochemical analysis method for determining the amount or amount of We also provide the equipment.

Brief description of the drawing Figure 1 (a) and (b) show the proton-added molecule ion peak appearing at 532 of HF. In the mass spectrum, Figure 2, the maximum peak appears at approximately 186 and the second peak appears at approximately 223. Figure 3 shows the polar lipid properties of HF.72. 8 5. Mass spectrum with peaks appearing at 99.105, 273, 291 and 346 Tor, Figure 4 is an HPLC graph showing the presence of HF-1 and HF-2; Figures 5A and 5B show the changes in systemic vascular resistance (SVR) after HF injection in sheep. The average SVR values were plotted over time over the experimental period to show the effect of injection Graphs, Figures 6A and 6B, show that SVR increases within 5 minutes after Sodium fractional excretion after HF injection (hereinafter referred to as FE Na (!racjio nal (also referred to as Na), that is, an increase in sodium excretion Showing increased excretion, FE N! Groups suggesting that the response is slower than the SVR response rough, Figure 7 shows the vasoconstriction effect of HF in arterioles treated with ultrafiltrate containing HF. Comparing the vasoconstrictive effects in arterioles treated with ultrafiltrate containing control substances other than Comparison: Among the four substances, the HF reagent induced the greatest contraction of the anterior placental artery in humans. Figure 8 shows the ultrafiltrate containing HF in one kidney of eight dogs. The fractional sodium excretion (FE) achieved by administering a control substance to the other kidney FE Na values were compared between kidneys administered with reagent (blank bars) and kidneys administered with control substance. The kidneys (shaded bars) are shown separately, with the period before kidney infusion as the baseline period. , a bar graph with the period during and after the injection as the experimental period; Figure 9 shows the concentration of administered HF and FE Na in 8 dogs.

i.e., showing a relationship between increased sodium excretion and In relation to the difference in HF levels in the ultrafiltrate, the difference between kidneys administered with reagent and kidneys administered with control substance Graph depicting the maximum FENa increase difference, Figure 10 shows the effect of HF on the dynamics of 4SCa2-accumulation in dog kidney cells. Graph showing, Figure 11 shows the effect of HF on the dynamics of 45Ca2- accumulation in monkey smooth muscle cells. A graph showing, Figure 12 shows the dynamics of 22Na- and 45Ca2m transport in monkey smooth muscle cells. Figure 13 is a graph showing the effect of HF-1 on monkey smooth muscle cells. Graph showing the effect of HF-2 on + and 4sCa2 transport, Figure 14 shows human Na in in vitro experiments on red blood cells.

K-ATPase and Ca, Mg-ATPBse inhibitory effect, HF-1 and HF-2 produces a significant inhibitory effect on both oxygen levels approximately proportional to the dose administered. A graph showing, Figure 15 shows the fast atom bombardment mass spectrum (FAB MS) of HF-2. Figure 16 is a collisionally activated combined mass spectrum (CAD MS) of HF-2. Figures 17A-G show the quality observed in the mass spectra of Figures 15 and 16. structure of all fragments with an amount greater than 100, Figure 18 shows pump A to Na of HF (semi-purified) in isolated human RBC membranes. TPase inhibition in relation to dosage, IH% activity = 7.5 nmol Pi Graph shown as 7mg protein/min, Figure 19 shows Ca pump A of HF (purified mixture) in isolated human RBC membranes. TP asc and calmodulin-activated Ca pump A TPase inhibition 100% activity of calmodulin-activated ATP age in relation to dosage. =66.7nmol Pi/min Graph shown as 7mg protein, Figure 20 is an isolation protocol for HF obtained from plasma of hypertensive patients.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS It is believed that natriuresis factors or hormones are involved in the causes of essential hypertension. It has been a long time since the hypothesis was formulated.

In a broad sense, the present invention is also referred to as digoxin-like immunoreactive substance (DLIS). These factors have been isolated, purified, and structurally characterized. This factor has one characteristic In order to avoid overemphasizing only this factor, this factor is referred to herein as a hypertension factor or will be called "HF".

HF isolated from animal tissues or body fluids and purified has a molecular weight of 52 It is characterized as a polar lipid of 1 to 541. In the ultraviolet spectrum of HF, approximately The maximum peak is at 186 nm, the second peak is at about 223 nm, and the minimum value is at about 203 nm. be.

HF can be isolated from tissues or body fluids of various animals, for example It can be extracted from filtered blood obtained from dialysis patients suffering from hypertension. Separation of HF There are various methods of purification, some examples of which will be described later as Examples 1 to 3, and The flowchart is shown in Figure 0. Typically a body fluid or tissue sample obtained from a patient Their HF separation involves extracting the sample with an alkaline solution in the presence of ammonium ions. by a method that includes the steps of extracting and then extracting with an ether/acetone solvent. is achieved.

Once HF has been isolated and purified, it can be characterized by its physicochemical properties and It can be distinguished from other natriuresis factors previously reported. 1st As shown in the figure, the results of fast atom bombardment (FAB) mass spectrum analysis of HF , with a molecular weight of about 521 to 541, more specifically about 531 in the proton-deprived form. A single dominant component was obtained. Relatively small peaks were also observed at approximately 363 and 345. I can't stand it. In the ultraviolet spectrum of HF, the maximum value is at about 186 nm, and the maximum value is at about 223 nm. A smaller peak than that, with a maximum value at 203 nm, is observed.

Other analyzes have shown that HF is a compound that takes the form of unsaturated phospholipids or sulfolipids. It is suggested that it is a polar lipid compound. As shown in Figure 3, HF is used as a lipid. We characterized 72. of polar lipid molecules. 85. 99. 105.273. Based on mass spectral data showing peak characteristics at 291 and 346 This was the result of the analysis.

After determining the lipid properties, back calculation from the known molecular weight of the backbone structure reveals that HF is unsaturated. It turns out to be a phospholipid or a sulfolipid.

HF can also be characterized by its biological or biochemical properties. Book According to the theory of the etiology of systemic hypertension, HF suppresses the action of Na-ATPase. Can be done. Na+urc of Hamlyn et al., 300: 650-652 ( A modification was made to the Na-ATPage inhibition analysis method described in (1982). It was confirmed by the method. The inventors also reported that HF isolated from patient plasma is Ca, Mg-AT Pa5e and calmodinilin-activated Ca-ATPas c, thus altering calcium transport across the cell membrane. discovered that it increases intracellular calcium concentration, thereby making cells hypersensitive. did. The present inventors investigated Na/N-ATPase in red blood cell (RBC) membrane, Ca.

Mg-ATPase and calmodulin (Cam) activated Ca”ATPxse The influence of HF on the action was investigated. Purification (after HPLC) HF with different concentrations Using I was able to do that. As is clear from the measurement results shown in Figures 18 and 19, HF IC6° is Na, 10ngSCam activated Ca for -ATPase -15 ngs inactivated Ca, mg-ATPas for ATPxse! against It was IOB. Cam activated Ca-TPxs! Ca, M than for g-ATPas! The high IC5o for HF is due to the interaction between HF and Cam. This is thought to be due to interaction with the pump (calmodulin binding part).

HF can also form complexes with anti-digoxin antibodies. This complex formation Reagents purchased from New England Nuclear (Rainen D igorin 2J KN, Ncv England Nuclear, vsa Chusset County (11862, North Billerica) according to manufacturer's instructions. Depending on the use, two or three cases were carried out. However, 140 m mol/L A solution of 2 (10 g/L bovine γ-globulin dissolved in NaCg) 10 μL was supplemented. Comparison of antigen-antibody binding of unknown substances with digoxin standard in serum did.

HF increases the concentration of sodium and It can also be characterized by increased calcium absorption. This experiment The procedure described in Example 7 was followed, but Na, -ATPase and Ca, Mg- The results supported the ATPase inhibitory function. in individual platelets due to the action of HF. Platelet aggregation can be activated in response to the increase in intracellular calcium that occurs. This is also a feature of HF.

As a result of structural analysis of HF isolated from the plasma of patients with essential hypertension, HF-2 was found to be α It has a 19:4 carbon to carbon (19 carbon:4 double bond) fatty acid side chain and has a molecular formula of C25H4. It was found that it is a novel phosphatidylserine derivative represented by 209NP. Ta. The chemical structure of endogenously produced HF-2 isolate is 1; CH20CCl8H29 H-OH A similar compound to the above compound was synthesized as described later in Example 10-12. , showed similar biological effects to endogenously produced HF isolated from patients. Therefore, the book The invention also relates to compounds having the following general formula.

CH20R30R4CH Rl is CIO to C26 unsaturated alkyl having at least three double bonds or is Asil, R2 is -OH, -H, -CH3 or CIO to 026 unsaturated alkyl ester , R3 is S or Pl R4 represents C1 to C6 saturated alkyl, and R5 represents NH or alkylamine. vinegar.

Without wishing to be bound by any theory, the biologically active portion of the HF molecule is glycerol. It is thought that it is an unsaturated fatty acid side chain at the alpha carbon of the molecule skeleton. Therefore, α carbon The R1 part in the element is unsaturated from CIO to C26, preferably from C14 to C22. Table 1,3-501850 (7) characterized as alkyl or acyl I get kicked. In the most preferred embodiment, R1 is C18-C20 unsaturated alkyl. or acyl. Furthermore, biological activity is associated with at least three double-bonded fatty acids. Seems to require unsaturation. Preferably, the double bond is conjugated. follow Therefore, the upper limit of conjugated double bonds in the C26 side chain is 13.

For the other moieties, namely R2 to R5, the conditions limiting the selection are those of the fatty acid side chain. The only condition is that it does not cause steric hindrance that would interfere with biological effects. minutes from the patient The R2 moiety at the β carbon of the released HF is often -OH, but in the present invention Also included within the scope are compounds having -H or -CH3 moieties.

R2 may be a C10 to C26 unsaturated alkyl ester. Regarding the R3 part , the present invention contemplates sulfur and phosphorus. R4 linkage group is C1 to C6, preferably or C1 or C2 saturated alkyl, R5 moiety is amine or alkylamine group But that's fine.

Representative examples of compounds within the scope of the invention include lysophosphatidylserine (14: 3), (lit: 4), (19: 4).

(20:4), (22:4) and (2,6:4) (number of carbons in parentheses: double ether and ester analogs (number of bonds), and lysosulfolecithin (19: 3) nityl and ester-like compounds are mentioned.

During the separation and purification process of HF, the HFs have the same molecular weight and similar physical properties. However, it is a mixture of compounds HF-1 and HF-2 that have different physiological or biological effects. It was discovered that it exists. That is, HF-1 induces increased sodium excretion. HF-2 can induce vasoconstriction, whereas HF-2 can induce vasoconstriction. Chrollo Thin layer chroma using form:methanol:R20 (volume ratio 65:35:5) as solvent HF-1 and HF-2 can also be distinguished from each other based on the results of toography (TLC), and R The f values were 0.90 and 0.85, respectively. It's just that the physiological effects are different. Specifically, HF-1 and HF-2 are related to their respective relative Na, -ATP, and e-suppressing abilities. can be distinguished even if The relative ATPase inhibition ratio of both is approximately 1:20 (HF-1:HF-2). In Example 6, high pressure liquid chromatography (H Explaining the method of separating a mixture of HF-1 and HF-2 using PLC Ru. The method of the present invention for separating HF-1 and HF-2 mixtures by HPLC Preconditioning the column with extract from the sample (e.g. , preparing a pooled lipid from a sample extract) and a mixing ratio of about 15-20 + 15-25: 55-75 methanol: acetonitrile: water solvent separating a pair of compounds.

Once HF and separated pairs HF-1 and HF-2 have been separated and purified, they can be used in various Those skilled in the art can easily foresee applications for therapeutic and diagnostic purposes.

Furthermore, as mentioned above, HF derivatives or analogous compounds can inhibit the biological effects of HF. It can be chemically synthesized so that it can be maintained. For ease of explanation, the following explanation , the expressions "HF", "HF-1" and ``HF-2'' refer to these Synthesized derivatives or analogous compounds are also included.

Once the role of HF in the pathogenesis of essential hypertension is clarified, pharmaceutically effective doses of H. By administering F, HF-1 or HF-,2 to animals, their hypertension can be controlled. can be adjusted. Specifically, the administration of HF-1 causes vasoconstriction. It can independently regulate natriuresis and diuresis. Administration of HF-1 It acts as a diuretic and only increases the excretion of sodium and water in the urine. It also has the significant effect of not increasing lium excretion. Taking conventional diuretics Calirum loss is a serious problem for patients with

Vasoconstriction and the resulting high blood pressure are often seen as negative physiological conditions, but Vasoconstriction and relatively high blood pressure may be desirable. For example, if you go into a state of shock By administering HF-2 to patients who have become hypotensive due to this, blood pressure can be reduced. It can constrict the tubes and increase blood pressure. Emergency treatment for dangerously low blood pressure It is a condition often seen in hospital rooms, intensive care units, and cardiac care units. follow to return the patient's blood pressure to a more normal range by immediately administering HF-2. Can be done.

The factors of the invention have potentially valuable pharmaceutical properties.

Administration of HF-1 and HF-2, either separately or in various combinations, results in can regulate hypertension, increased sodium excretion, and vasoconstriction. Therefore, HF.

A drug consisting of a combination of HF-1 or HF-2 is prepared and a specific factor is administered to a mammal. All you need to do is instruct them to administer the drug.

Prominence of HF-1 in promoting sodium excretion without potassium loss This ability makes HF-1 a particularly promising diuretic. conventional diuretics Potassium loss as a result of HF-1 administration is a serious problem and It can be overcome by pharmaceutical action. Causes both increased sodium excretion and vasoconstriction If desired, the HF mixture before separation or a predetermined mixture of separated HF-1 and separated HF-2 It will be clear to those skilled in the art that the following may be administered.

In addition, by administering an appropriate amount of HF, platelet formation can be induced, and platelets can be HF can also be applied therapeutically by treating it in vitro with F and then injecting it back into the patient. can be done.

Compounds of the invention are suitable for use in animals such as mammals, birds, fish, especially humans, livestock and pets. It can be widely administered to animals such as

Following conventional pharmaceutical methods for manufacturing drugs for administration to humans and other animals It is sufficient to process HF. HF is an oral, parenteral, or oral drug that does not react adversely with the active compound. or any known excipient suitable for topical administration, i.e., pharmaceutically acceptable organic or inorganic It can be used in combination with a carrier material. A suitable pharmaceutically acceptable carrier For example, water, saline, alcohol, vegetable oil, benzene alcohol, Liethylene glycol, gelatin, carbohydrates (e.g. lactose, amylose) or starch), magnesium, stearate, talc, silicic acid, viscous para Finn, perfume oil, fatty acid mono- and diglycerides, pentaerythritol , fatty acid esters, hydroxyl methylcellulose, pyrrolidone, etc. Ru.

Sterilize the drug and add auxiliary agents as necessary, e.g. lubricants, preservatives, stabilizers, wetting agents , emulsifiers, salt to control osmotic pressure, buffers, colorants, seasonings and/or aromatics. It can be mixed with substances that do not react adversely with the active compound, such as fragrance substances. Ma It can also be combined with other therapeutic agents, e.g. vitamins, if necessary. Ru.

For internal use, tablets, sugar-coated tablets, drops, drops, tablets, and capsules are particularly suitable. Yes, and if a sweetener can be used, it may be in the form of syrup, elixir, etc.

sterile injectable solutions (preferably oily or aqueous solutions) for parenteral administration; Particularly suitable are suspension, emulsion, or waste-containing implant formats. fixed Ampules are convenient for dosage administration. In addition, liposomes, transdermal patches, or active If the compound is coated with different grades, e.g. sustained release or direct release, such as compositions that are protected by heavy coatings or other methods. They can also be formulated in release compositions. Freeze-dry the HF compound and use this as an example. For example, it can also be used in the preparation of injection drugs.

For topical dosing, a carrier suitable for topical dosing, preferably with a migration velocity greater than that of water. Non-spray formulated compositions, used as sticky to semi-solid or solid compositions containing Ru. Suitable formulations include, for example, solutions, suspensions, emulsions, creams, soft There are salves, powders, liniments, and two-sip sol. For example, it may be mixed with preservatives, stabilizers, wetting agents, buffers, or salt for osmotic pressure control. match. Sprayable aerosols are also suitable for topical administration, in which case The active ingredient is preferably pressurized in combination with an inert carrier material, either solid or liquid. The mixture is mixed with a volatile propellant such as fluorocarbon and filled into a squeezing container.

Dosage depends on the specific compound used, composition, mode of administration, and specific organ to be treated. It's different. The dosage for a given patient is determined in a known manner, e.g. Compare the individual effects of component compounds, e.g. by known pharmacological protocols. You can decide by doing this.

The present invention provides antibodies specific to HF, HF-1 and HF-2 that can be used for diagnosis and treatment. The purpose is to develop.

Polyclonal antibodies can be prepared using known techniques, such as in goat or rabbit antibodies. It can be isolated from the serum of immunized mammals. Noutre, 2 56: 495-97 (1975) by Kohler and Milsfein. By utilizing the basic methods reported, one skilled in the art can determine whether HF, HF-1 or developed a hybridoma cell line that produces monoclonal antibodies specific to HF-2. You will be able to do so. The production method for such monoclonal antibodies is based on mouse or mice. Other suitable mammals are immunized with HF, HF-1 or HF-2 to produce antibodies. The organ that produces the product, such as the pancreas, is removed from the animal, and a cell homogenate is obtained from the removed organ. This cell homogenate is then fused with cultured cancer cells, e.g. myeloma cells. and select hybrid cells that produce monoclonal antibodies specific to the HF immunogen. or to screen and sustainably produce hybrid cells, i.e. The hybridoma is cloned and the HF immunogen produced by the hybridoma is Collecting unique monoclonal antibodies.

The type of antibody produced may be IgM or IgG antibody. HF, HF-1 or if HF-2 is not sufficiently immunogenic in the immunized animal, this can be characterized as a hapten, and the hapten molecule interacts with the carrier molecule at 1 lux. This causes an immunogenic reaction.

1. Methods for linking haptens with carriers are known, and methods for linking haptens with HF are known. There are various carrier molecules that can be used, such as ovalbumin and thyglobulin. monoclone The anti-HF antibody was published by Ranch et al. in the European Journal of Immuno. Published in u+ology, 14:529-534 (In2), T, ko ik! etc. are C11nical Expe+imcnts bHr, Immun , 57: 345-350 (1984), and also E. Har. Yama et al. Cl1n, Lab, Immun, 16: 1-6 (1985 ) can be cultured according to the method that examined cross-reactivity.

Continuous cell lines, i.e. hybridomas, were screened to obtain the antibodies of interest. In vitro detection methods for detecting the presence or amount of HF factor in patient samples Monoclonal antibodies may be used in the method. In vitro detection method for detecting HF and If a sample from a patient is mixed with HF, HF-1 or HF-2, Contact with at least one type of antibody having reactivity, and detect the antibody and HF by immunoassay. to measure the binding of Alternatively, the sample can be mixed with HF, HF-1 or HF-2. is contacted with at least one type of antibody having specific reactivity to the antibody associated with the factor. By measuring the amount of antibodies and correlating this amount with the amount of factor present in the sample. Therefore, the amount of HF in the sample can be quantitatively measured. These measurements require mono Both clonal and polyclonal antibodies can be used. In addition, antibody fragments and anti- Genetically engineered proteins that target different regions of the body are also available. For example, Loiuu et al. is Cl1n, Exper, Im+mcn,, 67: 738-745 ( Regarding the EL I SA method published in 1985), P. Co5tello and F. G+aen is Int! ct, and Immun.

56: The nitrocellulose binding method reported in 173g-1742 (19H) Therefore, immunoassays regarding the factors of the present invention can be performed. Furthermore, anti-jigo In view of the cross-reactivity of HF to digoxin antibodies, anti-digoxin antibodies should be used during immunoassays. The body can be used as an antibody.

The presence or amount of antibody associated with the factor can be determined by labeling the antibody with a detection marker. Therefore, it can be detected.

The labeled antibodies used in the present invention are labeled with the same label as used in known immunoassays. That's fine. Known labels include those disclosed in U.S. Pat. No. 3,940,475. Fluorescent labels used in fluorescence analysis such as those described in US Pat. Examples include enzyme markers such as those disclosed in No. 5.090. Also, for example, B unjer and G+! envood is Nature, 144:945 ( Biochemist B, 13: According to the method published in 1014-1021 (1974), Such radioisotopes may be used as labels.

The present invention detects the presence or amount of endogenously produced HF in body fluids or tissues obtained from a patient. It is also related to the receptor analysis method used. Receptor analysis is basically based on patient samples. HF, HF-1 or HF-2, and a known amount of a synthetic HF analog compound. It is a competitive analysis method that includes a step of contacting with an enzyme that can also complex with similar compounds. . Examples of the enzymes include Na, Ni-ATPase, Ca, Mg-ATPase, etc. or calmodulin-activated Ca-ATPage. high blood in the sample Pressure factors and HF-like compounds compete for a limited number of binding sites on the enzyme side, and each Enzymes have only one binding site. Specifies competition for enzyme binding sites After a period of time, the enzyme is stopped and HF, HF-1 or HF-2 is present in the sample. Determine whether it is connected to. This can be a qualitative or yes/no analysis. be. In the case of quantitative analysis, the amount of HF-like compound bound to the enzyme is measured, This is proportionally correlated to the amount of hypertensive factor present in the patient sample.

This correlation is achieved by labeling HF-like compounds with a detectable marker, free or bound. This is usually done by measuring the amount of HF-like compounds. sample enzyme Samples or HF-like compounds should not be reacted simultaneously with samples or HF-like compounds. It is preferable to culture in advance with the material.

As already mentioned, HF contains Ca, Mg-ATPAN and calmodulin-activated C. a-ATPas! It also has the ability to inhibit calcium transport across cell membranes. You can also. Therefore, competition between HF and Ca-ATPa+e type enzyme substrate Based on this, receptor assays can be developed. Na, ni-ATPas! based on This Ca-ATPxsc-based assay has unique advantages compared to . In other words, there are many substances that suppress Na-ATPase, but Ca, Mg -Specific inhibition of ATPage or camoshlin-activated Ca-ATPasa The present inventors are not aware of any endogenously produced substances. Therefore, Ca-ATPas! based on suppression This analysis allows for a more accurate quantitative analysis of HF in the sample. A person skilled in the art can As can be easily understood, the analytical method of the present invention can be qualitative or quantitative. Any product containing HF, HF-1 or HF-2 that has been separated or decomposed is It can be used for analysis of unprocessed patient samples or their extracts.

, HF, HF-1 and HF-2 are preferably detected in liquid samples; It can also be detected in textile samples. Liquid samples utilized according to the invention These include whole blood, serum, plasma, urine, sweat, tears, and saliva. Less specific to the HF in question Both HFs contain one type of antibody or enzyme.

A diagnostic device can be constructed to detect the presence or amount of HF-1 or HF-2. Wear.

In addition to the therapeutic use of the factor of the present invention, the hormonal action of the factor can be to block HF concentration by administering monoclonal antibodies specific for this factor. There are treatments for patients who are too sensitive. administering specific monoclonal antibodies to patients The ability to block HF, HF-1 or HF-2 can be used to treat high blood pressure, It can regulate increased excretion and vasoconstriction.

Furthermore, once the monoclonal antibody against the hypertension factor of the present invention is isolated, H Known methods that detect and block the F, HF-1 or HF-2 receptor site Anti-idiotypic antibodies can be developed by using this technology.

Not only antibodies but also receptor sites for HF, HF-1 and HF-2 can be blocked. Other substances that can be used may also be used, such as dibuxin and ouabain. That is, for the factor Modulate the pharmacological effects of endogenously produced factors by blocking receptor sites be able to. For example, patients with preeclamptic toxemia have high levels of a known vasoconstrictor. DLIS and Angiotensin n have been observed and are thought to be related to its pathogenesis. (Am, L 0bsL Gyn, of Gorejebener et al., 101:397-400 (1968) and Gudson et al. 0bsL Gyn, 150:83-85 (1984)). Therefore, Antibody Flag, a monoclonal antibody that can bind to and inactivate HF or antibody derivatives, or anti-idiolytic agents that can block the HF receptor site. Ip antibodies are promising for the treatment of pregnancy toxemia.

Example I Separation and purification of HF Separation and purification from tissue Fresh tissue, such as kidneys removed during surgery or cadaver dissection; The cultured tissue may be processed immediately at λ or stored at -70°C. Thaw the tissue Then, the mixture was chopped finely into small pieces, and a rotating cutting blade was operated for a short time to homogenize the mixture at a low temperature. centrifugal The precipitate is removed by separation, the supernatant is filtered as described below, and the supernatant is filtered with benzene etc. Extract. Tissue culture media are treated similarly to plasma samples.

Separation and purification from blood or urine Separate low molecular weight compounds by ultrafiltering fresh whole blood or urine. Let go. Plasma or serum may be separated from blood prior to ultrafiltration. Screeni The ultrafiltration step can be omitted for laboratory samples. cut off Ultrafilter with a diameter of 50,000 to 2,000 daltons, such as YM-2 shear Flow membrane (Amicon Corp.) can be used, and Gi bco filters (kiniprophane) are also suitable.

For mass production, urea is broken down by adding urease to blood ultrafiltrate or urine. . Taking into account the action of the enzyme and the urea content in the filtrate, this step is carried out at approximately 25°C. Steps should be short steps. This step may be omitted, but in that case, Separation yields are reduced. When the pH increases with the production of NH, IMHI Titrate to p, H7, o by adding . Urea content is 15 mg/dL or more In the above case, this step is desirable. Urea in clinical serum screening tests Ze treatment may be omitted.

In mass production using liquid matrices with low HF content, samples, e.g. The volume is reduced by lyophilizing the liquid ultrafiltrate. Then this drying The final volume (mL) of the residue was 50% N used to titrate to pH 8.7. Dissolve the residue (g) in water to make it 3 times the amount of the residue (g) including the volume of H4OH and make it liquid again. Make it. Add dry NacQ to saturate the solution.

1.0 to 3M ammonium acetate for urine or serum laboratory tests.

Adjust the pH to 8.6 with sodium chloride. If using Tris or other buffers , ammonium salts or ammonium hydroxide must be added. optimal Dilution should not exceed 3 times to achieve recovery. To achieve maximum extraction Ammonium ion is required. If the pH is set between 7.2 and 9.2 Good, but maximum recovery occurs between 8.6 and 8.8.

Solvent extraction is carried out in two stages. In the first step, add an equal volume of benzene to the sample. Remove non-polar lipids with a tube.

Discard the organic layer. The second step separates the HF. For purification, use the same volume as the sample. ether:acetone (1:1) is used. This depends on the length of time step, and the longer the solvent exposure time, the greater the amount recovered. 1 to 2 before separation 0 hours of mixing is required; 1 hour is sufficient for extraction for clinical screening serum tests. be. To handle large amounts of samples, use ether:acetone (5:7). good. Type of solvent along with HF for other solvent systems with similar polarity and solubility Extract the proportion of pollutants according to the Repeat the extraction 2-3 times and save the organic layer. . Centrifugation is required to separate the layers. If antioxidants are not used, The solvent must be evaporated under N2 atmosphere as quickly as possible. For sample drying Freeze drying is required. After this step, exposure is limited.

For clinical screening analyses, organic residues should be dissolved in saline or N20. lysed to the original sample volume or used for immunoassays or receptor assays. Reduce the volume to less than the original sample volume if necessary due to certain sensitivity limitations. To redissolve Requires at least 30 minutes and thorough stirring. Clinical serum or urine determination For quantitative factor analysis, organic residues are separated into HPLC solvents, e.g. methanol:acetonitrile. Trill: Dissolve in water (15:15:70). In semi-quantitative separations, residual chloroform: methanol: water (H: 35: 5) or methanol: Isopropyl alcohol:water (15:15 near 0), dissolved in TLC solvent .

For mass production, an additional extraction step is added.

Dissolve the residue in a small amount of chloroform:methanol:H20 (60:35:5) let Insoluble residues are separated by centrifugation, washed and discarded. Solvent in N2 atmosphere Remove under ambient air and lyophilize if necessary. The residue was then dissolved in HPLC solvent. Let me understand.

When handling a large number of samples containing nine or more types of contaminants (for example, B10 -Rid Laboratories Inc, 100-200 mesh B If you use silicic acid column chromatography (equipped with io+il A) stomach. The column (2X 27cm) was heated at approximately 25°C with chloroform:methanol: Formed using N20 (60:35:5') as a solvent. sample in this solvent Add chloroform:methanol:N20 mixture of 60:35:2. Elute. The two factors elute before the majority of the contaminating lipids. Enzyme inhibition or immunoassay, by thin layer chromatography (TLC) as described below. It is more convenient to monitor the ingredients by All extraction steps without exception This is done in the dark under an atmosphere of N2 and/or vacuum. and removed at a temperature of 50°C or less. Freeze the residue, which may have a greasy appearance. Do not over-dry in condensation dryer.

Preferred separation methods for use with immunoassays are as follows. Each test at room temperature 0.3 in a test tube (polypropylene conical centrifuge with screw cap) Inject mL of serum. 0.3 mL in each test tube 1. OM, acetic acid pH 8.6 Inject ammonium buffer. Inject 0.3 mL of benzene into each test tube and Stir for 0 seconds. The tubes were then centrifuged at room temperature for 10 minutes with a force of approximately 1500 g. Separate.

Remove the benzene with a pipette, discard, and remove the remaining by using a N2 stream in a 44°C water bath. Blow off the organic layer and about 50% aqueous layer. 6, OmL of ether in a test tube Le: Inject acetone and stir gently. Place the test tube in a locker and store it in the dark for 1. Leave it for 5 hours. Wash each test tube with 0.5 mL of ether:acetone mixture; Stir, pipette, and add this to the solvent kept in the dark. 44 Evaporate to dryness with N2 at °C. Again 0.3 mL of saline or N20 Dissolve in. Stir well and solvate for at least 30 minutes, preferably Leave it for 120 minutes.

Thin layer chromatography (“TLC”) purification steps of hypertensive factors as described above. or can be applied to extracts of blood, urine or tissue culture to monitor the progress of TLC can be used as a fast semi-quantitative separation system.

As an adsorbent, a glass fiber sheet impregnated with silica gel (ITLC- SG, Gelman 5sciences, lnc) or LHP-KD high performance Use 200 micro thick glass reinforcement plate (Wxtmann) etc. Can be done. The solvent/system was a chloroform:methanol:H20 mixture (60:35 :5). Solvent systems with similar polarity, e.g. ethyl acetate, acetone or Satisfactory separations can also be obtained with acetonitrile or acetonitrile. At 25°C (e.g. 2 After separation (for 0 min), plate 50% H) P O or 25% T Develop with CA and heat to 100°C. on a plate of digoxin standard 0.77 The factors showed typical Rf values of 0.8 and 0.76. HF-1 and HF-2 are They can be discriminated based on their respective Rf values of 0.90 and 0.85.

Example 2 Separation and purification of 8F Hollow fiber artificial kidney ji (e.g., 8m1con, F+eciniua, Normotensive and hypertensive dialysis using Gambro and Trarenol) Hemofiltrate was prepared by ultrafiltration of the patient's blood. The filtrate is in the initial stage Contains 0.18 to 0.78 μg/L digoxin equivalent, solid after drying and average It contained 13 g/L. Urea is removed by treating the filtrate with urease, Neutralized and lyophilized. The resulting powder was dissolved in water again to make a volume four times its weight. The mixture was then alkalized to pH 8.8 with 8M NH4OH. 1 hour later, sura The organic layer was extracted with benzene and the organic layer was discarded.

The aqueous layer was then extracted with an equal volume of ether:acetone (5:7). organic layer After storage, dry Na(l) was added to saturate and the extraction was repeated.The organic layer was stored. and evaporated to dryness.

The extraction residue was extracted again with acetonitrile:methanol:water (25:20:55) was dissolved in the first HPLC mobile phase. Variable this (set to 223nm) C18 reversed phase column (25 cm x 5 cm, Wanrs, Milford, MA). and partially purified. Two HF-containing fractions separated under equal conditions (retention times are 2.4 and 2.7 m1n) were pooled, evaporated to dryness, and then transferred again to the second mobile phase (acetate). It was dissolved in nitrile:methanol:water=15:15:70). next HF-1 and HF-2 (retention time 14.4 and 15.7 m1n, respectively) were collected separately. Both chromatographs The flow rate for the fee separation was 1.0 mL/min.

HPLC grade solvents were used throughout this protocol. Water is deionized did. Isopropyl alcohol replaces acetonitrile in one of the HPLC steps. can be used. When reducing the volume of the solvent after HPLC purification, reduce the volume by 40% or more at this stage. Freeze drying of samples must be avoided as losses of 60% can occur. frozen As an alternative to drying, dry under a nitrogen atmosphere (e.g. salt, alcohol, acetonate). After removing the solvent (such as tolyl), the anti-digoxin antibody or Na-ATP HF is recovered using affinity chromatography using ase. You can also

Example 3 Separation and purification of HF By adjusting the pH to 8.6 and extracting with benzene, it can be used for immunoassay. Blood from dialysis patients or its ultrafiltrate (0.3 mL) was treated. Then water-based The phase was extracted twice with ether:acetone (1:1).

After evaporating the pooled organic layer, the residue was again diluted with 0.3 mL of 140 mmol/L N aC! ! It was dissolved in The manufacturer's instructions are to collect 100 μL of this tick sample. According to Engineering 125 digoxin radioimmunoassay (Ri!n!n digoxin Kit, New English+l Nuclea+, NoNh B111e riea, MA01862). However, the buffer is 140 mm. ol/　10μ solution of 200g/L bovine γ-globulin dissolved in LNaCl L was added. Comparing the antigen/antibody binding of unknown components with digoxin standards in serum, The results were calculated as +BDE/mL. The sensitivity of this method is 0.08-4 ngD It was E/mL. Repeated analysis of a single pool over 20 days The degree was 0.28±0.04DE/mL (x + SD).

Since the partially purified HF agent has already been extracted with benzene and ether/acetone, the above No extraction step is employed, from the pooled chromatographic eluate under the action of a nitrogen flow. Remove the solvent from the 5 μL and 25 μL aliquots and reconstitute 140 mmol/ml for immunoassay. Dissolved in L NaCl.

Ultrafiltrate from dialysis patients is treated with urease until free of urea, then Lyophilized. Dissolve 15 to 41 g of the dried powder in water again, pH 8, 6 and the solvent was extracted as described above. Ether: Residue of acetone extraction After drying, add again to chloroform:methanol:water (volume ratio 60:35:1). Dissolve and 1.5X32C! Added to O silicate chromatography column. 1m Chloroform:methanol:water (C60:35:5) was used to elute the L fraction. used. Transfer 10 u of the eluate onto a silicic acid thin layer chromatography plate in the same solvent. The elution profile was followed by fractionating the L fraction. Major components by TLC Lamb fractions >0.690 minutes were pooled. After solvent removal, the residue was passed through four sheep. For the first experiment, 5 mL of 140 mmol/L N a was used for injection. C1:L tanol mixture (2:1) and 3 mL for injection in other experiments. 140 mmol/L N:aCl Nidimethyl sulfoxide (1:2) Each was dissolved. The former is a cloudy or oily solution, the latter is a clear solution Met.

Eluate pool from silicic acid column chromatography crosses with digoxin antibody It contained two substances that reacted. Both have nominal exclusion limits of 1,000 molecular weight. Am1con YA-2 filter (Amicon, Darve+ s, MA). Na, ni-ATPa+e inhibitory function is also similar to digoxin. There were also 1 to 6 contaminants that were not immunoreactive. Among the 6 types of HF agents In the two types, there are only three components in thin layer chromatography, two of which are HF. It was. This means that the other contaminants in the remaining four HF agents are organisms that can be attributed to HF. This suggests that there was no scientific response.

Example 4 Characteristic analysis of 8F - Physical properties Immunoreactivity of HF obtained from blood or ultrafiltrate was measured after solvent extraction. Ma Additionally, the ultrafiltrate extract of the HPLC fraction from the previous protocol was evaporated and the solvent was evaporated. It was again dissolved in 0.8 NaCA and then measured. In both cases, precipitation is facilitated. Add 10μL0200mg/mL bovine γ-globulin fraction■ to No changes were made other than the addition of 125I digoxin radioimmunoassay. A kit (New England Nuclea+) was used. Analysis result The results are linear from 0.1 ng/mL to 8 ng/mL digoxin equivalents, with an error of The level is 0.28±0.04 part g digoxin equivalent/mL (X+SD, n=20) Met. Therefore, HF cross-reactivity with anti-digoxin antibody was confirmed.

Bamlyn et al. Fed, P+oc, 44: 2782-8 (19 85), with the exception of Tris(2-amino-2( HEPES (N- buffer with 2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid). The inhibition of canine Na-ATPase was measured spectroscopically at 340 nm. injection Sample a 5 μL aliquot of the concentrated HF agent on the day of the test and analyze immediately or The absorption spectrum of purified HF agent stored at 0°C for 72 hours and dissolved in HPLC solvent is Measurements were made at 25°C with a variable wavelength recording spectrometer. As shown in Figure 2, approximately 186 The maximum value of the UV spectrum was observed at nm.

Kra! Using os MS-50 triple analyzer, 8Kv accelerating voltage Fast atom bombardment (FAB) mass spectra were obtained at pressure. FAB no ta Argon was used as the primary bombardment gas and the helium was allowed to decay at 25°C. Figure 1 As shown in , the largest peak is at 532, and relatively small peaks are at 345 and 363. was recognized.

Factors were separated and/or quantified using high pressure liquid chromatography. 22 A C18 reverse phase column (25 cm x 5 +nm, Wajer 5 Milfod, MA) was used. Column After thorough washing with tanol, the bulk extract was extracted under these chromatographic conditions. Precondition with a preparation obtained from the pooled lipids eluted at a retention time of 16 minutes. I nged. Without this conditioning of the column, the baseline method The two factors separate as a single peak rather than two peaks based on . Figure 4 shows H Two peaks associated with F-1 and HF-2 are shown. 15 in this column: 15:70 in solvent with retention times of 14.4 and 15.7 minutes, respectively. Factors can be separated under equal conditions. Required purity and starting material is 2 If this suggests that two HPLC steps are more convenient, methanol: Setonitrile: H2O is separated more quickly with 20:25:55 parts. (retention times for factors HF-1 and HF-2 are 2.4 and 2.0, respectively) ．． 7 minutes). When employing two HPLC steps, first The active fractions were collected using a solvent, the respective solvent was removed, and then the H2O The factor is again chromatographically analyzed in a portion of solvent.

A place for semi-quantitative systems to replace serum or urine clinical screening and TLC fractionation. Unless otherwise specified, HPLC is used to measure factors. After separation, the active fraction is subjected to nitrogen flow and Freeze dry.

Example 6 Vasoconstriction and Natriuresis Promotion An experiment was conducted on a man in his prime weighing 50 kg. neck By inserting a catheter into the artery and jugular vein, the arteriovenous catheter is exposed to the outside. Nures were transplanted and experiments were performed at least 1 week after transplantation.

During all experiments, the animals were kept in an upright position and their necks were gently restrained in the cage. . The arteriovenous cannula was attached and detached at its midsection. to the pulmonary artery via the venous cannula An intracardiac catheter was inserted. After confirming correct positioning.

A catheter was left at this site to measure cardiac output (CO) by thermodilution. . Mean arterial pressure (MAP) was measured via the carotid cannula. venous cannula Connect the injection tube to 72.5 mmol/L NaCj! using the injection pump. a constant speed normal losses were compensated for by replenishing with An indwelling catheter was inserted into the bladder. bladder Baseline data collection began after the bladder was completely emptied.

Urine, blood and hemodynamic data (cardiac output and mean Control conditions were established by collecting pulse pressure). 1 hour baseline - After data review, 3-5 mL of HF was injected into the carotid artery. After 5 minutes, 2 more Urine, blood and hemodynamic data were collected every 15 minutes over time.

Systemic vascular resistance was calculated from cardiac output and mean arterial pressure values. creatinine Calculate the glomerular filtration rate (GER) as the clearance (CICr), and Effective renal plasma flow was calculated based on minohippurate (PAH) clearance values. Experimental interval for at least one week. A total of 6 experiments were conducted on 2 sheep.

Urine and blood chemistry Astrg automatic plasma and raw electrolytes, urea nitrogen, creatinine and glucose Clinical analyzer (Backm2n 1nst+umcnls, Fullcrt on, CA) using known electrochemical and spectroscopic methods. measurement Values are accurate to within 1% and have a dispersion rate of 2.5 over the physiological range of the samples. Richt that was %! +ich Cl1nical CbemisHy: Theory and Prnejice, New York: S, Kargcr, Inc., PP, 479-81 (1969). PAH was measured by the Brajjon-Marshall method using spectrophotometry. .

hemodynamic effect Injected HF induced hemodynamic responses in all six experiments. Table IA and Table IB are average hemodynamic measurements obtained during the baseline data collection phase. Values are compared to the average of all measurements after concentrated HF injection. t-test and variance (p<0.Hl) analysis showed that the increase in SVH was extremely significant. . Other significant changes were decreased cardiac output and increased mean arterial pressure.

Table IA Baseline phase and after HF injection Hemodynamic data. Values are above-average (SEM) experimental conditions: mean arterial pressure, cardiac output, total Body vascular resistance mmHg I/min Baseline 74.9 (1,9) 8.2 (0,23) 749 (5,9)H After F injection 78.3 (1,5) 6.9 (0,12) 921 (13) 1) <　0.02　0.0001　0. OHI baseline stage and after HF injection Hemodynamic data. Values are averages (SEM) Experimental conditions Average arterial pressure Heart rate 8 Systemic vascular resistance mmHg! /min Baseline 76.6 (1,5) 7.6 (0,23) 836 (37)HF After injection 80.6 (1,1) 6°6 (0,13) 1011 (32) Figure 5 shows the average SVR values over time over the experimental period. of SVR 5 minutes after HF injection. An increase was observed. SVR was always above the baseline value throughout the 2-hour experimental period. Met. Variance (1)<0. As a result of analyzing H2), extremely significant systemic vascular resistance An increase was observed.

Glomerular filtration as calculated from creatinine clearance even if HF is injected There was no effect on rates.

Similarly, effective renal plasma flow (PAH clearance) is significantly altered by HF. That never happened.

Figure 6 shows the FE Na over time for control and testing for all experiments. The average of As shown, the increase in FENa is slower than the systemic vascular resistance response. won. However, similar to systemic vascular resistance, FENa increases throughout the 2 hours post-injection. persisted. This increase after HF injection was significant (p<0.01. Analysis of variance ).

Effect on kidneys Table 2 shows the baseline data collection phase over 1 hour before HF injection and 2 hours after injection. Renal data in stages over time are presented based on eight experiments. It is shown in Table 2. In addition, an increase in FENa equivalent to four times the baseline value appeared after HF injection. . Urine flow rate increased by more than 50% as FE Na increased. water clearance circulation Ring revealed that water absorption by renal tubules was significantly reduced. In other words, After HF injection, water clearance increased from baseline. of water and sodium Unlike clearance, fractional excretion of potassium decreased after HF injection.

A decrease in potassium fractional excretion with an increase in urinary flow rate and sodium excretion has not been observed until now. This is a surprising finding as it has never been reported in studies on factors that increase thorium excretion. . Normally, the fractional excretion of potassium increases as urine flow increases. However Inhibition of Na-ATPase in terminal nephrons reduces potassium excretion. It is thought that it can be done. Recent studies have reinforced the importance of potassium in essential hypertension. Potassium in the body is known to alter the ability of cardiac glycosides. ing. It is interesting that HF alters potassium handling by the kidneys.

Example 7 HF effect material on monkey smooth muscle and dog kidney cells “CaCl2 (1 mC i/mL) and “N a C1 (100 uCi/mL) are Amersham C Purchased from organization.

Ouabain is from Sigma Chemical Company (SLLoui Minimum essential medium, Denilbecco's modified Eagle's medium, bovine Fetal serum and Hanks Balanced Salt Solution (HBSS) were purchased from Gibco (Long 15 LAND, NY).

cell culture Durbecco's degenerative tissue supplemented with 5% fetal bovine serum was prepared using a 35 mm tissue culture dish. Monkey aortic smooth muscle cells (SMC) were plated in Google's medium. 5% fetal bovine serum Canine kidney cells (CKC) were cultured in minimal essential medium supplemented with C.I.

Keep the cells in a 37°C incubator in a humid atmosphere of 5% CO2 and 95% air. It was heated and used near the fusion stage.

Observation over time SMC and CKC cells in a confluent state were washed as detailed below, and quantitatively determined. HF was added and cells were incubated at 37°C for 30 minutes. 10μCi/in each dish mL of 450ac12 was added and the reaction was allowed to proceed for various specified times.

At the end of each specified time, the reaction is stopped and the cells are exposed to the relevant radioactivity as described below. It was measured.

22N　a　　” and J5Ca24 constant labeled SMC cells close to a fusion state in 3 mL Washed three times with HBSS. Purified HF-1 or HF-2 at various concentrations was added to cells ( Duplicate) and add 1 mL 45Ca” (10 μCi/m L) was added and kept warm for 2 hours until cell radioactivity was measured. 2 hours have passed Aspirate the solution with a roller, immediately transfer the cells to ice, and immediately add 3 mL of cold HBSS. Washed 5 times with After the final aspiration, add 2 mL of 0.5% SDC to each plate. The cell layer was lysed and transferred to a scintillation bottle. Then 1 in each bottle Add 5 mL of scintillation cocktail (Dimi fume) and After vigorous stirring, use a liquid scintillation counter to measure cell radioactivity. I moved it to the tar.

In parallel experiments, identical SMC cells were labeled with 2.5 μCi/mL”Na= The effect of HF on Na content was measured using a 60 mm tissue culture dish and 5% fetal bovine serum. CKC cells were plated to near confluence in minimal essential medium supplemented with . After washing with warm HBSS, 2 mL of MEM was added to each dish. 20 Cells were pretreated with μL of purified HF or 20 μL of ETOH for 30 min at 37°C. , then added Ht, tL of 45Ca C12 and stirred for 1.5 to 10 minutes. The reaction proceeded. At each time point, quickly remove the medium and The reaction was stopped by washing the cells with 5×3 mL of cold MEM. then Extract the cell layer by adding 2 mL of 0.5% SDS and add 15 mL of syringe. Add scintillation cocktail and collect cells in a liquid scintillation counter. Radioactivity was measured. The final HF concentration in each dish was 2.5 ng/mL, total release The radiation/dish was 6.21X 1106 cp.

The initial net 45% of CKC cells treated with 2 ng/mL HF as shown in Figure 10. Although the Ca2-accumulation rate was the same, the amount of 45(:, a2' associated with cells was unknown). It was 1.5-2 times that of treated cells.

Effect of HF on 45Ca2 accumulation trend in SMC cells Durbecco's degenerative tissue supplemented with 5% fetal bovine serum was prepared using a 35 mm tissue culture dish. SMC cells were plated in Google's medium. 1.05ng/cells near fusion state Pretreated with mL purified HF or equivalent volume of 0.9% NaCj' for 30 min at 37 °C. .

Add 10 μL of “CaC12” and monitor the reaction for 5-30 minutes. Ta. Cells were processed as described above for radioactivity measurements. Added reactivity for each dish Total reactivity was 3°45×10 6 cpm.

As shown in Figure 11, there was a slight difference in the initial Ca accumulation rate in experiments using SMC cells. There is. As is clear from these experiments, the effects of HF on SMC and CK Accumulation of 45Ca2'' is possible in both C cells.

SMC cells in a confluent state were washed with HBSS and treated with various concentrations (140-790 pg/mL) of HF-1 was added, and the mixture was kept warm for 1 hour. a5CaC12 or 22 N a C1 was added and steady cell radioactivity was measured as described above.

As shown in Figure 12, HF-1 increases the ratio of Ca and Na as the dose increases. As an example, the increase in 45Ca2- is slight in the case of high concentration HF-1. there were.

This is a parallel experiment to the experiment shown in Figure 12, but here, 390 pg/mL H F-2 was used. SMC cells were cultured as described above. Figure 13 and As is clear from Fig. 14, similar patterns were observed in HF-2 regarding Na and Ca accumulation. A turn was observed, but in the case of HF-2, the accumulation reached a plateau after 1100 p/mL. It became. In both HF-1 and HF-2, the response to dose was higher for Ca than for Na. was also sensitive.

Example 8 Receptor analysis for HF detection in patient samples 100 μL HF extract (For example, for total HF screening, use an ether:acetone (1:1) solution. (separated HPLC fractions for HF-1 or HF-2 quantification) by evaporation to dryness. , redissolved in H2O. Add this solution to 1 [10 μL of dog kidney Na, -AT Pgs! (Sigma 0.6mg/mL) and 0.25mM Na2E 700 μL containing DTA, 5 mM MgCl2 and IHmM (DNaCl) Incubate at 37°C for 100 minutes with 0.05M Tris-C1 (pH 7,2). Ta. Control with 1mM ouabain in 100μL and test substance with 100μL of water alone. Blanks were prepared separately and incubated simultaneously in the same buffer/enzyme mixture. .

Add 0.4 uCi of 3H-ouabain in 100 μL to the mixture and incubate for 30 minutes. did. Stop the reaction with water-cooled buffer and immediately quench by filtration or centrifugation The substance/enzyme complex was collected on a glass fiber filter. with water cooling buffer Retained radioactivity was counted with a liquid scintillation counter. Uaba It was quantified by comparing with In. The quantitative unit is expressed as 0-100% bond. .

Example 9 Structural analysis of HF-2 Blood filtrate of renal dialysis patients by alkaline solvent extraction and reverse phase Cl HPLC as described above. HF was separated from. A, Cl1n of DaBupta, Chcm, 3 3:890 (1987). -ATPase inhibition and immunoreactivity in Na of the purified substance by geoimmunoassay analyzed.

HF-1:HF-2 (DL I S-1: DL) in the blood filtrate depending on the patient There was a difference in the I5-2) ratio. Large amounts of HF while maintaining immune and suppressive ability It was difficult to analyze. This fact indicates that the molecular structure is inherently unstable, or suggested that it is easily altered. Therefore, only HF-2 was extensively analyzed. . The substance described here is equivalent to canine Na-K-ATPase/ng digoxin. Immunoreactive substances were inhibited by 20%.

Mass spectrometry using dithiothreatol/dithioerythretre matrix Fast atom impact mass spectrum from the Le Triple Analyzer (Kra+uMS-50) (FAB MS) was obtained. The sample was introduced at 25°C and an accelerating voltage of 8kv was used. , using argon gas as the fast atom source. The mass spectrometer is a cation detection model. It was operated using the code.

Using the same equipment, a collisional activation solution for cations was performed using helium as the collision gas. Performed mass spectrometry/mass spectrometry (CAD MS/MS) .

In the FAB MS spectrum of HF-2 (Figure 15), m/z is 363.345. ．． Daughter fragments appear at 329.221.195.179.161 and 119. It will be done.

As a result of examining the compound by CAD MS/MS, the fragment shown in Figure 16 was found. tion pattern was obtained. The high peak that appeared at m/z 346 is phosphorus. This is interpreted to be due to the leading group of serine and molecules that remain unchanged after proton loss (first 7A, B). Presence of phosphoserine group in the molecule i;! HCOH2CH(NH3- )(COO−)+: supported by the large peak at m/z 105 attributed to (Figure 17C). Figures 17A-G are observed in two mass spectra. Structural interpretation of all fragments with masses greater than 100 is shown.

The two fragments shown in Figure 16 indicate the presence of new components in the parent compound. It matches. First, the peak at m/z 291 is the proton of fatty acyl fragment. This is thought to be due to addition (Figure 17D). If so, at m/z 273 The larger peak is formed by H20 loss from the same fatty acid fragment. (Figure 17E). There are fatty acids in the human body that match the molecular weight of the structure of this factor. are not known to form.

The molecular structure of this fatty acid was obtained from mass spectral data as described below. . The peak at m/z 123 is a hypothetical fatty acid allele between C10 and C11. This is thought to be caused by division (Fig. 17F). Relatively small at m/z 170 The higher peaks indicate that the fragments are located in the glycerol backbone and not in the phosphoserine head group. Allyl between C6 and C7 of the same fatty acyl chain while retaining the hydroxyl group in position 2 This is thought to be due to the occurrence of fission (Figure 17G). Ratio at m/z170 The relatively small peak suggests a single allele split rather than a double allele split. Therefore, the possibility of a Δ5,8 configuration is higher than a Δ4,7 configuration. implied (Δ indicates the carbon position of the double bond). Two different fatty acyl chains The presence of two allele splits arising from the moiety means that there is one in the fatty acyl chain. There are four double bonds instead of two double bonds and two triple bonds suggests that. Δ5. Double bonds exist in Δ8 and Δ11 in C6 and 07 Fragments were formed by splitting between CIO and C11. This is thought to be caused by.

Although it agrees with the experimental mass spectrometry data, it is only a tentative figure. As a theory, the compound corresponding to HF-2, which is reconstructed and shown in Figure 17, is related to human physiology. It should be noted that this is new to Japan.

The synthetic procedure begins with 1,3-benzylidene glycerol, which is a) 1. P, Phumaxie of Kertsche+, 38: 421-422 (198 This is a variation of the approach described in 3). Benzoyl chloride, potassium hydroxide 1,3-benzylidene Glycerol is 2-benzoyl 1,3-glycerol (this is called compound A). ) was converted to

Commercially available araki using N-bromosuccinimide and triphenylphosphine Arachitonyl bromide was prepared from donic acid. Then arachitonyl bromide and sodium Compound A was converted to an arachitonyl ether derivative using a methane solution. H, Eibl Chem, Phys, Lipids, 26: 405-429 (19 Liberation of position 3 of the glycerol backbone using the approach described in 80) The hydroxyl group was converted to a phosphoserine head group.

Example 11 Synthesis of lysophosphatidylserine (20:4) ew, Pbys, Lipids, IT: 257-270 (1985) commercially available phosphatase using phospholipase D and L-serine as published in Zircholine (20:4.2 (1:4)) and phosphatidylserine (20:4 ．． 20:4).

Phospholipase A in ether as a solvent in the presence of Ca as a conjugate 2, phosphatidylserine (20:4.H:4) is easily lysed. Converted to phosphatidylserine (20:4).

Pentanal and 3-bromopropanoic acid are separated using the standard Witzteig reaction. Δ5・7・10・”-19:4 fatty acids (double bonds at positions 5, 7, 10, 14) A fatty acid with 19 carbon atoms and 4 double bonds was synthesized. using benzene as a solvent from 2-bromopropanoic acid and triphenylphosphine. Formed salt. After adding aldehyde to dry Wittstein salt, tetrahydrof Witsty using n-butyllithium in ran-dimethyl sulfoxide medium Protons were removed from arsenate.

The final product acid was then purified by base extraction.

Lithium aluminum hydride reduction followed by pyridinium chlorochromate oxidation The product Δ3-caprylic acid was converted to the corresponding aldehyde by . then again 3 - Witzteich reaction using triphnicol phosphobromide of bromopropanoic acid The final product Δ3-octanal is converted to Δ36-undecenecarboxylic acid by reaction. I changed it.

The steps in the same order as mentioned above, e.g. reduction, oxidation and Witzteig reaction. Repeatedly, the final product obtained from the same Witzteich salt was Δ3·6·’-14:3 acid. This acid is reduced and then oxidized using the method described above to produce Δ3.6.9-14:3. Obtained aldehyde. This aldehyde is converted into triphenylphosphonate of 5-bromovaleric acid. By reacting with Umbromide salt, the desired Δ5.8.1]・)4-IQ, 4 fatty acids I got it.

Djera++i etc. are Chem, Phy(Lipid+, 37: 257- 270 (1985), fatty acids as side chains. The fatty acid was converted into a phosphatidylcholine compound containing the fatty acid. As a starting material , the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine Cadmium salt of 1,2-glycero5n-3 phosphocholine and 19:4 fatty acids were used. The obtained phosphocholine molecules were treated with phospholipase D and L-separate. Phosphorus was used to convert it into a phosphoserine molecule. In the presence of Ca”, phosphoseri incubate with phospholipase A2 to incubate the glycerol skeleton at position 2.4. The final product was obtained by splitting the fatty acids.

Minimize the amount of HF used for each analysis and reduce the time required for analysis. To shorten the time, Thomz + Hinds and Ho5sein 5adrx adeh is A using 115% of the amount of HF (or reagent) normally used in analysis. A microplate assay was developed to measure TPxse. This analysis The total volume used for is IHmL. Tissue culture with 96 wells (wells) Using a culture plate, first add HF to the plate and evaporate the solvent under N2. .

Add Na, Mg and Ca (for -Ca and Cam analysis) to all wells. Add the reagents containing each well to all wells except the Na and K-ATPase wells. Add ouabain (0.01 mM) to the solution. Then add the membrane sample. (Protein concentration: 0.0045 mg) and incubate the plate at 37°C for 15 minutes. Next Add ATP (3mM) to all plates and incubate the plates at 37°C for 1 hour. do. Stop the reaction by adding 20 μl of 5% SDS. Arco A mixture of rubate, acid molybdate and SDS (130 mL) was added to the plate. The developed blue color is measured at 81 Onm using a plate reader.

Blank = well containing ATP, membrane and ouabain Mg”=well containing ATP, membrane and ouabain Na,/to=ATP and membrane and contains no ouabain. Well containing Ca=ATP, membrane and ouabain Cam=ATP, membrane and calcium Wells containing modulin Specific activity: Mg ATPaae = Mg - ATP (blank) / 60mn/mg Na/K ATPase = Na, -MgCa ATPase = Ca-Mg Cam ATPase = Cam-Mg In the present invention, micro ATPase is Check HFO specific activity (biological activity) using TP asc analysis, This was compared with digoxin antibody analysis (RIA). This allows the purification process to Monitor HF activity and consider whether there is a step to inactivate HF. We were able to. Furthermore, ATPas! Compare the results of the analysis with the digoxin antibody analysis. Since the above results are related to biologically active HF by comparison, HF We were able to confirm the accuracy of this analysis for measurements.

Although the invention has been described above in connection with preferred embodiments, those skilled in the art will appreciate that the invention Various modifications may be made to the above methods and compositions based on the following. subordinate Therefore, the scope of the patent granted for the present invention is the scope of the claims and their equivalents. It is limited only by.

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Claims (20)

[Claims] 1. C10 to C26 unsaturated alkyl in which R1 has at least 3 double bonds or acyl, and R2 is -OH, -H, -CH3 or C10 to C26 is a saturated alkyl ester, R3 is S or P, R4 is C1 to C6 saturated alkyl, R5 is NH3 or alkylamine Given that the expression ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound represented by 2. R1 is C19 (number of double bonds 4) acyl, C20 (number of double bonds 4) alkyl , or C20 (number of double bonds: 4) acyl, R2 is -OH, and R3 is P and R4 is CH2 or CH2CH2 and R5 is NH3. The compound according to claim 1, wherein the compound has the following characteristics. 3. Isolated from animal tissues or body fluids, associated with essential hypertension, molecular The amount ranges from about 521 to 541 and about 186 nm in the ultraviolet spectrum. Compounds with biological activity of internally generated factors showing maximum peak. 4. If the compound is capable of forming a complex with an anti-digoxin antibody or with the enzyme Na, K -ATPase, Ca, Mg-ATPase, and calmodulin-activated Ca- Claim 1 or 2, further characterized in that at least one of ATPases can be suppressed. or a compound according to item 3. 5. Claim 1 or 2, further characterized in that said compound is capable of causing vasoconstriction. Compound according to paragraph 3. 6. Claim 1, further characterized in that said compound is capable of causing increased sodium excretion. A compound according to item 1 or item 3. 7. administering a pharmaceutically effective amount of a compound of claim 1 or 3. Characteristic methods for regulating hypertension in animals. 8. Capable of specifically binding to the compound of claim 1, 3, 5 or 6 An antibody characterized by: 9. 9. The antibody according to claim 8, wherein the antibody is a polyclonal antibody. antibody. 10. Claim 8, wherein the antibody is a monoclonal antibody. antibodies. 11. The patient sample is treated with a small amount capable of forming a complex with the compound of claim 1 or 3. contacting with at least one antibody and determining complex formation of said antibody by immunoassay; In vitro detection of essential hypertension-related factors in patient samples characterized by How to get out. 12. A patient sample can be complexed with a compound according to claim 1 or 3. contact with at least one antibody capable of forming a complex with an essential hypertension-related factor. measuring the amount of said antibody present in said sample and comparing said amount with the amount of said factor present in said sample. 1. An in vitro method for measuring the amount of essential hypertension-related factors. 13. Patient samples and the compound according to claim 1 or 3 are The receptor is contacted with a blood pressure related factor and an enzyme capable of forming a complex with said compound. Essential hypertension-related, characterized in that complex formation of the enzyme is determined by analysis. Methods for in vitro detection of factors. 14. A patient sample and a compound according to claim 1 or 3 are combined with said factor. and contacted with an enzyme capable of forming a complex with the compound to form a complex with the enzyme. The amount of said compound is measured and the amount of said compound that has formed a complex is determined from the amount of said compound in said sample. An in vitro study of the amount of essential hypertension-related factors, characterized by correlating with the amount of the factors listed above. b) Measurement method. 15. The enzymes are Na, K-ATPase, Ca, Mg-ATPase and Ca. Claim 1, characterized in that it is selected from modulin-activated Ca-ATPase. The method according to item 3 or item 14. 16. Claim 1, characterized in that the compound is labeled with a detectable marker. The method according to item 3 or item 14. 17. has specific reactivity with the compound according to claim 1 or 3. to animals characterized by administering a monoclonal antibody to an animal in an amount effective as a drug. How to control high blood pressure. 18. Producing an antibody capable of specifically binding to the compound according to claim 1 or 3. hybridoma cell line. 19. (a) Extracting the sample with alkaline solution in the presence of ammonium ions death, (b) a patient's body fluid characterized by extraction with ether and acetone solvents or A method for isolating hypertensive factors from tissue samples. 20. (a) Precondition the column with extract from the patient sample. Google, (b) Methanol:acetonitrate at a mixing ratio of about 15-20:15-25:55-75 RIL: Separating hypertensive factor mixtures in an aqueous solvent system A method for separating hypertension factors using high pressure liquid chromatography.