WO1989003836A1 - Factors associated with essential hypertension - Google Patents

Abstract

Novel factors isolated and purified from animal tissue or fluid that are associated with essential hypertension are disclosed as well as analogues and derivatives thereof. Novel isolation and resolution methods. Biochemical assay methods for detecting the presence or amount of the hypertension factors in patient samples. The use of the hypertension factors, or derivative thereof in the regulation of essential hypertension, vasoconstriction and natriuresis.

Description

FACTORS ASSOCIATED WITH ESSENTIAL HYPERTENSION

Field of the Invention

The present invention relates to the discovery of novel factors that are associated with essential hypertension and analogues of such factors. More specifically, the present invention relates to qualitative and quantitative biochemical assay methods of detecting hypertension factors ("HF") in patient samples. Additionally, the present invention relates to the therapeutic use of these hypertension factors and analogues, including regulation of hypertension, ' vasoconstriction, and natriuresis. Further, the present invention relates to the therapeutic use of blocking agents to the novel factors.

Background of the Invention

Essential hypertension is a leading cause of morbidity and mortality in the United States and affecting over 60 million individuals, yet its pathogenesis remains unclear. Several years ago, Blaustein proposed the theoretical framework relating a hypothetical natriuretic hormone or factor to the pathogenesis of essential hypertension. (See Blaustein, et al., Annuals of Internal Medicine, 98: (part 2) 785-792 (1983).) First, a large proportion of essential hypertensives have an inherited defect in their renal sodium transport system so that they excrete less sodium than normotensives. This creates a tendency for affected individuals to retain more body water. The extracellular fluid volume (ECV) would gradually expand with the increasing sodium buildup, except that fluid volume expansion induces the release of a circulating factor which limits the sodium retention and causes natriuresis (the excretion of sodium in the urine). The postulated factor does so by inhibiting Na,K-ATPase, the enzyme responsible for renal tubular reabsorption of sodium from urine. Since it is alleged to cause loss of sodium (natriuresis) and water (diuresis) from the kidney, this factor has been called a natriuretic hormone. Effectively, a new homeostasis is put in place so that sodium content and body water are apparently "normal," but the inhibiting factor circulates at greater concentrations in blood. Unfortunately, Na,K-ATPase activity is not confined to kidney cells, but is widely distributed in smooth muscles; e.g., blood vessel walls, heart, and nerve tissue. Accordingly, the circulating factor also causes vasoconstriction in these tissues, resulting in hypertension.

There are a number of reports linking circulating Na,K-ATPase inhibitors induced by volume expansion and/or hypertension. Using enzyme inhibition assays based on a diminished substrate consumption by purified enzyme in vitro or on altered Rb transport (a K substitute) into intact cells, numerous investigators have described significant differences between plasma from hypertensives and that .from normotensives. Plasma electrophoresis has been used to separate ten inhibitory fractions in serum. Increased circulating inhibitor was also associated with increased intraeellular sodium in erythroeytes and intracellular calcium in platelets. Though not first to have done so, Cloix et al. have isolated a heat- stable, low molecular weight, anϊonic substance from human urine and plasma with Na,K-ATPase inhibitory properties which increased blood pressure when injected intracerebroventricularly. (See Cloix, J.F., et al., Advances in Nephrolog , 14:161-171 (1985).) Because the inhibition of Na,K-ATPase by ouabain occurs at hormone-like concentrations, some investigators sought an endogenous cardiac glycoside-lϊke substance. For example, unidentified interferences with the digoxϊn radioϊmmunoassay are widely reported. Digoxin-like immunoreactive substances ("DLIS") are found in the plasma of newborns, renal patients, and preeclamptics. (See Valdes, et al., Journal of Clinical Endocrinology and Metabolism, 60:1135- 1143 (1985).) Uncompensated matrix effects, drugs, and antibody cross-reactivity may explain some of these reports. DLIS(s) have been extracted from plasma, urine, brain, liver, and adrenal tissues. Attempts to characterize DLIS(s) by acid, base, heat, and solvent extractions have yielded one to five low molecular weight (200-700 daltons), very polar substances which are cross-reactive with anti-digoxin antibodies and are Na,K-ATPase inhibiting. However, little else is known about their physical characteristics and isolation protocols have varied greatly. There are no published investigations of comprehensive enzymatic inhibition or binding by such putative natriuretic hormones, but those studies have begun. (See Haupert, et al., Am J. Physiol., 247 16.-F919-F924 (1984).)

The presence of false positive tests for a DLIS in the plasma of hemodialysis patients has been described. (See Grave et al., Ann. Intern. Med., 99:604-608 (1983).) At that time, this observation was believed important because of the interference which DLIS imposed on digoxin immunoassays of plasma from these patients. However, elevated plasma DLIS levels also have been noted in volume expanded dogs. (See Gruber, et al., Nature, 305:6646 (1983).)

Various biochemical assays have been proposed to measure the so-called natriuretic hormone or factor. Hamlyn et al., U.S. Patent No. 4,665,019, disclose a method for measuring plasma levels of an inhibitor of Na,K-ATPase associated with hypertension. The method of Hamlyn is based on the inhibition of Na,K-ATPase by deproteinized plasma and does not identify or characterize the specific natriuretic factor. Nardi, et al. claim to have developed a method for diagnosing the presence of hypertension, or a predisposition to hypertension, in mammals, which is based on the detection of at least one protein associated with hypertension with a molecular weight of 10,000 to 17,000 daltons. (See Nardi et al., U.S. Patent No. 4,321,120.) More recently, a radioimmunoassay (RIA) method for determining the presence of a natriuretic factor in urine using anti-digoxin antibody has been reported, but does not identify the factor. (See Morise, et al., Endocrinol, 32(3):405-ll (1985).)

Most recently, Kuske, et al., Klin Wochensiher, 65:53-59 (1987), have attempted to better understand the "conflicting situation" with respect to the nature of the natriuretic factor. The state of the "conflicting situation" in the art is well described by Kuske, et al.: "A considerable diversity of factors inhibiting

(Na⁺ + K⁺)-ATPase in serum, urine, and tissue extracts has been described." (See

De Wardener, et al., Physiol. Rev. 65:658-759 (1985).) Conflicting results have been observed with regards to the immunoreactivity of these factors from different sources toward digoxin antibodies and their inhibitor action on (Na +

K⁺)-ATPase in serum (See, Crabos, et al., FE BS Lett, 176:223-228 [year?], and

Kelly, et al., J. Biol. Chem., 260:11396-11405 (1985)) and in partially purified fractions of serum, urine and from tissue fractions. (See, Buckalew et al., Ann

Rev. Physiol, 46:343-358, (1984); Crabos, supra-, DeWardener, Physiol. Rev.,

65:658-754 (1985); and Kelly, supra.) The low molecular weight substance has been suggested to be very polar (see DeWardener, Ann. Clin. Biochem., 19:137-140

(1982)) of peptide nature (see Buckalew, supra; Gruber, et al., Proc. Soc. Erker

Biol. Med., 159:463-467 (1978); Klingmuller, et al., Klin Wochensiher, 60:1249-

1253 (1982); Kramer, et al., Renal Physiol., 8:80-89 (1983); Morgan, et al., J. Biol.

Chem., 260:13595-13600 (1985)), or of steroidic nature (see Cloix, et al., Biochem.

Biophys. Res. Commun., 131:1234-1240 (1985).) Moreover, unsaturated fatty acids

(see Bidard, et al., Biochem. Biophys. Acta, 769:245-252 (1984), and Tamura, et al., J. Biol. Chem., 260:9672-9677 (1985)) and dehydroepiandrosterone sulfate have been ascribed digitalis-like properties (see Vasdev, et al., Res. Commun. Chem. Pathol. Pharmacol., 49:387-399 (1985).) Despite Kuske's efforts to clarify the situation, he was unable to purify and characterize the supposed natriuresis hormone.

Accordingly, numerous theories exist with respect to the nature of the postulated natriuresis hormone or factor; however, prior to the present invention, no one has succeeded in isolating, purifying, and characterizing a factor closely associated with extended hypertension. Moreover, the inventors have identified the general chemical structure of a class of these hypertension factors, which allows the chemical synthesis of derivatives or analogues of the indigenous hypertension factors. Identification, isolation, and synthesis of such hypertension factors and their derivatives allows the development of numerous therapeutic and diagnostic applications.

Summary of the Invention

The present invention is based upon the discovery that particular factors in animal tissue or fluid are associated with essential hypertension. These endogenous hypertension factors ("HF") have been isolated, purified, and their chemical structure elucidated. Accordingly, HF can be characterized by its physical and biological properties, as well as the general chemical structure of the endogenous HF and synthetic analogues thereof. HF is physically characterized as a polar lipid, e.g., unsaturated phospho- or sulfo-lipid, having a molecular weight within the range of about 521-541 and an ultraviolet spectrum maximum at about 186 n . Biologically, HF inhibits at least one of the Na,K-ATPase, Ca,Mg- ATPase and calmodulin-activated Ca-ATPase enzymes and can complex with anti- digoxin antibodies.

As defined by chemical structure, HF and analogues thereof include compounds of the formula:

CH₂ - 0 - Rl

^C!^{H " R2} 0 R5⁺

I ii /

CH, - 0 - R3 - 0 - R4 - CH

² • \ -

OH ^XC00

wherein:

Rl is a CIO to C26 unsaturated alkyl or acyl having at least three double bonds; R2 is -OH, -H, -CH₃, or a CIO to C26 unsaturated alkyl ester;

R3 is S or P;

R4 is a Cl to C6 saturated alkyl; and

R5 is a NHg or alkyl amine.

Moreover, the inventors have discovered that HF exists as a mixture of two compounds having the same molecular weight, HF-1 and HF-2, each of which is associated with essential hypertension. Surprisingly, the inventors have also discovered that HF-1 and HF-2 each have different primary physiological effects that are associated with essential hypertension. In resolved and purified form, administration of HF-1 primarily causes natriuresis, while HF-2 primarily causes vasoconstriction. Accordingly, as used hereinafter, the term HF shall mean a natural mixture of HF-1 and HF-2 or a predetermined mixture of isolated HF-1 and isolated HF-2.

Also provided in the present invention are methods for regulating hypertension, natriuresis, and vasoconstriction by administration of HF, HF-1, HF-2, or their analogues to an animal host. Additionally, the present invention provides methods for producing antibodies, monoclonal and polyclonal, specific for HF, HF-1, or HF-2, and the fused cell lines, i.e., hybridomas, producing the monoclonal antibodies.

The present invention also provides in vitro biochemical assay methods and kits for detecting the presence or determining the amount of HF, HF-1, or HF-2 in a patient sample.

Brief Description of the Drawings

FIGURE 1 depicts the mass spectrum of HF showing a protonated molecular ion peak at 532;

FIGURE 2 depicts the ultraviolet spectrum of HF with a maximum at about 186 and a second peak at about 223 nm;

FIGURE 3 demonstrates the polar lipid character of HF by showing mass spectrum peaks at 72, 85, 99, 105, 273, 291, and 346;

FIGURE 4 depicts the HPLC graph indicating the presence of the HF-1 and HF-2;

FIGURES 5A and 5B demonstrate the effect on systemic vascular resistance (SVR) following injection of HF in sheep. The mean SVR values are plotted against time for the duration of the experiments. The increase in SVR was seen within five minutes of injection; FIGURES 6A and 6B demonstrate the increase in fractional excretion of sodium (FE Na), i.e., natriuresis, following injection of HF in sheep. Notably, the FE Na response was slower than the SVR response;

FIGURE 7 demonstrates the vasoconstrictive effect on arterioles treated with ultrafiltrates containing HF versus other controls. Of the four substances tested, HF test produced the greatest contraction of human placenta pre- arterioles;

FIGURE 8 demonstrates the effect on fractional excretion of sodium (FE Na) resulting from administration of ultrafiltrate containing HF to one kidney and a control to the other kidney for a group of eight dogs. Values for FE Na are plotted for test (open bars) and control (shaded bars) kidneys separately. The period before renal infusion is labeled as baseline during and after infusion is labeled as experimental;

FIGURE 9 demonstrates the relationship between the concentration of HF administered, and the increase in FE Na, i.e., natriuresis in a group of eight dogs. The dif erence in maximum increase in FE Na between test^' and control kidney is plotted against the difference between the HF levels in test and control ultrafiltrates;

FIGURE 10 demonstrates the effect of HF on kinetics of ⁵CA²* accumulation in canine kidney eells;

F FIIGGUURREE 1111 ddeemmoonnssttrraatteess tthe effect of HF on kinetics of ⁴⁵Ca ⁺ accumulation in simian smooth muscle cells;

FIGURE 12 demonstrates the effect of HF-1 on ²²Na⁺ and ⁴⁵Ca²⁺ transport in simian smooth muscle cells;

FIGURE 13 demonstrates the effect of HF-2 on ²²Na⁺ and ⁴⁵Ca²⁺ transport in simian smooth, muscle cells;

FIGURE 14 depicts the Na,K-ATPase and Ca,Mg-ATPase inhibition activity in an in vitro study of human red blood eells. HF-1 and HF-2 produced significant inhibition of both of the enzymes in almost a linear fashion with increasing dosage;

FIGURE 15 depicts the Fast Atom Bombardment Mass Spectrum (FAB MS) of HF-2;

FIGURE 16 depicts the Collisionally Activated Association Mass Spectrometry (CAD MS) mass spectrum of HF-2;

FIGURE 17 depicts the structural interpretation of all fragments with mass greater than 100 observed in the mass spectra of FIGURES 15 and 16; FIGURE 18 demonstrates the HF (semi-pure) inhibition of the Na,^"K pump ATPase in isolated human RBC membranes in a dose-dependent fashion. 100% activity = 7.5 nmol Pi/min/mg protein;

FIGURE 19 demonstrates HF (pure mixture) inhibition of Ca pump ATPase and calmodulin activated Ca pump ATPase in isolated human RBC membranes in a dose-dependent fashion. 100% activity of the calmodulin activated ATPase = 66.7 nmol Pi/min/mg protein;

FIGURE 20 depicts an isolation protocol for HF from the plasma of hypertensive patients.

Detailed Description of the Preferred Embodiments Of the Invention A natriuretic factor or hormone has long been postulated to be involved in the pathogenesis of essential hypertension. In its broadest aspect, the present invention has isolated, purified, and structurally characterized such a factor that has sometimes been referred to as digoxin-like immunoreactive substance (DLIS). To avoid overemphasis of only one characteristic of this factor, we have referred to the factor throughout this text as hypertension factor or "HF".

HF, isolated and purified from animal tissue or fluid, is initially characterized as a polar lipid having a molecular weight within the range of 521 to 541. HF's ultraviolet spectrum indicates a maximum at about 186 nm and a second prominent peak at about 223 nm, with minimum values at about 203 nm.

HF can be isolated from numerous types of animal tissue or fluid; for example, HF may be extracted from hemofiltrates obtained from dialysis patients known to have essential hypertension. The isolation and purification of HF can be carried out in numerous ways, examples of which are described below as Examples 1-3 and depicted by the flow chart of FIGURE 20. Generally, isolation of the hypertension factors from fluid or tissue samples obtained from a patient is accomplished by a method that includes the steps of: extraction of the sample with an alkaline solution in the presence of ammonium ion; and a subsequent extraction with an ether:acetone solvent.

Once HF has been isolated and purified, its physiochemical properties permit its characterization and differentiation from other reported natriuretic factors. As shown in FIGURE 1, fast atom bombardment (FAB) mass spectral analysis of HF yields a single dominant component with a molecular weight within the range of about 521 to 541, more specifically at about 531 in the deprotonated form. Lesser peaks at about 363 and about 345 were also noted. An ultraviolet spectrum of HF indicates a maximum at about 186 nm with a less intense peak at about 223 nm and the minimum value at 203 nm. Further analysis indicates that HF is a polar lipid compound, most likely an unsaturated phospho- or sulfo-lipid. As shown in FIGURE 3, the lipid characterization of HF was the result of analysis using mass spectroscopy data indicating peaks characteristic of a polar lipid molecule at 72, 85, 99, 105, 273, 291, and 346. Solvent solubility studies further implicate HF as a lipid molecule. Once the lipid character was determined, back calculation from the molecular weights of the known backbone structure indicates that HF is an unsaturated phospho- or sulfo-lipid.

HF can be further characterized by its biological or biochemical properties. Consistent with the theoretical pathogenesis of essential hypertension, HF is capable of inhibiting Na,K-ATPase activity. This characteristic was confirmed by use of a modification of the Na,K-ATPase inhibition assay described by Hamlyn, et al. in Nature, 300:650-652 (1982), the disclosure of which is incorporated herein by reference. Applicants have also noted that HF isolated from plasma of patients is capable of inhibiting Ca,Mg-ATPase and calmoduliπ- actϊvated Ca-ATPase, and, therefore, would modify the transport of calcium across the cell membrane, thereby increasing the intracellular calcium concentration making the cell hyper-responsive. Applicants have studied the effect of HF on the activity of the Na/K-ATPase, Ca,Mg- ATPase, and calmodulin (Cam)-aetivated Ca-, ATPase, in red blood cell (RBC) membranes. Using different concentrations of purified (post HPLC) HF, applicants have been able to measure the ICJJQ of HF for these membrane-bond enzymes. The results, depicted in FIGURES 18 and 19, indicate that HF has an IC₅₀ of 10 ng for Na,K-ATPase, 15 ng for Cam activated Ca-ATPase, and 25 ng for unactivated Ca,Mg ATPase. The reason for higher ICcn for Ca,Mg ATPase versus Cam-activated Ca ATPase may be due to interaction of HF with Cam or interaction with pump (the part where calmodulin binds).

HF is also capable of complexing with anti-digoxin antibodies. This was performed in duplicate or triplicate with reagents purchased from New England Nuclear (Rainen Digoxin I Kit, New England Nuclear, North Billerica, MA 01862), and used according to the manufacturer's instructions, except that the buffer was supplemented with 10 yL of a 200 g/L bovine gamma globulin in 140 mmol/L NaCl. Antigen antibody binding of unknowns was compared to digoxin standards in serum.

HF can be further characterized as being capable of causing increased sodium and calcium uptake in cultured simian aortic (smooth) muscle cells and in cultured canine kidney cells. This experiment was carried out according to the procedure described in Example 7, and further supports the Na,K-ATPase and Ca,Mg-ATPase inhibition properties of HF. HF can also be characterized as being capable of activating platelet aggregation in response to the increased intraceliular calcium induced by HF in individual platelets.

Structural analysis of HF isolated from plasma of patients with essential hypertension indicates that HF-2 is a novel phosphatidyl serine derivative with a 19:4 (19 carbons:4 double bonds) fatty acid side chain on the A carbon and has the molecular formula C25H42O9NP. The chemical structure of endogenous HF-2 isolate is:

Analogues of the above-noted compound have been synthesized as described in Examples 10-12 and have shown biological activity similar to endogenous HF isolated from patients. Accordingly, the present invention is also directed to compounds having the general formula:

wherein:

Rl is^" a CIO to C26 unsaturated alkyl or acyl having at least three double bonds;

R2 is -OH, -H, -CH₃ or a CIO to C26 unsaturated alkyl ester;

R3 is S or P;

R4 is a Cl to C6 saturated alkyl; and

R5 is a NHn or alkyl amine.

Although applicants do not wish to be bound by any theory, it is postulated that the biologically active moiety of the HF molecule is the unsaturated fatty acid side chain on the A carbon of the glycerol backbone. Accordingly, the Rl moiety on the A carbon is characterized as a CIO - C26, preferably C14 - C22, unsaturated alkyl or acyl. In a most preferred embodiment, Rl is a C18 - C20, unsaturated alkyl or acyl. Also, biological activity appears to require a degree of fatty acid unsaturation of at least three double bonds. It is preferred that the double bonds be conjugated. Accordingly, an upper limit for conjugated double bonds for a C26 side chain would be 13.

With respect to the other moieties, R2 - R5, there selection is only limited by the requirement that they do not cause steric hindrance with the fatty acid side chain that would effect its biological activity. Although the R2 moiety on the B carbon of the HF isolated from patients is most likely -OH, derivatives within the scope of the present invention include compounds with a -H or -CHg moiety. Alternatively, R2 can be a CIO to C26 unsaturated alkyl ester. With respect to the R3 moiety, the present invention contemplates sulfur as well as phosphorus. The R4 linkage group may be a Cl to C6, preferably Cl or C2, saturated alkyl. The R5 moiety may be an amine or alkyl amine group.

Representative examples of compounds within the scope of the present invention include: the ether and ester analogues of lysophosphatidylserine (14t3), (18:4), (19:4), (20:4), (22:4), and (26:4); and the ether and ester analogues of lysosulfolecithin (19:3).

During the isolation and purification of HF, it was discovered that the HF exists as a mixture of compounds having the same molecular weight, HF-1 and HF-2, with similar physical properties, but separate and independent physiological or biological effects; i.e., HF-1 is capable of causing natriuresis, while HF-2 is capable of causing vasoconstriction. Thin layer chromatography (TLC) in a chloroform :methanol:H θ (65:35:5 by volume) solvent also distinguished HF-1 and HF-2 as having R_* values of 0.90 and 0.85, respectively. In addition to having different physiological effects, HF-1 and HF-2 can be distinguished by their relative Na,K- ATPase inhibition potency. A relative ATPase inhibition ratio of about 1:20 has been found for HF-l:HF-2. Example 6 describes a method for resolving the mixture of HF-1 and HF-2 using high pressure liquid chromatography (HPLC). The novel features of resolving the mixture of HF-1 and HF-2 by HPLC include the steps of preconditioning the column with extracts from a patient sample, e.g., a preparation of pooled lipids from a sample extract, and separating the pair of compounds with a methanol:aeetonitrile:water solvent having about a 15-20:15-25:55-75 mixture ratio.

Once HF and the resolved pair, HF-1 and HF-2, have been isolated and purified, one of skill in the art can appreciate the numerous therapeutic and diagnostic applications. Also, as discussed above, derivatives or analogues of HF may be chemically synthesized that retain HF's biological activity. For simplicity of discussion, "HF", "HF-1", and "HF-2" as used hereinafter shall include synthetically produced derivatives or analogues thereof.

Given the role of HF in the pathogenesis of essential hypertension, regulation of hypertension in an animal host can be achieved by administering a pharmaceutically effective dose of HF, HF-1, or HF-2 to a host. More specifically, natriuresis and diuresis may be regulated independently of vasoconstriction by administering HF-1 to a host. The administration of HF-1 acts as a diuretic with the additional and significant advantage that HF-1 selectively increases the excretion of sodium and water in the urine, but does not cause an increased loss of potassium. Potassium loss is a significant problem for patients taking conventional diuretics.

Although vasoconstriction and the resulting high blood pressure are generally viewed as negative physiological conditions, there are situations where vasoconstriction and higher blood pressure are desirable. For example, HF-2 can be _.administered to patients suffering from shock, and the associated low blood pressure, to cause vasoconstriction and elevate blood pressure. Dangerously low blood pressure is a frequently encountered condition in emergency room situations, intensive care units, and coronary care units. Accordingly, immediate administration of HF-2 can restore a patient's blood pressure to a more normal range.

The factors of the present invention possess potentially valuable pharmacological properties. HF-1 and HF-2, administered either individually or in various combinations, are capable of regulating hypertension, natriuresis, and vasoconstriction in an animal host. Accordingly, a medicament may be formulated which comprises HF, HF-1, or HF-2 in combination with instructions for administering the selected factor to a mammalian host. HF-l's unusual ability to enhance excretion of sodium without the loss of potassium makes it a particularly promising diuretic. Potassium depletion resulting from the administration of conventional diuretics is a serious problem that can be overcome by the selective pharmacological activity of HF-1. Of course, in situations where it is desirable to induce both natriuresis and vasoconstriction, one skilled in the art would appreciate that an unresolved mixture of HF, or a predetermined mixture, of isolated HF-1 and isolated HF-2, can be administered to the host.

Additional therapeutic applications of HF include the administration of HF dosage forms to induce platelet formation or the in vitro pre-treatment of platelets with HF and subsequent reinjection into the patient. The compounds of the present invention are generally administrate to animals, including, but not limited to, mammals, birds, and fish, and especially to humans, livestock and household pets.

HF can be processed in accordance with conventional methods of pharmacy to produce the agents for administration to humans, patients, and other animal hosts. HF can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, or topical applications that do not deleteriously react with the active compounds. Suitable pharmaceutical acceptable carriers include, but are not limited to, water, salt solutions, alcohols, vegetable oils, benzene alcohols, polyethylene glycols, gelatins, carbohydrates, such as lactose, amylose or starch, magnesium, stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid monoglyeerides and diglycerides, pentaerythritol, fatty acid esters, hydroxyl methyl cellulose, pyrrolidone, etc. Pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously act with the active compounds. They can also be combined where desired with other therapeutic agents, e.g., vitamins.

For internal applications, particularly suitable are tablets, άragees, liquids, drops, suppositories, or, capsules, a syrup, an elixir, or the like, can be used wherein a sweetened vehicle can be employed. For parenteral applications, particularly suitable are injectable, sterile solutions (preferably oily or aqueous solutions), as well as suspensions, emulsions, or implants including suppositories. Ampules are convenient unit dosages. Sustained or direct release compositions can be formulated, e.g., liposomes, transdermal patches, or compositions wherein the active compound is protected with differential grading coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the HF compounds and use the lyophilizates obtained, for example, for the preparation of products for injection.

For topical applications, they are employed as nonsprayable forms, viscous, to semi-solid or solid forms comprising a carrier compatible with topical application, and having a dynamic velocity preferably greater than water. Suitable formulations include, but are not limited to, solutions, suspensions, ^"emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents; e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical applications, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normal propellant; e.g., a freon.

The dosages administered in any specific case will vary according to the specific compounds being utilized, the particular compositions formulated, the mode of application, and the particular organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds of a known agent, e.g., by means of appropriate, conventional pharmacological protocols.

An additional aspect_., of the present invention is the development of antibodies specific for HF, HF-1, and HF-2 for utilization in diagnostic and therapeutic applications. Polyclonal antibodies can be isolated from serum of immunized mammals, for example, goats or rabbits, using conventional techniques. Using the basic method developed by Kohler and Milstein, reported in Nature, 256:495-97 (1975), the disclosure of which is herein incorporated by reference, a skilled artisan may develop hybridoma cell lines producing monoclonal antibodies specific for HF, HF-1, or HF-2. The method for producing such monoclonal antibodies includes: immunizing the mouse or other suitable mammalian hosts with HF, HF-1, or HF-2; harvesting an antibody producing organ, e.g., spleen, from the host of choice; preparing a cellular homogenate from the harvested organ; fusing the cellular homogenate with cultured cancer cells, e.g., myeloma cells; selecting or screening for hybrid cells that produce monoclonal antibodies specific for the HF immunogen; cloning the hybrid eells, i.e., hybridomas, so that they produce perpetually; and, harvesting monoclonal antibodies specific for the HF immunogen produced by the hybridomas.

The class of antibodies produced may be either of the IgM or IgG variety. Where HF, HF-1, or HF-2 is not sufficiently immunogenic in the host selected for immunization, it can be characterized as a hapten, and an immunogenic response induced by linking the hapten molecule to a carrier molecule. Methods of linking haptens to carriers are well known in the art, and numerous carrier molecules are available for coupling with HF, e.g., ovalbumin and thyrogiobulin. Monoclonal anti-HF antibodies can be raised according to the method described by Rauch et al., European Journal of Immunology, 14:529-534 (1984) and cheeked for cross-reactivity as suggested by Koike, T. et al., Clinical Experiments Exper. Immu., 57:345-350 (1984), and Harris, E. et. al., Clin. Lab. Immun. 16:1-6 (1985). Once the continuous cell lines, i.e., hybridomas, are screened for the appropriate antibody production, the monoclonal antibodies can be utilized for in vitro methods for detecting the presence or amount of HF factors in patient samples. A method for the in vitro detection of the presence of an HF includes contacting a sample obtained from a patient with at least one antibody having specific reactivity with HF, HF-1, or HF-2, and determining the complexing of the antibody to the HF by means of an immunoassay. Alternatively, a quantitative measurement of the amount of HF in a sample may be made by contacting the sample with at least one antibody having specific reactivity with HF, HF-1, or HF-2, determining the amount of the antibody associated with the factor, and correlating the amount of the association with the amount of factor present in the sample. Both monoclonal and polyelonal antibodies can be utilized in these assays. Moreover, antibody fragments and genetically engineered proteins corresponding to the variable region of the antibody can be employed. For example, immunoassays can be performed for the factors of the present invention according to the cellulose nitrate binding method described by Costello P. and Green, F., infect, and Immun., 56:1738-1742 (1988) for the ELISA method of Loizou et al., Clin. Exper. Immun., 62:738-745 (1985). Also, because cross reactivity of HF with anti-digoxin antibodies is a characteristic of HF, anti- digoxϊn antibodies may be utilized as an antibody in the immunoassay method.

The presence or amount of antibodies associated with the factor being assayed can be achieved by labeling the antibody with a detectable marker. The labeled antibody used in the present invention may be provided with the same labels used in prior art immunoassays. Among these may be mentioned fluorogenic labels for detection by fluorometry, as described in U.S. Patent No. 3,940,475, and enzymatic markers, as described in U.S. Patent No. 3,645,090. The label may also be a radioisotope, such as I , using, for example, the procedure of Hunter and Greenwood, Nature, 144:945 (1962), or that of David et al., Biochemistry, 13:1014-1021 (1974).

The present invention is also directed to a receptor assay method for detecting the presence or amount of endogenous HF in fluid or tissue obtained from a patient. The receptor assay is basically a competition assay which includes the steps of contacting a patient sample and a known quantity of a synthetic analogue of HF, with an enzyme capable of complexing with HF, HF-1, or HF-2 and also capable of complexing- with the HF analogue. Examples of such an enzyme would be Na,K-ATP-ase, Ca,Mg-ATPase or calmodulin activated Ca- ATPase. The hypertensive factor in the sample and the HF analogue compete for the limited number of binding sites on the enzyme, each enzyme having only one binding site. The competition for the enzyme binding sites is stopped after a predetermined amount of time and a determination is made whether any HF, HF-1, or HF-2 is present in the sample and complexed with the enzyme. This would be a qualitative or simple yes/no assay. For a quantitative assay, the amount of HF analogue complexed with the enzyme is determined and is proportionally related to the amount of hypertension factor present in the patient sample. Generally, this correlation is achieved by labeling the HF analogue with .a detectable marker, and measuring the amount of free or bound HF analogue. Rather than react the enzyme with the sample and the HF analogue simultaneously, it is preferred to preincubate either the sample or the HF analogue with the enzyme.

As previously noted, HF also exhibits the ability to inhibit Ca,Mg- ATPase and calmodulin activated Ca-ATPase, and may generally modify calcium transport across the cell membrane. Accordingly, a receptor assay can be developed based on the competition of HF and a substrate for the^" Ca-ATPase type enzyme. There are specificity advantages to such a Ca-ATPase-based assay over a Na,K-ATPase- based assay; namely, there are a number of substances that inhibit Na,K-ATPase, but the applicants are unaware of any endogenous substances that specifically inhibits Ca,Mg-ATPase or calmodulin activated Ca-ATPase. Accordingly, an assay based on Ca-ATPase inhibition can provide a more quantitatively accurate measure of HF in a sample. One of skill in the art will appreciate that the assay methods of the present invention may be qualitative or quantitative in nature and may be employed to test untreated patient samples or extracts of patient samples containing isolated or resolved HF, HF-1, or HF-2.

Although HF, HF-1, and HF-2 are preferably detected in fluid samples, they also may be determined in tissue samples. Fluid samples utilized according to the present invention include whole blood, serum, plasma, urine, sweat, tears and saliva. A diagnostic kit for detecting the presence or amount of HF, HF-1, or HF- 2, which includes at least one antibody or enzyme specific for the HF of interest, can be assembled.

An additional therapeutic application of the discovery of the factors of the present invention is the treatment of a patient with an excessive HF titer by the administration of a monoclonal antibody specific for one of the factors to block its natural hormonal activity. The ability to block HF,, HF-1, or HF-2 by administration to a host of a specific monoclonal antibody may be utilized to regulate hypertension, natriuresis, and vasoconstriction. Additionally, once the monoclonal antibody to the hypertension factors of the present invention has been isolated, anti-idiotype antibodies can be developed using conventional techniques that will recognize and block the receptor site of HF, HF-1, or HF-2. In addition to antibodies, other agents that are capable of blocking the receptor sites for HF, HF-1, and HF-2 may be utilized; e.g., digoxin and oubain. Thus, the pharmacological affects of the endogenous factors may be regulated by blocking the receptor site for the factors. For example, elevated DLIS levels and Angiotensin II, a known vasoconstrictor, have been observed in patients with toxemia d pregnancy and implicated in its pathogenesis. (See, Goretelehner, et al., Am. J. Obst. Gyn., 101:397-400 (1968) and Gudson, et al., Am. J. Obst. Gyn., 150:83-5 (1984).) Accordingly, a monoclonal antibody, antibody fragment or antibody derivative capable of complexing with HF and deactivating it, or an anti- idiotype antibody that is capable of blocking the HF receptor site, may offer a treatment for toxemia of pregnancy.

Example 1 - Isolation and Purification of HF From tissues:

Fresh tissue, e.g., kidney, removed in surgery or at autopsy, or cultured tissues, may be processed immediately or stored at -70°C. Tissue was thawed, minced, and^" mixed with 2 equal volumes of physiological saline, and homogenized in the cold with short bursts of rotary cutting blades. Sediment is removed by centrifugation. The supernatant is filtered and extracted with benzene, etc., as described below. Tissue culture media are treated like plasma samples. From blood or urine:

Fresh whole blood or urine is filtered through an ultrafilter to separate the low molecular weight compounds. Alternatively, the plasma or serum can be separated from the blood prior to ultrafiltration. In samples for a screening clinical assay, the ultrafiltration step can be omitted. Ultrafilters with cutoffs from 50,000 to 2,000 daltons can be used, e.g., YM-2 diaflo membranes (Amicon Corp.), and Gϊbeo filters for renal patient hemodialysis (cuprophane) are also appropriate.

When the substances are being prepared in bulk, urease is added to the blood ultrafiltrate or to urine to eliminate urea. The specific activity of the enzyme and the urea content of the filtrate are taken into account so that this step, which takes place at about 25°C, is of short duration. This step can be omitted, but separation is. then less reproducible. As the pH rises with NHo production, it is titrated to pH 7.0 by the addition of 1M HC1. This step is desirable when the urea content exceeds 15mg/dL. Urease treatment may be omitted in the clinical serum screening assay. For bulk preparations using a fluid matrix with low HF contents, the volume is reduced by lyophylizing the sample, e.g., an ultrafiltrate of blood. Then the dried residue is reconstituted in HnO so that the final volume in mL is 3 times the weight of residue in grams, including the volume of 50% NH^OH used to titrate to pH 8.7. Dry NaCl is added to saturate the solution.

For a clinical urine or serum assay, the pH is adjusted to 8.6 with 1.0 to 3M ammonium acetate. If tris or other buffers are used, ammonium salts or ammonium hydroxide should be added. The dilution should not exceed 3 fold for optimum recovery. Ammonium ions are needed for maximum extraction. The pH can vary from 7.2 to 9.2, but maximum recovery is obtained at 8.6 to 8.8.

Solvent extraction is accomplished in two steps. In the first, non-polar lipids are removed with benzene in a volume equaling the sample volume. The organic layer is discarded. The second extraction is to isolate the HF. The purest preparations are accomplished with etheπacetone (1:1), used in equal volume with the sample. This is a time-dependent step; recoveries increase with longer solvent exposure. One to twenty hours mixing before separation is satisfactory, with one hour adequate for the extraction of clinical screening serum tests. Ether:acetone (5:7) may be used for bulk work. Other solvent systems with comparable polarity and solubilizing properties will extract the HF and different proportions of contaminants. The extraction is repeated two to three times, and the organic layers pooled. Centrifugation is needed to separate layers. Unless an antioxidant is used, the solvents should be evaporated under N2 as rapidly as possible. Lyophylization is needed to dry the sample. From this step forward, exposure to light is restricted.

For the clinical screening assay, the residue of the organic phase is reconstituted in physiological saline or H2O to the original sample volume or less, if required by the sensitivity limits of the immunoassay or receptor assay in use. Resolubilization requires at least 30 minutes and adequate vortexing. For the clinical serum or urine quantitative factor assay, the organic residue is reconstituted in HPLC solvent, e.g., methanol:acetonitrile:water (15:15:70). For semiquantitative separation, the residue is dissolved in the TLC solvent of chloroform:methanol:water (60:35:5) or methano isopropyl alcohokwater (15:15:70)

For bulk preparation, an additional extraction step is added. The residue is dissolved in a minimal amount of 60:35:5 chloroform:methanol:H2θ. The insoluble residue is separated by centrifugation and discarded after washing. The solvent is removed under N2 and lyophylization as necessary. The residue is then dissolved in HPLC solvent. Silieic acid column chromatography (e.g., with Biosϊl A, 100-200 mesh, Bio-Rad Laboratories Inc) may be used for bulk preparations with more than nine contaminants. A column (2 x 27 cm) is prepared in chloroforπ methanokHgO (60:35:2) at about 25°C. The sample is applied in this solvent and eluted with it or a gradient to a chloroforπnmethanokHgO mixture of 60:35:5. The two factors are eluted before most of the contaminating lipids and near the void volume of the column. Fraction content is conveniently monitored by thin layer chromatography (TLC),- as described below, rather than enzyme inhibition or immunoassay. As in all extraction steps, this solvent is removed under N2 and/or vacuum, in the dark, and at temperatures less than 50°C. The residue, which may be oily in appearance, should not be dried excessively in the lyophylizer.

A preferred isolation procedure for use in connection with immunoassays is as follows. At room temperature, pipet 0.3 mL serum into each test tube (15 mL polypropylene conical centrifuge with screw caps). Pipet 0.3 mL 1.0 M ammonium acetate buffer at pH 8.6 into each test tube. Pipet 3.0 mL benzene into each test tube and vortex for 30 seconds. Then, centrifuge the test tubes for 10 minutes at room temperature, at about 1500 g-force.

Remove the benzene with a pipet, discard, and blow off remaining organic layer and approximately 50% of the aqueous layer using a Ng stream in 44° C water bath. Dispense 6.0 mL of ether:acetone, into the test tubes, and vortex gently. Place the test tubes on a rocker for 1.5 hours in the dark. Remove ether:acetone solvent with pipette and save in the dark at 4°C. Rinse each test tube with 0.5 mL ether:acetone mixture, vortex, remove with pipette and add to the solvent retained and stored in the dark. Evaporate to dryness with N at 44°C. Reconstitute in 0.3 mL physiological saline or H₉O. Vortex well, allowing at least a 30 minute, preferably a 120 minute, solvation time. Thin Layer Chromatography ("TLC") of Hypertension Factors

TLC may be used to monitor the progress of the purification step, as noted above, or as a rapid semiquantitative separation system applicable to extracts of blood, urine, or tissue culture media.

The media may be silica gel impregnated glass fiber sheets (ITLC-SG, Gelman Sciences, Inc) or LHP-KD high performance 200 micro thick glass-backed plates (Watmann), for example. The solvent . system is the ehloroforππmethanokHgO mixture (60:35:5). Solvent systems with similar polarity render acceptable separation; e.g., ethyl acetate., acetone, or acetonitrile. After separation at 25°C (e.g., for 20 mϊn), the plates are developed with 50% H₃P0₄, or 25% TCA and heated to 100°C. Typical Rf values of 0.8 and 0.76 are achieved for the factors on a plate showing 0.77 for a digoxin standard. HF-1 and HF-2 can be distinguished based on their Rf values of 0.90 and 0.85, respectively. Example 2 - Isolation and Purification of HF

Hemofiltrates were prepared by ultrafiltrating blood of normotensive and hypertensive dialysis patients with hollow fiber, artificial kidneys (available ,for example, through Amicon, Fresinius, Gambro, and Travenol). The filtrate contained 0.18-0.78 ug digoxin equivalents per liter initially and averaged 13g of solids/L when desiccated. Hemofiltrates were treated with urease to remove urea, neutralized, then lyophylized. The resulting powder was reconstituted in water to a volume four times its weight, then alkalinized to pH 8.8 with 8M NH^OH. After one hour, the slurry was extracted with benzene and the organic layer discarded. The aqueous layer was then extracted with an equal volume of etheπacetone (5:7). After reserving the organic layer, dry NaCl was added to saturation and the extraction repeated. The organic layers were pooled and evaporated to dryness.

The extraction residue was reconstituted in the first HPLC mobile phase of acetonitrile:methanol:water (25:20:55). It was applied to a C18 reverse phase column (25 cm x 5 cm, Waters, Milford, Ma) coupled to an HPLC instrument fitted with a variable wave length detector (set at 223 nm) to desalt and partially purify. The two HF containing fractions (retention times of 2.4 and 2.7 min.), which separated under isocratic conditions were pooled, evaporated to dryness, and reconstituted in the second mobile phase (acetonitrile:methanol:water, 15:15:70). The solution was then subjected to HPLC, and HF-1 and HF-2 (retention times of 14.4 and 15.7 min., respectively) were collected separately for characterization. The flow rate for both chromatographic separations was 1.0 mL/min.

HPLC grade solvents were used throughout the protocol. Water was deionized. Isopropyl alcohol can be substituted for acetonitrile in one of the HPLC steps. When reducing solvent volume after HPLC purification, it is important to avoid lyophylizing the sample to dryness, as a 40% to 60% loss may be encountered at this stage. Affinity chromotography with anti-digoxin antibody or sodium potassium ATPase may be used to recover the HF after solvent (e.g., salt, alcohol, and acetonitrile) removal under nitrogen as an alternate to the lyophylization step. Example 3 - Isolation and Purification of HF Blood or ultrafiltrates of blood (0.3 mL) from dialysis patients were prepared for immunoassay by adjusting the pH to 8.6, then extracting with benzene. The aqueous phase was subsequently extracted twice with ether:aeetone (1:1). After evaporating the pooled organic layer, the residues were reconstituted in 0.3 mL of 140 mmol/L NaCl. Duplicate 100 μL aliquots of this were measured with a I 125 digoxin radioimmunoassay (Rianen digoxin kit, New England Nuclear, North

Billeriea, MA ^"01862) performed according to the manufacturer's instructions, except that the buffer was supplemented with 10 μL of a 200 g/L bovine gamma globulin in 140 mmol/L NaCl. Antigen-antibody binding of unknowns was compared to digoxin standards in serum, with results reported as ng DE/mL. The method was sensitive from 0.08 to 4 ng DE/mL. Its repeatability, determined by analysis of a single pool over 20 days, was 0.28 ± 0.04 DE/mL (x± SD).

Partially purified HF preparations were already extracted with benzene and ether acetone, thus the extraction step described above was not used. Instead the solvents of 5 and 25 μL aliquots from the pooled chromatography eluates were removed under a nitrogen stream, then reconstituted in 140 mmol/L NaCl for immunoassay.

The ultrafiltrates from dialysis patients were treated with urease^" until urea free, then lyophylized. Dried powders in 15-47 g batches were reconstituted with water, adjusted to pH 8.6, and solvent extracted as described above. After the residue of the ether:acetone extraction was dried, it was reconstituted in chloroform:methanol:water (60:35:1 by volume), and applied to 1.5 x 32 cm silicic acid chromatography columns. Chloroform:methanol:water (60:35:5) was used to elute 1 mL fractions. The elution profile was followed by fractionating 10 μL aliquots of eluate on silicic acid thin layer chromatography plates with the same solvent. Column fractions in which the primary constituents had > 0.69 by TLC were pooled. After solvent removal, the resulting residues were reconstituted in 5 mL of 140 mmol/L NaCl: ethanol mixture (2:1) for injection in the first of four sheep experiments, and in 3 mL of 140 mmol/L NaCl:dimethyl-sulfoxide (1:2) for injection in other experiments. The former solutions were turbid or oily; the latter were clear.

Eluate pools from the silicic acid column chromatography contained two substances which cross reacted with the digoxin antibodies. Both pass through an Amicon YM-2 filter (Amicon, Darvers, MA) with a nominal 1,000 molecular weight exclusion limit. One to six contaminants with neither Na,K- ATPase inhibition nor digoxin-like immunoreactive properties were also present. In two of the six HF preparations there were only three constituents by thin layer chromatography, two of which were HFs. This suggests that the other contaminants in the remaining preparations were extraneous to the biological responses ascribed to HF. Example 4 - HF Characterization - Physical Properties The immunoreactivity of HF from blood or ultrafiltrates was measured after solvent extraction. The extracts of ultrafiltrate of the HPLC fractions from the preceding protocols were measured after evaporating the solvent and reconstituting in 0.8 NaCl. In either case, a ⁵I digoxin radioimmunoassay kit (New England Nuclear) was used without modification except that 10 μL of 200 mg/mL bovine gamma globulin fraction II was added to facilitate precipitation. The assay was linear from 0.1 ng/mL to 8 ng/mL digoxin equivalents while the imprecision was 0.28+/-0.04 ng digoxin equivalents/mL @C ± SD, n=20). Accordingly, HF cross-reactivity with anti-digoxin antibodies was confirmed.

Inhibition of canine Na,K-ATPase was assayed spectrophotometrically at 340 nm using the coupled enzyme system described by Hamlyn et al., Fed. Proc, 44:2782-8, 1985, except that the buffer was prepared with HEPES (N-2-hydroxy- ethylpiperazine-N'-2-ethane-sulfonic acid) instead of Tris (2-amino-2(hydroxy- methyl)-l,3-propandiol) as originally formulated. Five yL aliquots of concentrated HF preparations were sampled on the day of infusion and assayed immediately or stored at -20°C for as long as 72 hours.

The absorption spectrum of purified HF preparations in HPLC solvent were measured at 25 °C with a variable wavelength recording spectrophotometer. As shown in FIGURE 2, a UV spectrum maximum was observed at about 186 nm.

Fast atom bombardment (FAB) mass spectrometry spectra were obtained using the Kratos Ms-50 triple analyzer at 8Kv acceleration voltage. Helium was used for collisionally activated decomposition at 25°C using argon as the bombarding gas for FAB. As shown in FIGURE 1, a major peak at 532 was observed, as well as lesser peaks at 345 and 363.

Example 5 - Resolution of Mixture of HF-1 and HF-2 High pressure liquid chromatography is used to separate and/or quantitate the factors. A C18 reverse phase column (25 cm x 5 mm, Waters, Milford, MA) is used coupled to a spectrophotometric detector set at 223nm. The column is thoroughly washed with methanol then preconditioned with a preparation prepared from the pooled lipids which are eluted from the bulk extract under these chromatrographic conditions and having retentions >16 minutes. Unless the column is so conditioned, the two fractions will separate as a single peak, rather than two peaks with baseline resolution. FIGURE 4 shows the two peaks associated with HF-1 and HF-2. With this column, the factors can be separated under isocratic conditions in the 15:15:70 solvent with retentions of 14.4 and 15.7 minutes. More rapid separation can be accomplished with a 20:25:55 mixture of methanol:acetonitrile:H₂O (retention of 2.4 and 2.7 minutes for factors HF 1 and 2, respectively), when the needed purity and starting material suggest two HPLC steps are appropriate. If two HPLC steps are used, the solvent with less water is used first, the active fractions collected, theiF solvent removed, and- then the factors rechromatogrammed in the solvent with 70 parts of HgO.

HPLC is used for all variations of the factor measurement, except the clinical screening of serum or urine and the semiquantitative system which substitutes TLC fractionation. After separation, the active fractions are reduced to dryness under Ng and lyophylization.

Example 6 - Vasoconstrictive and Natriuretic Properties

Female sheep weighing 50 Kg were used for the experiments. Exteriorized arterio venous eannulae were surgically implanted by catheter izing the carotid artery and jugular vein in the neck. The actual experiments were conducted at least one week after surgery.

For each experiment the standing animal was placed in gentle neck restraint in a cage. The arteriovenous eannulae were clamped and separated in the middle. A Swan-Ganz cardiac catheter was passed into the pulmonary artery through the venous cannula. After verifying proper positioning, the catheter was left in situ to measure cardiac output (CO) by the thermodilution method. Mean arterial pressure (MAP) was measured through the carotid artery cannula. An infusion line was connected to the venous cannula and 72.5 mmol/L NaCl was delivered at a constant rate with an infusion pump to replace normal losses. An indwelling catheter was passed into the urinary bladder. The bladder was emptied completely and baseline collections initiated.

Urine, blood and hemodynamic data (cardiac output and mean arterial pressure) were collected every fifteen minutes for one hour to establish control conditions. After one hour of baseline study a 3-5 mL bolus of HF was injected into the jugular vein. Urine and blood specimens and hemodynamic data were collected at five minutes and then every fifteen minutes thereafter for a period of 2 hours.

Systemic vascular resistance was calculated from the cardiac output and mean arterial pressure values. Glomerular filtration rate (GFR) was calculated as creatinine clearance (ClCr) and para-amino hippurate (PAH) clearance was used to measure effective renal plasma flow. At least one week elapsed between experiments. A total of six studies were conducted on two different sheep. Urine and Blood Chemistry

Plasma and urine electrolytes, urea nitrogen, creatinine, and glucose were determined by common electrochemical and spectrophotometric methods on the Astra automated clinical laboratory analyzer (Beckman Instruments, Fullerton, CA). The measurements were accurate to within 1% and have <2.5% coefficient of variance over the physiological range of analytes. PAH was measured with the spectrophotometric Bratton-Marshall method, as described by Richterich, Clinical Chemistry: Theory and Practice, New York: S. Karger, Inc., pp. 479-81 (1969). Hemodynamic Effects

The injected HF material elicited hemodynamic responses in all six experiments. Tables 1A and IB compare average hemodynamic measurements obtained during the baseline period with the average of all values following the injection of concentrated HF. The increase in SVR was highly significant by t-test and analysis of variance (p<0.001). The other significant changes were a decrease in cardiac output and increase in mean arterial pressure.

TABLE 1A Hemodynamic Data At Baseline and Following The Infusion of HF. Values Are Mean + (SEM)

TABLE IB

Hemodynamic Data At Baseline and Following

The Infusion of HF. Values are Mean (SEM)

FIGURE 5 shows the mean SVR values plotted against time for the duration of the experiments. The HF injection was followed by an increase in SVR seen at five minutes. SVR remained elevated above baseline values throughout the two hour experimental period. The increase in SVR was highly significant by the analysis of variance (p <0.002).

Infusion of HF produced no effect on glomerular filtration rate as calculated from ereatinine clearance. Similarly, effective renal plasma flow (PAH clearance) was not significantly altered by HF.

Average values of FE Na for all experiments are plotted against time during control and study periods. As seen in FIGURE 6, the increase FE Na was slower to occur than was the SVR response. However, as with SVR, the increase in FE Na lasted throughout the two hour post-injection period. This increase following HF was significant (p <0.01, analysis of variance). Renal Effects

Table 2 shows the renal data at baseline for one hour prior to injection of HF and during the two-hour period following injection based on eight experiments. As shown in Table 2, there was a four-fold increase in FE Na following HF injection compared to baseline values. Associated with the increase in FE Na was an increase of over 50% in urine flow rate. Water clearance calculation revealed a significant decrease in reabsorption of water by the renal tubules. In other words, water clearance increased from baseline following the HF injection. Unlike water and sodium clearance, fractional excretion of potassium decreased following HF infusion.

TABLE 2

Renal Data at Baseline and Following Infusion of HF

Values are Mean (SEM)

Decreased fractional excretion of potassium which was associated with increased urine flow and natriuresis, has not been reported before in natriuresis factor related studies, and is unusual. Normally there is increased tubular secretion of potassium associated with increased urine flow rates. However, it is probable that the inhibition of Na,K-ATPase in the distal nephron causes a decrease in potassium secretion. Recent studies have emphasized the importance of potassium in essential hypertension, and body potassium is known to modulate cardiac glycoside potency. It is interesting that HF modifies the renal handling of potassium.

Example 7 - HF Effect on Simian Smooth Muscle and Canine Kidney Cells Materials

The ⁴⁵CaCl₂ (lmCi/mL) and ²²NaCl(100uCi/mL) were purchased from Amersham Corporation. Ouabain was purchased from Sigma Chemical Company (St. Louis, MO). Minimal Essential Medium, Dulbecco's modified Eagle's medium, fetal calf serum and Hanks balanced salt solution (HBSS) were purchased from Gibco (Long Island, NY). Cell Cultures

Simian aorta smooth muscle cells (SMC) were plated and cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum on 35 mm tissue culture dishes. Canine kidney cells (CKC) were cultured in minimal essential medium (MEM) supplemented with 5% fetal bovine serum. The cells were incubated in a 37°C incubator with a humidified atmosphere of 5% CO₂, 95% air, and used when near confluency. Time Course Studies

Nearly confluent SMC and CKC cells were washed, as detailed below, a constant amount of HF was added and the cells were incubated for 30 min. at 37°C. Then 10 u Ci/mL of 45CaCl₂ was added to each dish and the reaction was allowed to proceed for various specified times as indicated. At the end of each specified time, the reaction was terminated, and the cell-associated radioactivity determined as described below.

Steady-State Labeling of ²²Na⁺ and ⁴⁵Ca²*

Nearly confluent SMC cultures were washed three times with 3 mL of HBSS. Varying concentrations of purified HF-1 or HF-2 were added to cell cultures (duplicates) in 1 mL ⁴⁵Ca (10 u Ci/mL) was added to each culture dish and incubated for two hours prior to determination of cellular radioactivity. At the end of two hours, the solution was aspirated, cells were immediately placed on ice, and quickly washed five times with 3 mL of cold HBSS. After the last aspiration, 2 mL of 0.5% SDC was added to each plate to dissolve the cell layer, which were then transferred to scintillation vials. Then, 15mL of scintillation cocktail (Dϊmϊlume) was added per vial, and the contents were vortexed vigorously before placing in a liquid scintillation counter for determination of cell-associated radioactivity. In a parallel experiment, identical SMC cultures were labeled with 2.5 u Ci/mL ²²Na⁺ to determine HF effect on Na content. Effect of HF on Kinetics of pa— Accumulation in MDCK cells

CKC cells were plated on 60 mm tissue culture dishes and grown in minimal essential medium supplemented with 5% fetal bovine serum until near confluency. After^' washing with warm HBSS, 2 mL of MEM was added per dish. The cells were pretreated with 20 yL of purified HF or 20 yL ETOH for thirty min. at 37°C, and 20 yL of ⁴⁵CaCl₂ was then added and the reaction followed for 1.5 to 10 min. At each respective time point, the reaction was terminated by quickly removing the medium and washing the cells with 5 x 3 mL of cold MEM. Then, 2 mL of 0.5% SDS was added to extract the cell layer, 15 mL of scintillation cocktail added, and the cellular radioactivity was determined in a liquid scintillation counter. The final HF concentration for each dish was 2.5 ng/mL, and the total radioactivity added/dish was 6.21 x 10° cpm.

As shown in FIGURE 10. CKC eells treated with 2 ng/mL HF showed an identical initial rate of net ⁴⁵Ca accumulation; however, the extent of cell associated ⁵Ca was greater by 1.5-2 fold over untreated cells. Effect of HF on Kinetics of ^Ca— Accumulation in SMC Cells

SMC cells were plated on 35 mm tissue culture dishes and grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Nearly confluent cells were pretreated with either 1.05ng/mL purified HF or an equal amount of 0.9% NaCl for thirty minutes at 37°C; 10 yL of ⁴⁵CaCl₂ was added and the reaction monitored for five to thirty minutes. The cells were processed for determination of radioactivity, as described above. Total reactivity added per dish was 3.46 x 10⁶ cpm.

As shown in FIGURE 11, in the experiments using SMC cells, there appears to be a slight difference in initial rate of Ca accumulation. From these experiments it appears that the effect of HF is to allow the content of Ca in both SMC and CKC eells. Effect of HF-1 on ---Na.- and ϋca^± Transport in SMC Cells

Nearly confluent SMC cells were washed with HBSS, and HF-1 of varying concentrations (140-790 pg/mL) was added and preincubated for one hour at 37°C. ⁵CaCl₂ or ²²NaCl was added and the steady-state cellular radioactivity was determined as described above.

As shown in FIGURE 12, HF-1 caused a linear increase in Ca as well as Na content with increased dosage; however, at higher concentrations of HF-1, there appeared to be a lesser increase in ⁴⁵Ca content. Effect of HF-2 on ^Na± and ca²^ Transport in SMC Cells

This is a parallel experiment to that shown in FIGURE 12, except that 40- 390pg/mL of HF-2 was used. The SMC cells were of the same generation. As shown in FIGURES 13 and 14, a similar pattern was also observed with HF-2 for both Na and Ca accumulation; however, with HF-2 (accumulation reached a plateau after 100 pg/mL). For both HF-1 and HF-2, the Ca dose response was more sensitive than the Na dose response.

Example 8 - Receptor Assay for Detection of HF in Patient Sample 100 yL of HF extract (e.g. ether:acetone (1:1) solvent extract for total HF screening or isolated HPLC fractions for HF-1 or HF-2 quantitation) is evaporated to dryness, and reconstituted in H2O. The reconstituted solution is then preincubated with 100 yL canine kidney Na, K-ATPase (Sigma 0.6 mg/mL) and 700 yL of .05 M Tris-Cl, pH 7.2, containing 0.25 mM Na₂EDTA, 5mM MgCl₂, and 100 mM NaCl for 100 minutes at 37°C. Controls with ImM ouabain in 100 yL and blanks with 100 μL H2O only as the test substance are prepared and incubated simultaneously in the same buffer enzyme mixture. 3H - ouabain, 0.4 u Ci in 100 μL, is added to the mixture and incubated for 30 minutes. The reaction is stopped with ice cold buffer and the inhibitor-enzyme complex is immediately collected on glass fiber filters by filtration or centrifugation. The radioactivity retained after following with cold buffer is counted in a liquid scintillation counter. Quantϊtation is made by comparison to ouabain. The quantitative units are defined as 0-100% binding.

Example 9 - Structural Analysis of HF-2

HF's were isolated from hemofiltrate of renal dialysis patients by alkaline solvent extraction and reverse phase C18 HPLC as described above. The purified materials were assayed for Na,K-APase inhibition and immunoreactivity in a digoxin radioimmunoassay using the procedure described in Dasgupta, A. et al., Clin. Chem., 33:890 (1987), the disclosure of which is hereby incorporated by reference.

The ratio of HF-l:HF-2 (DLIS-l:DLIS-2) varied between hemofiltrates from different donors. The HFs were also difficult to isolate in large quantities while retaining their immunologϊeal and inhibitory properties. This suggested that the molecular structure is either inherently labile or susceptible to degradation. Consequently, only HF-2 was extensively analyzed. The material described herf In produced 20% inhibition of canine Na-K-ATPase/nanogram of digoxin equivalent immunoreactϊve material.

Fast atom bombardment mass spectra (FAB MS) were obtained from a mass spec triple analyzer (Krator MS-50) using a dithiothreatol/dithioerythretol matrix. The sample was introduced at 25°C and argon gas used as the fast atom source using a 8kv acceleration potential. The mass spectrometer was operated in positive ion detection mode.

Collisionally activated dissassociation mass spectrometry/mass speetrometry (CAD MS/MS) of positive ions were performed using the same instrument and helium was used as the collision gas.

The FAB MS spectrum of HF-2 (FIGURE 15) shows daughter fragments at m/z 363, 345, 329, 221, 195, 179, 161, and 119.

When the compound was examined by CAD MS/MS, the fragmentation pattern shown in FIGURE 16 was obtained. The strong peak shown at m/z 346 can be interpreted as resulting from the intact molecule after loss of a phosphoserine head group and a proton (FIGURES 17a, b). The presence of a phosphoserine group in the molecule was further supported by the intense peak at m/z 105 ascribed to HOCH₂CH(NH₃₊)(COO^") (FIGURE 17). FIGURE 17 illustrates the structural interpretation of all fragments with mass greater than 100 observed in the two mass spectra. Two fragments shown in FIGURE 16 are consistent with the proposal of a novel component in the parent compound. First, the m/z 291 peak could be ascribed to protonation of a fatty acyl fragment (FIGURE 17d). The intense peak at m/z 273 would then be formed by loss of H₂0 from the same fatty acid fragment (FIGURE 17e). No fatty acids known to occur in humans fit the molecular weight of this factor's structure.

The molecular structure of this fatty acid was derived from the mass spectral data as fellows. The m/z 123 peak could be due to the allylic cleavage of the hypothetical fatty acid between C₁₀ and C_n (FIGURE 17f). The relatively weak peak at m/z 170 could be due to allylic cleavage between Cg and C, on the same fatty acyl chain while the fragment retained the glycerol backbone and hydroxyl group at position 2 but not the phosphoserine head group (FIGURE 17g). The relatively weak peak at m/z 170 points towards an allylic cleavage rather than a double allylic cleavage, suggesting that a Δ5, 8 configuration is more likely than a Δ4, 7 arrangement. The presence of two such allylic cleavage fragments arising from two different parts of a fatty acyl chain point to the presence of four double bonds rather than to one double bond and two triple bonds in the fatty acyl chain. The presence of double bonds at Δ5, Δ8, and Δll is likely because fragments were formed by cleavage between Cg and C- and also between CJQ and CJJ carbons.

It is important to note that the reconstructed compound hypothesized for HF-2 in FIGURE 17, while consistent with the experimental mass speetrometric data, is novel to human biology.

Example 10 - Synthesis of Lysophosphatidylserine (20:4) Ether

The synthesis scheme starts with 1,3-Benzylidene glycerol and is a modification of the approach described by Kertscher, H.P., Pharma∑ie, 38:421-422 (1983) the disclosure of which is hereby incorporated by reference. The 1,3- Benzylidene glycerol is converted to 2-Benzoyl 1,3-glycerol ("Compound A") by addition of benzoyl chloride, potassium hydroxide, and finally acidification with H₂S0₄.

Arachidonyl bromide was prepared from commercially available arachidoncic acid using N-bromosuccinimide and triphenylphosphine. Compound A is then transformed to an arachidonyl ether derivative using arachidonyl bromide and sodium. The free hydroxyl group of position 3 of the glycerol backbone is converted to a phosphoserine head group using the approach of Eibl, H., Chem. Phys. Lipids, 26:405-429 (1980) the disclosure of which is hereby incorporated by reference. Example 11 - Synthesis of Lysophosphatidylseriπe (20:4)

Commercially available phosphatidylcholene (20:4, 20:4) is converted into phosphatidylserine (20:4, 20:4) using phospholipase D and L-serine as described by Djerassi et al., Chem. Phys. Lipids, 37:257-270 (1985). The phosphatidylserine (20:4, 20:4) is easily converted to lyso phosphatidylserine (20:4) using phospholipase-Ag in ether medium in the presence of Ca²⁺ as cofactor.

Example 12 - Synthesis of Lysophosphatidylserine (_Δ5.7.10.14. _19;4)

The A"*'⁷'¹⁰' -19:4 fatty acid is synthesized starting from pentanal and 3- bromopropanoic acid using the standard Wittig reaction. The Wittig salt is formed from 2-bromopropanoie acid and triphenylphosphine using benzene as solvent. The aldehyde is added to dried Wittig salt after which the Wittig salt is deprotonated using n-butyllithium in tetrahydrofuran dimethylsulfoxide medium. The final product acid is then purified by base extraction.

The product Δ°-octanoϊc acid is converted to corresponding aldehyde by lithium aluminum hydride reduction followed by pyridinium chlorochromate oxidation. The final product, Δ -octanal is converted into Δ°" -undecenoic acid using Wittig reaction where triphenylphosphobromide salt of 3-bromoprαpanQΪe acid is used again.

Following the same sequence of steps described above, e.g., reduction, oxidation, and Wittig reaction, again the final product with same Wittig salt yields Δ 3 ^> 6 ' 9 -14:3 acid. Reduction of that acid and subsequent oxidation method as described above yields Δ^3,6'⁹-14:3 aldehyde. Reaction of that aldehyde with triphenylphosphonium bromide salt of 5-bromovaleric acid yields its desired _Δ5,8,ll,14_₁₉._{4 fatty aeid}

The fatty acid is converted to a phosphatϊdylcholine compound containing that fatty acid as a side chain using the approach of Djerassi et al. Chem. Phys. Lipids 37:257-270 (1985). The starting materials are a cadmium salt of 1,2- glycero Sn-3 phosphoeholϊne and the 19:4 fatty acids in presence of dicyclohexylcarbodiimide and 4-(dimethylamϊno)pyridine. The resulting phosphocholϊne molecule is converted into phosphoserine molecule using phospholipase D and L-serine. The final product :s obtained by incubating phosphoserine with phospholipase A2 in presence of Ca~⁺ to cleave the fatty acid in position 2,4 glycerol backbone.

Example 13 - ATPase Assay

In order to minimize the amount of HF used in each assay, also to shorten the time needed to comlete the assay, Thomas Hinds and Hossein Sadrzadeh, have developed a microplate assay for measuring ATPase using 1/5 of the amount of HF (or reagent) used in the standard assay. The total volume is 100 mL for this assay. A 96-well tissue culture plate is used and HF is added to the plates first and the solvent is evaporated under ₂. Then reagents with Na,K, Mg and Ca (for Ca, and Cam assay) is added to all wells. Ouabain (.OlmM) is added to all wells except those for Na,K ATPase. Then a membrane sample is added (absolute protein concentration in .0045 mg) and plates are pre-incubated at 37°C for 15 minutes. Then ATP (3mM) is added to all plates and plates are incubated at 37°C for one hour. The reaction is topped by the addition of^' 20 ul of 5% SDS. A mixture of Ascorbate, acid molylidate, SDS is added to plates (130 mL of the mixture) and the blue color developed is measured at 810 nm in a plate reader.

Accordingly:

Blank = wells with ATP, membrane, ouabain

Mg++ = wells with ATP, membrane, ouabain

Na/K ^•= wells with ATP, membrane, no ouabain

Ca = wells with ATP, membrane, ouabain

Cam = wells with ATP, membrane, calmodulin.

Specific activity:

Mg ATPase = Mg-ATP (Blank)/60 mn/mg

Na/K ATPase = Na,K - Mg

Ca ATPase = Ca - Mg

Cam ATPase = Cam - Mg

Applicants use the micro ATPase assay to check the activity (biological activity) of HF during the purification process and compare that with Digoxin antibody assay (RIA). By so doing, applicants are able to monitor the activity of HF through the purification process and see whether some steps in the process inactivate HF. Also, by comparing the results of ATPase assay with Digoxin antibody assay, applicants can determine the accuracy of this assay for measuring HF since they are concerned with biologically active HF.

While the present invention has been described in conjunction with the preferred embodiment, one of ordinary skill after reading the foregoing specification will be able to effect various changes, substitutions of equivalents and alterations to the methods and compositions set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.

Claims

ClaimsThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of the formula:

CH₂ - 0 - Rl

?^{■ " R2} 0 R5⁺

CH₂ - 0 - R3 - 0 - R4 - CH

OH COO^"

wherein:

Rl is a CIO to C26 unsaturated alkyl or acyl having at least three double bonds;

R2 is -OH, -H, -CH₃ or a CIO to C26 unsaturated alkyl ester;

R3 is S or P;

R4 is a Cl to C6 saturated alkyl; and

R5 is a NH« or alkyl amine.

2. The derivative of Claim 1, wherein Rl is a C19:4 acyl, C20:4 alkyl, or C20:4 acyl; R2 is -OH; R3 is P; R4 is CH₂ or Ch₂CH₂; and R5 is NH3.

.

3. A compound having the biological activity of an endogenous factor associated with essential hypertension isolated from animal tissue or fluid, said factor characterized as a polar lipid having a molecular weight within the range of about 521-541, and an ultraviolet spectrum maximum at about 186 nm.

4. The compound of Claim 1 or 3, wherein said compound is further characterized as being capable of complexing with anti-digoxin antibodies or inhibiting at least one of the following enzymes: Na,K-ATPase; Ca,Mg-ATPase; and calmodulin-activated Ca-ATPase.

5. The compound of Claim 1 or 3, wherein said compound is further characterized as being capable of causing vasoconstriction.

6. The compound of Claim 1 or 3, wherein said compound is further characterized as being capable of causing natriuresis.

7. A method for regulating hypertension in an animal host, comprising administering to said host a pharmaceutically effective dose of the compound of Claim 1 or 3.

8. An antibody capable of specifically binding with the compound of Claim 1, 3, 5, or 6.

9. The antibody of Claim 8, wherein said antibody is a polyclonal antibody.

10. The antibody of Claim 8, wherein said antibody is a monoclonal antibody.

11. An in vitro method for the detection of the presence of a factor associated with essential hypertension in a patient sample comprising contacting said sample with at least one antibody capable of complexing with the compound of Claim 1 or 3 and determining the complexing of said antibody by means of an immunoassay.

12. An in vitro method for determining the amount of a factor associated with essential hypertension present in a patient sample comprising contacting said sample with at least one antibody capable of complexing with the compound of Claim 1 or 3, determining the amount of said antibody complexed with said factor and, correlating the amount of said factor complexed with the amount of said factor present in said sample.

13. An in vitro method for detection of the presence of a factor associated with essential hypertension in a patient sample comprising contacting said sample and the compound of Claim 1 or 3 with an enzyme capable of complexing with said factor and with said compound, and determining the complexing of said enzyme by means of a receptor assay.

14. An in vitro method for determining the amount of a factor associated with essential hypertension present in a patient sample comprising contacting said sample and the compound of Claim 1 or 3 with an enzyme capable of complexing with said factor and with said compound, determining the amount of said compound complexed with said enzyme, and correlating the amount of said compound complexed with the amount of said factor in said sample.

15. A method of Claim 13 or 14, wherein said enzyme is selected from the group consisting of Na, K-ATPase, Ca,Mg-ATPase and calmodulin activated Ca-ATPase.

16. A method according to Claim 13 or 14, wherein said compound is labeled with a detectable marker.

17. A method for regulating hypertension in an animal host, comprising administering to said host a pharmaceutically effective dose of a monoclonal antibody having specific reactivity with the compound of Claim 1 or 3.

18. A hybridoma cell line producing antibodies capable of specifically binding with the compound of Claim 1 or 3.

19. A method for isolation of hypertension factors from a fluid or tissue sample obtained from a patient comprising:

(a) extraction of the sample with an alkaline solution in the presence of ammonium ion; and

(b) extraction with an ether:acetone solvent.

20. A method for resolving a mixture of hypertension factors utilizing high pressure liquid chromotography comprising:

(a) preconditioning the column with an extract from a patient sample; and

(b) separation of said mixture with a methanol:acetonitrile:water solvent system having about a 15-20:15-25:55-75 mixture ratio.