GB2308594A - Sodium pump inhibitor compounds and their binding partners - Google Patents

Description

SODIUM PUMP INHIBITOR COMPOUNDS AND THEIR BINDING PARTNERS The present invention relates to newly identified, naturally occurring sodium pump inhibitor compounds and derivatives thereof. It also relates to immunological binding partners for such compounds, and to methods of analysis of such compounds in biological samples.

There is now considerable evidence that certain kinds of hypertension in mammals, especially humans, are related to increased intracellular sodium levels. It is thought that the high intracellular sodium levels are due to inhibition of the normal cellular sodium pump in susceptible individuals. In hypertension, inhibition of the sodium pump increases renal sodium excretion (natriuresis) and restores vascular volume while at the same time leading to hypertension by increasing intracellular sodium content by potentiating existing vasoconstriction, thereby initiating a new cycle in the pathogenesis of hypertension.

Certain naturally occurring steroidal sodium pump inhibitors are known. These include the cardenolides, which are widespread in the plant kingdom. The cardenolides include Ouabain and Digoxin. The bufadienolides are similar, naturally occurring sodium pump inhibitors. The best known bufadienolide is Bufalin, which is prepared from the dried venom of the Chinese toad. The cardenolides and bufadienolides have the following general formula:

In which R2 is CH3 or CH20H; R3 is H or OH; R4 is H, or R3 and R4 together form a double bond or epoxy group; R5 and R6 are each independently H or OH; and R7 is OH or a glycoside residue. The principal difference between the cardenolides and the bufadienolides is that, in the cardenolides, R7 is a glycoside and R1 is a butenolide (abutenolactone, furone) ring. In bufadienolides, R7 is OH and R1 is a 5-a-pyrone ring. The cardenolides and bufadienolides are used as cardiotonic agents.

GB-A-1362383 describes and claims processes for the synthesis of cardenolide and bufadienolide compounds for use as cardiotonic agents.

The possible role of naturally occurring cardenolides and bufadienolides in mammalian sodium pump inhibition is controversial. There is no conclusive evidence that mammals produce endogenous cardenolides or bufadienolides. In spite of this, W094/12210 describes and claims antibodies raised against the natural toad venom bufadienolides, and the use of such antibodies to treat hypertension. The antibodies are administered to human patients, and are alleged to bind "endogenous digoxin-like factors", thereby reducing natural sodium pump inhibition and relieving acute myocardial ischemia. This method appears to rely on non-specific binding between the antibody raised against toad venom bufadienolides and naturally occurring, bufadienolide-like compounds.

It has now been found that there is a steroidal sodium pump inhibitor compound present in human placentas that has a novel structure distinct from the previously known cardenolides and bufadienolides.

Accordingly, the objects of the present invention include providing the newly identified sodium pump inhibitor compounds in substantially pure form, use of such compounds to raise antibodies, and the provision of binding partners, including immunological binding partners that bind to the newly identified compounds.

The objects of the invention include the provision of tests, including test kits, to identify individuals who may be at risk from diseases mediated by sodium pump inhibitors, including certain types of hypertension, and to identify the causes of hypertension in already hypertensive-patients.

It is a further object of the present invention to provide pharmaceutical compositions comprising the binding partners and/or blocking compounds that mimic the newly identified sodium pump inhibitor compounds and to treat hypertension and related pathological conditions such as acute myocardial ischemia, pre-eclampsia, coronary, cerebral and renal vascular diseases.

The present invention provides a compound of formula I

wherein R1 is a-dihydropyrone; R2 is CH3, CH2OH or esterified CH20H; R3 is H, OH or esterified OH; R4 is H, or R3 and R4 together form a double bond or an epoxy group; 4 and R6 are each H, OH or esterified OH; and R7 is OH, esterified OH or a glycosidic residue.

The characterising feature of these compounds is the presence of the a-dihydropyrone group at the 17 position of the steroid framework. The carbon-carbon double bond of the a-dihydropyrone group may be located across the 3,4 or 4,5 or 5,6 positions of the pyrone ring. The a dihydropyrone may be joined to the steroid through the carbon atom at the 3,4,5 or 6 position of the pyrone ring.

However, preferably, the a-dihydropyrone is a-3,4 dihydropyrone linked to the steroid framework through the 5 position on the pyrone ring (i.e. Dara to the carbonyl group of the dihydropyrone ring).

Preferably, the steroid framework of the compound of Formula I is unsaturated. More preferably, the steroid framework contains one carbon-carbon double bond, most preferably at the 4:5 or 5:6 position of the framework.

Preferably, R2 is CH3. Preferably, R3 is H. Preferably, R4 is H. Preferably, R5 and R6 are H. Preferably R7 is OH.

Most preferably, R1 is 5-a-3,4-dihydropyrone, R2 is CH3, R3, R4, 4 and R6 are all H, and R7 is OH.

The most preferred compound enumerated above is thought to correspond to a natural sodium inhibitor compound actually isolated from human placental tissue. The main novel feature of this compound is the a-dihydropyrone substituent R1. The other substituent definitions for R2 R7 enumerated above are commonplace substituents for the known cardenolide and bufadienolide sodium pump inhibitor compounds, and accordingly it is reasonably predictable that substantially all of the compounds in the claimed group having the a-dihydropyrone substituent as R1 will show useful activity.

The compounds of Formula I can have any stereochemical configuration. However, preferably the stereochemical configuration is the same as that of the corresponding naturally occurring bufadienolide and/or cardenolide compounds, in particular the stereochemistry at the 3, 5, 14 and 17 position of the steroid framework.

Preferably, the compounds are provided in substantially pure form. In certain pharmaceutical compositions the compounds are provided in combination with one or more pharmaceutical excipients.

The present invention also provides the use of the compounds according to the invention in the preparation of standards for the analysis of the same compounds in a biological sample.

The present invention also provides the use of the compounds according to the invention in the preparation of an immunogenic agent.

The present invention also provides a binding partner for binding a compound according to the present invention.

The binding partner is preferably an immunological binding partner more preferably an antibody.

The term "antibody" as used herein comprises monoclonal antibodies, polyclonal antibodies or fragments thereof. The antibody fragments may be fragments such as Fv, Fab and F(ab')2 fragments or any derivatives thereof, such as a single chain Fv fragment. The antibodies or antibody fragments may be non-recombinant, recombinant or humanised.

The techniques for producing such antibodies or antibody fragments are well known to those skilled in the art and are described in EP-A-0120694 and EP-A-0125023.

The binding partner is preferably coupled to a detectable marker, such as an enzyme, a fluorescent marker, a radionuclide or a spin label. The present invention further provides use of a binding partner according to the invention as a diagnostic agent. The present invention yet further provides pharmaceutical compositions comprising a binding partner according to the present invention, and the use of a binding partner according to the present invention for the preparation of medicaments for use in the treatment of medical conditions mediated by a sodium pump inhibitor.

Such conditions include hypertension and pre-eclampsia.

The present invention further provides a test kit for determining the presence of a natural sodium pump inhibitor in a biological sample, the kit comprising a binding partner according to the present invention.

The present invention also provides a method of determining the level of sodium pump inhibition in a mammal, the method comprising quantitating the concentration of compounds according to the present invention in a sample of mammalian tissue or body fluid. Preferably, the method is carried out on a sample of blood or serum. Preferably, the quantitating comprises contacting the sample with an immunological binding partner according to the present invention.

Specific embodiments of the present invention will now be described in detail.

(a) Isolation of a Natural Sodium Pump Inhibitor The natural sodium pump inhibitor was isolated from human placentas. Isolation of the active material was achieved by homogenization of placentas in 10% (V/V) methanol in water and sodium azide (0.1% W/V) in a Waring blender. The homogenate was dialysed in Visking grade tubing for 48 hours at room temperature against 10% methanol in water containing 50g C8 ODS silica. The ODS silica was subsequently recovered by filtration, washed with water and eluted with 100% methanol. The eluate was evaporated to dryness, taken up in 100% methanol and subjected to reversed phase HPLC on a C18 ODS silica column, internal diameter 0.5cm and length 25cm (Thames Chromatography Spherisorb 5mm ODS 2) with a continuous gradient (10% methanol/90% water30% methanol/70% water over two hours, flow rate 2.5ml/min.) Fractions were collected at two minute intervals, evaporated to dryness and analyzed.

(b) Biological Activitv Measurements Biological activity was assessed by determining the inhibition of the sodium pump of leucocytes as described by P.J. Hilton et al. In Journal of Cell Phvsiolosv Vol. 109, pages 323-332 (1981). Briefly, the cells are separated from whole blood by dextran sedimentation and are then loaded with 22Na in the presence of the fraction under test. The loss of radioactivity over a 15 minute period is observed and pseudo-first order rate constant for sodium efflux is calculated from this. The residual eflux in the presence of ouabain is substracted from this to yield the ouabainsensitive rate constant that it is the characteristic of the sodium pump. Inhibition was observed in the HPLC fraction that eluted at 82 minutes in a nominal methanol concentration of 28%.

Further biological testing was undertaken on a fraction that showed 50% inhibition in the leucocyte assay. Ouabain binding studies were performed on leucocytes incubated at 37 0C for 20 minutes in the presence of the active fraction and 3H labelled Ouabain. This study demonstrated 54% inhibition of Ouabain binding, the effect being lost progressively over a ten-fold dilution.

The activity of the above fraction on the sodium pump of erythrocytes was also studied using the uptake of aRb in the presence and absence of 10pM Ouabain. Ouabain-sensitive aRb was inhibited by 36% by the isolated active fraction, which also inhibited canine kidney ATPase when studied by the technique of Hamlyn et al (Nature Vol. 300, pages 650652 (1982)).

(c) Phvsicochemical Characterisation The chemical structure of the active compound obtained above was determined by mass spectrometry and comparison with semi-synthetic compounds.

The principal ion seen on the positive ion fast atom bombardment (FAB) mass spectrometry of the active material was a potassiated species with an m/z of 409. Sodiated and protonated forms were also identified with m/z of 393 and 371, respectively. The accurate mass of the potassiated ion was 409.215, within the resolution limits imposed by the resolution of the instrument and the limited quantity of the material available. The low C:H ratio is in keeping with a cyclic compound. The deuteration of the molecule increased the mass by 1, indicating the presence of one exchangeable hydrogen suggesting a single hydroxyl group. Finally, both electrospray and FAB mass spectrometry in negative ion mode showed a prominent deprotonated molecule ion of m/z 369, which on MS/MS gave rise to a peak of m/z 97 with a product ion at m/z 80 consistent with hydroxyl radical loss.

Since the accurate mass of the parent compound precluded the presence of either sulphur or nitrogen, the fragment of m/z 97 seen on negative ion mass spectrometry must consist of carbon, hydrogen and oxygen only with an empirical formula of CsH602. To investigate further the origins of this fragment, model compounds were examined, derived principally from cardenolides and bufadienolides. Mass spectrometry of the cardenolides Ouabain, digoxin and digitoxin and their aglycones did not yield the fragment of m/z 97 in negative ion mass spectrometry. Under these conditions, bufalin yielded an ion of m/z 95, but this did not undergo a further 17 mass unit loss. This suggests that a dihydropyrone-substituted steroid is the most likely source of the fragment of m/z 97 seen in negative ion mass spectrometry of the active material.

To confirm the above conclusion, bufalin was hydrogenated by exposure to hydrogen at atmospheric pressure and room temperature for 20 minutes in the presence of platinum on charcoal. The resultant mixture of dihydro- and tetrahydro- bufalins was separated by reversed phase HPLC.

The NMR and positive ion mass spectral characteristics confirmed the structure of the products of the reaction, but none of the dihydro-bufalins gave a fragment of m/z 97 in negative ion mass spectrometry, although all inhibited the sodium pump of leucocytes.

To examine the possibility that the failure of these compounds to undergo fragmentation to 97 and subsequently 80 m/z product ions mass spectrometry was due to an effect of the OH at the 14 position of the steroid system, this possibility was eliminated by hydrogenation of bufalin as previously described, followed by dehydration at the site of the tertiary alcohol group with POC13. NMR spectrometry confirmed that the 14-OH group had been replaced by a double bond at the 14:15 position (some elimination towards C8 was also observed) and that a mixture of the dihydro- and tetrahydro- compounds had been formed. On mass spectrometry (both positive and negative ion) no intact molecule ion was detected, but electrospray mass spectrometry in negative ion mode showed fragments of m/z 97 and 80 as seen in the active material. Finally, in order to examine the fragmentation pattern induced by a hydrogen at C14, 3P-OH, 14 a20:21 bufenolide was synthesised from androsterone and this also gave the 97/80 fragments characteristic of the active material in negative ion mass spectromatory.

The experiments described above show that the fragmentation pattern of the active material can be reproduced in semi-synthetic pyrone-substituted steroids, and that this fragmentation does not occur when a hydroxyl group is present at C14, from which it may be predicted that the active material must have either a proton or a double bond at this site. The 8:14 and 14:15-enes of bufalin and dihydrobufalin are virtually insoluble in water and were found to have no detectable effects on the sodium pump of leucocytes, possibly as a result of their insolubility.

However, they must act as the ultimate precursors of the fragments of m/z 97 and 80 seen in negative ion mass spectrometry, the deprotonated molecule ion of which is clearly too unstable to be seen in the mass spectrometer.

Their apparent lack of effect on the sodium pump, which may be a result of their insolubility, precludes the possibility that the naturally occurring material contains a double bond at either of the sites. Accordingly, C14 of the natural sodium pump inhibitor must be hydrogen substituted. This was supported by the study of the compound 3p-OH,14a20:21 bufenolide, which not only gave the characteristic product ions of the active material, but also inhibited the sodium pump of human leucocytes.

It follows from the results presented above that the natural sodium pump inhibitor isolated from human placentas is a dihydropyrone-substituted steroid. Its empirical formula is C24HS03, and its structure is as recited in Formula I above, wherein R1 is 5-a-3,4-dihydropyrone, Rz is CH3, R3, R4, 4 and R6 are all H, and R7 is OH. The biological activity and fragmentation pattern of three - OH, 14a20:21 bufenolide support this view.

(d) Preparation of Isounological Binding Partners Immunological binding partners capable of specifically binding to the sodium pump inhibitor compounds according to the present invention can be prepared by methods well known in the art. By immunological binding partners as used herein is meant primarily antibodies and antibody fragments.

The antibodies may be monoclonal or polyclonal antibodies.

Suitable antibodies and antibody fragments may be prepared by methods well known in the art. Suitable techniques are exemplified in the publication Antibodies: A Laboratorv Manual (1988) Eds. Harlow & Lane, Cold Spring Harbour Laboratories Press, and US Patents Nos. 4,381,292, 4,451,570 and 4,618,577.

Antibodies may be raised against the specific sodium pump inhibitors of the present invention. However, in view of the relatively low molecular weight of these specific compounds, it is preferred to raise antibodies against the compounds conjugated to a carrier molecule. Suitable carrier molecules include bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpet haemocyanin (KLH). The sodium pump inhibitor compounds of the present invention may be conjugated to the carrier molecule, for example, by bonding through the hydroxyl or glycoside group at R7. This is preferred, since it would present the preferred epitope, namely the dihydropyrone substituent, to the primed antibody producing cells (e.g. B-lymphocytes). Methods for binding haptens to carrier molecules are described in detail in, for example, Laboratorv Techniques in Biochemistry and Molecular Biology, T. Chard, Vol. 6 (1987), Partz Elsevier, New York, incorporated herein by reference.

Preferably, the immunological binding partners according to the present invention comprise monoclonal antibodies or monoclonal antibody fragments. Monoclonal antibodies can be produced as follows. The spleen or lymphocytes from an immunised animal are removed and immortalised or used to prepare hybridomas by methods known to those skilled in the art and described in detail in the references above. To produce a human hybridoma, a human lymphocyte donor is selected. Epstein-Barr virus (EBV) can be used to immortalise human lymphocytes or a human fusion partner can be used for human-human hybridomas. Antibodies secreted by the immortalised cells are screened to determine the clones that secrete antibodies of the desired specificity.

(e) Use of immunological binding partners to detect natural sodium puma inhibitors.

The immunological binding partners according to the present invention can be used in diagnostic assays. Among the biologically useful information that can be obtained is a determination of natural sodium pump inhibitors for use in prediction and analysis of medical disorders mediated by endogenous sodium pump inhibitors, for example certain kinds of hypertension. The immunoassays using the immunological binding partners according to the present invention preferably use an antibody or antibody fragment coupled to a detectable marker. Examples of suitable detectable markers include enzymes, coenzymes, enzyme inhibitors, chromophores, fluorophores, chemiluminescent materials, paramagnetic metals, spin labels, and radionuclides.

Examples of standard immunometric methods suitable for measurement of natural sodium pump inhibitor levels in tissue or body fluid samples include, but are not limited to, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and sandwich immunometric assay (IRMA). Further details of these techniques can be found in the textbooks cited above.

In their simplest form, these immunoassays can be used to determine the absolute level of natural sodium pump inhibitor in a body fluid by simply contacting the body fluid with an immunological binding partner specific for the natural sodium pump inhibitor. Preferably, the body fluid is initially partially purified by techniques such as cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.

Test kits in accordance with the present invention contain immunological binding partners prepared as described above that specifically bind to natural sodium pump inhibitor molecules. It is preferred that the immunological binding partners are coupled to a detectable marker of the type described above.

(f) Therapeutic use of the immunoloqical binding partners The immunological binding partners according to the present invention can be combined with other pharmaceutically acceptable ingredients in medicaments for use in the treatment of medical conditions mediated by endogenous sodium pump inhibitors. These conditions include hypertension and cardiac arrythmia. Treatment is carried out by administering the compositions by any of the conventional routes, for example, oral, intramuscular, intravenous or rectally. Preferably, the compositions are administered intravenously. The actual dosage unit is determined by such generally recognised factors as body weight of patient, and, in particular, the severity of the pathological condition being treated. The dosage is also preferably correlated with the level of endogenous sodium pump inhibitor, as determined by an immunoassay technique as detailed above.

Claims (16)

1. A compound of formula (I):

wherein R1 is a a-dihydropyrone; R2 is CH3, CH2OH or esterified CH2OH; R3 is H, OH or esterified OH; R4 is H, or R3 and R4 together form a double bond or an epoxy group; RS and R6 are independently each H, OH or esterified OH; and R7 is OH, esterified OH or a glycoside residue.

2. A compound according to claim 1, wherein R1 is 5-a-3,4dihydropyrone.

3. A compound according to claim 1 or 2, wherein R3 and R4 are each H.

4. A compound according to claim 1, 2 or 3, wherein: R1 is 5-a-3,4-dihydropyrone; R2 is CH3; R31 R4, R5 and R6 are all H; and R7 is OH.

5. A pharmaceutical composition comprising a compound according to any of claims 1 to 4.

6. Use of a compound according to any of claims 1 to 4 in the preparation of a standard for the analysis of a biological sample.

7. Use of a compound according to any of claims 1 to 4 in the preparation of an immunogenic agent.

8. A binding partner for binding a compound according to any of claims 1 to 4.

9. A binding partner according to claim 8, comprising an antibody.

10. A binding partner according to claim 9, comprising an antibody or antibody fragment coupled to a detectable marker.

11. Use of a binding partner according to claim 8, 9 or 10 as a diagnostic agent.

12. A test kit for determining the presence of an endogenous sodium pump inhibitor in a biological sample, wherein the test kit comprises a binding partner according to claim 8, 9 or 10.

13. A pharmaceutical composition comprising a binding partner according to claim 8 or 9.

14. Use of a binding partner according to claim 8 or 9 for the preparation of a medicament for the treatment of a medical condition mediated by endogenous sodium pump inhibitors.

15. A method of determining the level of sodium pump inhibitors in a mammal, the method comprising quantitating the concentration of one or more compounds according to any of claims 1 to 4 in a tissue or body fluid sample.

16. A method according to claim 15, comprising contacting the said sample with an immunological binding partner according to claim 8 or 9.