Compounds as positive allosteric modulators for erythropoietin and erythropoietin receptor to treat erythropoietin deficiency diseases

Abstract

Disclosed are compounds which can act as a positive allosteric modulator for erythropoietin and erythropoietin receptor and have the activity in promoting erythropoiesis. Also disclosed are pharmaceutical compositions comprising said compounds and treatment methods utilizing said compounds.

Claims

1. A compound, which is a positive allosteric modulator for erythropoietin and erythropoietin receptor and has a formula selected from the group consisting of: ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##

2. The compounds of claim 1, having a formula selected from the group consisting of: ##STR00068## ##STR00069##

3. The compound of claim 1 for use in treating an erythropoietin deficiency disease by acting as a positive allosteric modulator for erythropoietin and erythropoietin receptor.

4. The compound of claim 2 for use in treating an erythropoietin deficiency disease by acting as a positive allosteric modulator for erythropoietin and erythropoietin receptor.

5. A pharmaceutical composition comprising a compound according to claim 1, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the compound has a formula selected from the group consisting of: ##STR00070## ##STR00071##

7. The pharmaceutical composition of claim 5 for treating an erythropoietin deficiency disease.

8. The pharmaceutical composition of claim 7, wherein the erythropoietin deficiency disease is selected from the group consisting of anemia, a chronic kidney disease, chronic heart failure, a neurodegenerative disease, age-related macular degeneration, a chronic obstructive pulmonary disease, an anemic cancer in a patient undergoing chemotherapy, dry eye, and aging related insomnia.

9. The pharmaceutical composition of claim 8, wherein the erythropoietin deficiency disease is anemia.

10. The pharmaceutical composition of claim 9, wherein the erythropoietin deficiency disease is anemia associated with a kidney disease.

11. A method for treating an erythropoietin deficiency disease comprising administering to a subject in need thereof an effective amount of a compound according to claim 1.

12. The method of claim 11, wherein the compound has a formula selected from the group consisting of: ##STR00072## ##STR00073##

13. The method of claim 11, wherein the erythropoietin deficiency disease is selected from the group consisting of anemia, a chronic kidney disease, chronic heart failure, a neurodegenerative disease, age-related macular degeneration, a chronic obstructive pulmonary disease, an anemic cancer in a patient undergoing chemotherapy, dry eye, and aging related insomnia.

14. The method of claim 13, wherein the erythropoietin deficiency disease is anemia.

15. The method of claim 14, wherein the erythropoietin deficiency disease is anemia associated with a kidney disease.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

(2) In the drawings:

(3) FIG. 1A shows the effect of EH202 on the formation of hemoglobin in bone marrow cells.

(4) FIG. 1B shows the effect of EH203 on the formation of hemoglobin in bone marrow cells.

(5) FIG. 1C shows the effect of EH204 on the formation of hemoglobin in bone marrow cells.

(6) FIG. 1D shows the effect of EH205 on the formation of hemoglobin in bone marrow cells.

(7) FIG. 1E shows the effect of EH206 on the formation of hemoglobin in bone marrow cells.

(8) FIG. 1F shows the effect of EH207 on the formation of hemoglobin in bone marrow cells.

(9) FIG. 1G shows the effect of EH222 and EH232 on the formation of hemoglobin in bone marrow cells.

(10) FIG. 2A is a schematic diagram showing the protocol for investigating the therapeutic effects of EH202 after cisplatin (CDDP)-induced nephropathic mice.

(11) FIG. 2B shows that EH202 accelerates the recovery from renal dysfunction in cisplatin-induced nephropathy in mice. The functional recovery of the kidneys of mice treated with EH202 on day 24 was measured by creatinine level in the peripheral blood. The values are presented as the meansSEM (n=3-6 animals each group). ***p<0.001, **p<0.01 versus control group.

(12) FIG. 2C shows that EH202 accelerates the recovery from renal dysfunction in cisplatin-induced nephropathy in mice. The functional recovery of the kidneys of mice treated with EH202 on day 29 was measured by BUN level in the peripheral blood. The values are presented as the meansSEM (n=3-6 animals each group). **p<0.01, *p<0.05 versus control group.

(13) FIG. 2D shows that EH202 accelerates the recovery from anemia in cisplatin-induced nephropathy in mice. The functional recovery of the RBC of mice treated with EH202 on day 24 was measured by hemoglobin (HGB) level in the peripheral blood. The values are presented as the meansSEM (n=3-6 animals each group). **p<0.01, *p<0.05 versus control group.

(14) FIG. 2E shows that EH202 accelerates the recovery from anemia in cisplatin-induced nephropathy in mice. The functional recovery of the RBC of mice treated with EH202 on day 29 was measured by RBC numbers in the peripheral blood. The values are presented as the meansSEM (n=3-6 animals each group). **p<0.01, *p<0.05 versus control group.

DETAILED DESCRIPTION OF THE INVENTION

(15) The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

(16) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

(17) As used herein, around, about or approximately shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximately, meaning that the term around, about or approximately can be inferred if not expressly stated.

(18) In one aspect, the invention provides a compound having a formula selected from the group consisting of:

(19) ##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##

(20) The chemical names of said compounds can be found in Table 1 below.

(21) TABLE-US-00001 TABLE 1 Chemical name and structure of the compound Name Structure Formula (1), also called EH202: (Naphthanol- 4fluoro- stilbenzene)- 1-O--D- glycoside Formula (2), also called EH203: (Naphthanol-4- fluoro- stilbenzene)-2- O--D-glycoside Formula (3), also called EH204: (3,5,4- trifluorophenol- stilbenzene)- 2-O--D- glycoside Formula (4), also called EH205: (Naphthanol-4- methoxy- stilbenzene)- 1-O--D- glycoside Formula (5), also called EH206: (3,5-difluoro- phenol- stilbenzene)- 2-O--D- glycoside Formula (6), also called EH207: (3,5-difluoro- phenol-4- methoxy- stilbenzene)- 2-O--D- glycoside Formula (7), also called EH208: (Naphthanol-2- fluoro- stilbenzene)-1- O--D-glycoside Formula (8), also called EH209: (3,5-difluoro- phenol-2- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (9), also called EH210: (3,5-difluoro- phenol-4- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (10), also called EH211: (Naphthanol-3- methoxy- stilbenzene)- 1-O--D- glycoside 0 Formula (11), also called EH212: (Naphthanol- 3,5-difluoro- stilbenzene)- 2-O--D- glycoside Formula (12), also called EH213: (3,5-difluoro- phenol-3- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (13), also called EH214: (Naphthanol-2- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (14), also called EH215: (Naphthanol-3- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (15), also called EH216: (Naphthanol- stilbenzene)- 1-O--D- glycoside Formula (16), also called EH217: (Naphthanol-2- fluorostilbenzene)- 2-O--D- glycoside Formula (17), also called EH218: (Naphthanol-4- (trifluoromethyl) stilbenzene)- 2-O--D- glycoside Formula (18), also called EH219: (Naphthanol-3- fluorostilbenzene)- 2-O--D- glycoside Formula (19), also called EH220: (3,5,4-triamino- stilbenzene)- 2-O--D- glycoside Formula (20), also called EH221: (3,5-dimethoxy- 2-fluoro- stilbenzene)-2- O--D-glycoside 0 Formula (21), also called EH222: (3,5-dimethoxy- 4-fluoro- stilbenzene)-2- O--D-glycoside Formula (22), also called EH223: (3,5-dihydroxy- 4-fluoro- stilbenzene)-2- O--D-glycoside Formula (23), also called EH224: (3,5-dimethoxy- 3-fluoro- stilbenzene)-2- O--D-glycoside Formula (24), also called EH225: (3,5-dimethoxy-2- nitrostilbenzene)- 2-O--D- glycoside Formula (25), also called EH226: (3,5-dimethoxy-3- nitrostilbenzene)- 2-O--D- glycoside Formula (26), also called EH227: (3,5-dimethoxy-4- nitrostilbenzene)- 2-O--D- glycoside Formula (27), also called EH228: (3,5-dimethoxy-2- cyanostilbenzene)- 2-O--D- glycoside Formula (28), also called EH229: (3,5-dimethoxy-3- cyanostilbenzene)- 2-O--D- glycoside Formula (29), also called EH230: (3,5-dimethoxy-4- cyanostilbenzene)- 2-O--D- glycoside Formula (30), also called EH231: (3,5,2-trimethoxy- stilbenzene)- 2-O--D- glycoside 0 Formula (31), also called EH232: (3,5,4-trimethoxy- stilbenzene)- 2-O--D- glycoside Formula (32), also called EH233: (3,5,3-trimethoxy- stilbenzene)- 2-O--D- glycoside Formula (33), also called EH234: (E)-2-(2- (naphthalen- 2-yl)vinyl) naphthaol-1- O--D-glycoside

(22) Preferably, the compound has a formula selected from the group consisting of:

(23) ##STR00054## ##STR00055##

(24) The compound of the present invention was found to be a positive allosteric modulator for erythropoietin and erythropoietin receptor and has the activity in promoting erythropoiesis. Accordingly, the compound of the present invention may be used in treating an erythropoietin deficiency disease.

(25) A positive allosteric modulator (PAM) or allosteric enhancer induces an amplification of the orthosteric agonist's effect, either by enhancing the binding affinity or the functional efficacy of the orthosteric agonist for the target protein (May, L. T. et al., Annual review of pharmacology and toxicology 47, 1-51 (2007)).

(26) The compound of the present invention may be chemically synthesized through a process known in the art.

(27) In another aspect, the present invention provides a pharmaceutical composition comprising a compound according to the present invention, and a pharmaceutically acceptable carrier.

(28) The pharmaceutical composition of the present invention may be used to treat an erythropoietin deficiency disease.

(29) In one further aspect, the present invention provides a method for treating an erythropoietin deficiency disease. The method comprises administering to a subject in need thereof an effective amount of a compound according to the present invention.

(30) According to the present invention, the erythropoietin deficiency disease includes but is not limited to anemia, a chronic kidney disease, chronic heart failure, a neurodegenerative disease, age-related macular degeneration, a chronic obstructive pulmonary disease, an anemic cancer in a patient undergoing chemotherapy, dry eye, and aging related insomnia.

(31) In certain embodiments of the present invention, the erythropoietin deficiency disease is anemia associated with a kidney disease. The kidney disease includes but is not limited to a chronic kidney disease, an acute kidney injury, renal ischemia, renal failure, and a combination thereof.

(32) Examples for a neurodegenerative disease include amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and Parkinson's disease.

(33) The pharmaceutical composition of the present invention can be manufactured by conventionally known methods with one or more pharmaceutically acceptable carriers. The term pharmaceutically acceptable carrier as used herein encompasses any of the standard pharmaceutical carriers. Such carriers may include, but are not limited to: saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, cremophor, nanoparticles, liposome, polymer, and combinations thereof.

(34) The pharmaceutical composition of the present invention may be constituted into any form suitable for the mode of administration selected. For example, compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for topical administration include cream, ointment, gel, suspension, drops, emulsions, skin patches.

(35) In addition to standard carriers, an oral pharmaceutical composition of the present invention may be supplemented with one or more excipients that are normally employed in oral formulations, such as surfactants, inhalants, solubilizers, stabilizers, emulsifiers, thickeners, coloring agents, sweetening agents, flavoring agents, and preservatives. Such excipients are well known to those skilled in the art.

(36) According to the invention, the pharmaceutical composition may be administered to a subject through any route, such as oral administration or parenteral injection.

(37) The term effective amount as used herein refers to a sufficient amount of a compound of the present invention to provide desired therapeutic effects, or the induction of a particular type of response. The effective amount required varies from subject to subject, depending on the disease state, physical conditions, age, sex, species and weight of the subject, etc. However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

(38) The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

EXAMPLES

Example 1: Computational Docking Simulations

(39) Computational docking simulations were carried out to predict the binding of EH202 analogs to EPO-EPO receptor (EPOR) complex. Among our designed over 200 chemical molecules docked into models of computation simulation system, thirty three chemical molecules are selected as positive allosteric modulators (EH202EH234) by free energy of binding to EPO-EPOR complex. We found that each EH202 analogs binds preferentially to the EPO-bound EPOR complex (EPO/EPOR) rather than the EPO-free nave EPOR (estimated free energy of binding for EH202 analogs were ranging from 6.92 to 10.03 kcal.Math.mol.sup.1, compared to 6.30 kcal.Math.mol.sup.1 for EPO-free nave EPOR). See Table 2 below.

(40) TABLE-US-00002 TABLE 2 Estimated free energy of binding for EH202 analogs Estimated free energy of binding Compound to EPO-bound EPOR complex EH202 10.03 kcal/mol EH203 9.06 kcal/mol EH204 8.96 kcal/mol EH205 8.78 kcal/mol EH206 8.24 kcal/mol EH207 7.65 kcal/mol EH208 9.91 kcal/mol EH209 9.88 kcal/mol EH210 9.73 kcal/mol EH211 9.73 kcal/mol EH212 9.64 kcal/mol EH213 9.52 kcal/mol EH214 9.45 kcal/mol EH215 9.38 kcal/mol EH216 9.34 kcal/mol EH217 9.31 kcal/mol EH218 9.29 kcal/mol EH219 9.18 kcal/mol EH220 8.97 kcal/mol EH221 8.87 kcal/mol EH222 8.64 kcal/mol EH223 8.54 kcal/mol EH224 8.51 kcal/mol EH225 8.04 kcal/mol EH226 7.92 kcal/mol EH227 7.84 kcal/mol EH228 7.69 kcal/mol EH229 7.60 kcal/mol EH230 7.57 kcal/mol EH231 7.52 kcal/mol EH232 7.31 kcal/mol EH233 7.18 kcal/mol EH234 6.92 kcal/mol

Example 2: Synthesis of EH222

1. Synthesis of (2-bromo-4,6-dimethoxyhydroxyphenoxy)-O-aceton--D-glycoside

(41) ##STR00056##

(42) The mixtures of 2-bromo-4,6-dimethoxyphenol (5 mmol), cesium carbonate (5.5 mmole) and Acetonbromo-alpha-D-Glucose (15 mmole) in acetonitrile were stirred for 16 hours at room temperature under nitrogen. The inorganic precipitate was filtered off, and the filtrate was concentrated under reduce pressure. The residue was diluted with water and extracted with dichloromethane three times. The combined organic solution was dried over magnesium sulfate and evaporated by Rota Vapor. The residue purification was chromatographed on silica gel with EA/n-hexane as the eluent. The compound was white solid and the yield was 72%.

2. Synthesis of (2-hydroxy-3,5-dimethoxy-4fluoro-stilbenzene)-2-O-aceton--D-glycoside

(43) ##STR00057##

(44) The Br-glycoside derivate as starting material was mixed with triethylamine (1.5 mmole) and 1-fluoro-4-vinylbenzene (1.5 mmole) in the presence of catalyst (Bis-triphenylphosphine) (5 mmole %) palladium dichloride to dissolve in dry/degas DMF at 110 degree over 20 hours. The reaction was monitored by TLC stain until the starting material was consumption. The solvent was removed by Rota Vapor and extracted by EA with water. The organic layer was dry magnesium sulfate. Then, the suspension was filtered and concentrated in vacuum. The residue purification was chromatographed on silica gel with EA/n-hexane as the eluent. The compound was white solid and the yield was 80%.

3. Synthesis of EH222

(45) ##STR00058##

(46) A solution of (2-hydroxy-3,5-dimethoxy-4fluoro-stilbenzene)-2-O-aceton--D-glycoside in anhydrous methanol was treated with methanolic sodium methoxide for 6 hours. The reaction was monitored by TLC stain until the starting material was consumption. The mixture was neutralized with Amberlite IR-120(H.sup.+) and the residue purification was through short column with methanol as the eluent. The compound was white solid and the yield was 95%.

(47) .sup.1H NMR (400 MHz, CDCl.sub.3, 25 C.): 7.78 (d, .sup.3J.sub.HH=16.5 Hz, 1H, CH), 7.63 (m, 2H, ArH), 7.11 (d, .sup.3J.sub.HH=17.2 Hz, 1H, CH), 7.08 (m, 2H, ArH)), 6.84 (s, 1H, ArH), 6.56 (s, 1H, ArH), 4.74 (dd, .sup.3J.sub.HH=7.6 Hz, .sup.4J.sub.HH=2.28H, 1H, Glycoside), 4.30 (s, 6H, OCH.sub.3), 3.78 (m, 1H, Glycoside), 3.69 (m, 1H, Glycoside), 3.58 (m, 1H, Glycoside), 3.47 (m, 2H, Glycoside), 3.20 (m, 1H, Glycoside); LRMS(ESI.sup.+): calculated for [M+H].sup.+: 459.15; Found: 459.2.

Example 3: Synthesis of EH232

1. Synthesis of (2-hydroxy-3,5,4-trimethoxystilbenzene)-2-O-aceton--D-glycoside

(48) The reagent was replacement for 4-vinylanisole and followed the procedure as describe in Example 2. The product yield was 71%.

2. Synthesis of EH232

(49) The procedure as describe in Example 2 was followed. The product yield was 98%.

(50) .sup.1H NMR (400 MHz, CDCl.sub.3, 25 C.): 7.55 (d, .sup.3J.sub.HH=16.4 Hz, 1H, CH), 7.42 (d, .sup.3J.sub.HH=8.8 Hz, 2H, ArH), 6.85 (d, .sup.3J.sub.HH=16.0 Hz, 1H, CH), 6.84 (d, .sup.3J.sub.HH=8.8 Hz, 2H, ArH), 6.65 (d, .sup.4J.sub.HH=2.8 Hz, 1H, ArH), 6.29 (d, .sup.4J.sub.HH=2.8 Hz, 1H, ArH), 4.50 (d, .sup.3J.sub.HH=8.8 Hz, 1H, Glycoside), 4.30 (br, 4H, Glycoside-OH), 3.79 (s, 3H, OCH.sub.3), 3.76-3.61 (m, 7H, Glycoside), 3.72 (s, 6H, OCH.sub.3); LRMS(ESI.sup.+): calculated for [M+H].sup.+: 471.17; Found: 471.2.

Example 4: Effects of the Compounds on the Formation of Hemoglobin in Bone Marrow Cells

(51) C57BL/6JNarl mice, 8-10 weeks of age, were purchased from National Laboratory Animal Center (NLAC, Taiwan) and used. Acute hemolytic anemia was induced by a single intraperitoneal (i.p.) injection phenylhydrazine hydrochloride (Sigma-Aldrich) at a dose of 100 mg/kg in phosphate buffered saline (PBS). Bone marrow cells from mice were isolated and cultured as reported earlier with minor modification 6 days after injection (Worthington et al., 1985 and Rosenthal et al., 1987). Cells were adjusted to about 610.sup.5 cells/ml in MEM alpha medium (-MEM, Gibco) containing 1% (v/v) bovine serum albumin (BSA, Sigma-Aldrich), 7.5 M 2-mercaptoethanol (Sigma-Aldrich), 1.4 mM L-glutamine (Sigma-Aldrich), 10 M ferric chloride (FeCl3, Sigma-Aldrich) and 50 mU/ml EPO (RecormonEpoetin, Roche), plated at approximately 1.510.sup.5 cells/well on 96-well plates (Costar) and then cultured at 37 C. in a humidified incubator of 5% CO.sub.2-95% air. Cells were treated with different concentrations of EH202EH207, EH222 and EH232 (0, 0.1, 1, 10, and optionally, 100 g/ml) the next day, and the relative level of hemoglobin were determined by DAF-based hemoglobin colorimetry assay (Kaihoand Mizuno, 1985 and Worthington et al., 1985) with minor modification 4 days later. In brief, cells were washed with PBS, lysed in 50 l/well of 0.01% (v/v) Nonidet P 40 (NP-40, Sigma-Aldrich) and added with 100 l/well of 100 g/ml 4,5-diaminofluorescein (DAF, Sigma-Aldrich) as well as 6 l/well of 30% hydrogen peroxide (Sigma-Aldrich). After incubation for 5-10 minutes, the absorbance at 620 nm was measured by a Victor 2 1420 Multilable Counter (Wallac, PerkinElmer). Results were expressed as relative indexS.E. (n=6) and statistical significance was evaluated by Student's t test (*P<0.05, **P<0.01, ***P<0.001 versus control group (0 g/ml)). The results shows that the compounds significantly promote the formation of hemoglobin at the concentration of 0.1100 g/ml (FIGS. 1A-1G).

Example 5: EH202 Ameliorates Anaemia and Renal Function in Cisplatin-Induced Nephropathy

1. Materials and Methods

(52) Forty six-to-seven-week-old C57Bl/6J male mice were i.p. injected with three doses of cisplatin (Sigma-Aldrich), following the scheme of 7, 6, and 6 mg.Math.kg-1 body weight, at 4 to 5 day intervals, and the normal group (n=4) was injected with saline (FIG. 1 scheme). On day 12, the collected serum samples were assayed for the urea nitrogen content (BUN). Mice with BUN values greater than 80 mg.Math.dL-1 were chosen for the experiment. An average seventy percent of injected mice were successful induced renal dysfunction, and the ineffective mice were excluded from the EH202 treating experiments. The mice were subsequently divided randomly into 4 cohorts comprising the control (Ctrl, n=6) and three EH202-treated groups (n=5 to 6 for each group) for an additional 2 weeks. Blood samples from all the mice were collected every 5 days. The RBC numbers were determined from the complete blood cell count using a Sysmex Kx-21 haematology analyser (Sysmex America), and the serum BUN and creatinine levels were determined using a commercial kit (Randox Laboratories Ltd. United Kingdom). All results are expressed as the meanSEM. The statistical analysis was performed using Student's t-test. One-way ANOVA was used to examine the differences across the animal experimental groups. The posthoc differences between the means of the experimental groups were determined via Tukey's test. P<0.05 was considered significant.

2. Results

(53) Because acute kidney injury may result from renal ischemia caused by the use of nephrotoxic agents and to examine the effect of EH202-induced EPO production on the anaemia with renal insufficiency, we adopted an established cisplatin-induced nephropathy mouse model (FIG. 2A). We observed significant impaired renal function from day 12 and anaemia from day 19 after the first injection of cisplatin. Notably, the administration of 30 and 90 mg.Math.kg-1 of EH202 for 10 days (on day 24, FIG. 2B) led to an almost complete recovery of renal dysfunction. Moreover, the BUN levels of the EH202 10, 30 and 90 mg.Math.kg-1 treatment groups were also significantly recovered (FIG. 2C). Furthermore, the administration of 30 and 90 mg.Math.kg-1 of EH202 (FIGS. 2D and 2E) led to an increasing recovery of anaemia. Collectively, these findings suggest that EH202 improved the recovery from cisplatin-induced anaemia and renal dysfunction.

(54) It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.