5-β, 14-β-androstane derivatives useful for the treatment of proteinuria, glomerulosclerosis and renal failure

Abstract

Compound of formula (I), wherein the symbol have the meaning reported in the text; for preparing a medicament for the prevention and/or treatment of proteinuria, glomerulosclerosis or renal failure.

Claims

1. A method for treating proteinuria, comprising the step of administering to a normotensive patient in need thereof a therapeutically effective amount of a compound of formula (I), ##STR00002## wherein: the symbol represents a single bond; Y is OR.sup.4 and has an alpha or a beta configuration; R is 3-furyl; R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen.

2. The method according to claim 1, wherein the compound is administered in a dose of from 0.05 mg to 20 mg per day.

3. The method according to claim 2, wherein the compound is administered in a dose of from 0.5 mg to 15 mg.

4. The method according to claim 3, wherein the compound is administered in a dose of from 5 mg to 10 mg.

5. The method according to claim 4, wherein the compound is administered) in a single dose schedule.

6. The method according to claim 4, wherein the compound is administered in a multiple dose schedule.

7. The method according to claim 1, wherein the compound is administered to the patient orally, intravenously, intramuscularly, intra-arterially, intramedullary, intrathecally, intraventricularly, transdermally, transcutaneously, subcutaneously, intraperitoneally, intranasally, enterally, topically, sublingually or rectally.

8. A method of antagonizing podocyte protein loss in a normotensive patient in need thereof, which comprises administering to said patient a therapeutically effective amount of a compound of formula (I), ##STR00003## wherein: the symbol represents a single bond; Y is OR.sup.4 and has an alpha or a beta configuration; R is 3-furyl; R.sup.1 is hydrogen; R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is hydrogen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents the level of urinary protein excretion (mg/6 h) in mice carrying the knockout (KO) of the beta adducin as compared with the wild type (WT) controls. Male mice were 11 month-olds and urinary protein excretion was measured on urine collected for 6 hours from each mouse housed in metabolic cage. Data are meansem of 15 WT and 19 KO mice. Statistical analysis was carried out by t Student's test. The figure shows that the 6 hour-urinary protein excretion was significantly decreased (by 30%) in KO mice for beta adducin as compared to WT controls.

(2) FIG. 2 represents the amount of podocyte proteins (nephrin, -actinin, ZO-1, podocin, -adducin and actin) expressed in cultured podocytes obtained from neonatal (<10-day-old) rats from the congenic NB and NA strains. Podocyte proteins were quantified on podocyte extracts by Western blotting with appropriate antibodies (see the representative traces on the top of bars). Data are reported as meant sem of several experiments ranging from 4 to 24 for each strain. Statistical analysis was carried out by t Student's test. The figure shows that the amounts of Nephrin, -Actinin, ZO-1, Podocin and -Adducin are significantly reduced in podocytes from NB normotensive rats carrying the mutant -adducin as compared to NA controls carrying the wild type variant, while the housekeeper protein actin is similar.

(3) FIG. 3 represents the expression of some podocyte proteins (Nephrin, Synaptopodin, -Actinin, ZO-1, Fyn and Vimentin) as detectable by immunofluorescence in renal glomeruli from NB normotensive rats carrying the mutant -adducin as compared to NA controls carrying the wild type variant. The figure shows that the expression of these proteins is drastically reduced in NB as compared to NA rats, while Vimentin, a microfilament localized in the podocyte cell body, is normally expressed in the two strains.

(4) FIG. 4 shows the progression of renal failure evaluated as the decay of glomerular filtration rate (GFR) over time (ml.min^.year 1) in patients affected by IgA nephropathy subdivided in 4 groups according to -adducin (ADD1, Gly460Tyr) and -adducin (ADD2, C399T) genotypes. The interaction between the two genes on the rate of decay was found significant.

(5) FIG. 5 represents the amount of podocyte proteins (nephrin, ZO-1, podocin, -adducin, synaptopodin and actin) expressed in cultured podocytes obtained from neonatal (<10-day-old) rats from the congenic NB strain and incubated for 5 days with or without Rostafuroxin 10.sup.9M. Podocyte proteins were quantified on podocyte extracts by Western blotting with appropriate antibodies. Data are reported as meansem of several experiments. Statistical analysis was carried out by t Student's test. The figure shows that the amounts of Nephrin, ZO-1, Podocin, -Adducin and Synaptopodin, but not Actin, are increased in podocytes cultured in the presence of 10.sup.9M Rostafuroxin.

(6) FIG. 6 represents the systolic blood pressure (SBP), urinary protein excretion and amount of Nephrin from renal cortex of rats chronically infused with ouabain (OS) and treated with vehicle as compared either to control saline infused rats or OS rats orally treated for 8 weeks with Rostafuroxin 100 g/kg/day. Data are reported as meansem of 8 rats for each group. Statistical analysis was carried out by t Student's test. The figure shows that Rostafuroxin significantly reduced SBP and urinary protein excretion while it increased Nephrin expression in OS rats, thus antagonizing the renal effects of ouabain.

(7) The following non-limiting examples further illustrate the invention.

EXAMPLE 1

(8) To test the activity of the compound of the invention for the prevention of loss of podocyte proteins, congenic NB rats carrying the beta adducin mutation (Tripodi G. et at Effect of Addl gene transfer on blood pressure in reciprocal congenic strains of Milan rats. BBRC 2004; 324: 562-568) were used. Said NB rats are non-hypertensive rats and are available at Prassis Research Institute, Sigma-tau, Italy.

(9) NB rats of 7 to 10 days of age were used for podocyte isolation and culture. Podocytes from NB rats were incubated for 5 days without (NB control, n=4) and with Rostafuroxin at 10.sup.9M (NB n=5). Podocyte proteins were quantified at the end of the 5 days of incubation by Western blotting. The quantification by Western blot was replicated two to three times for each podocyte marker. Tables IA and IB show the final number of podocyte samples analyzed for each condition, as mean values of the replicates (NB control, n=4; NB+Rostafuroxin, n=5). The densitometric analysis was quantified as optical density, in arbitrary units.

Podocyte Isolation and Protein Quantification in Cultured Podocytes

(10) Glomeruli were isolated from NB kidneys by sieving and further manually purification. Glomeruli were then seeded in culture flasks (Corning, Sigma-Aldrich, Milan, Italy), pre-coated with collagen type IV (Sigma-Aldrich) at 37 C. in 5% CO.sub.2 atmosphere. On days 4 to 5, podocyte growth started and, by day 8, glomeruli were detached using trypsin-EDTA. Second passage podocytes, which resulted in >90% pure as judged by light microscopy inspection, were seeded on flasks and chamber slides. Podocyte protein quantification (10 g protein/lane) was performed by Western blotting technique by using specific antibodies against nephrin, podocin, ZO-1, adducin, synaptopodin and actin.

(11) The results obtained are reported in the following Table 1A, 1B and in FIG. 5

(12) TABLE-US-00001 TABLE 1A Podocyte Sample NB Sample NB + % Marker number Controls number Rostafuroxin vs. Ctrl. Nephrin 1 19.21-19.27 1 24.53 2 27.06 mean 19.24 25.8 +34 ZO-1 1 7.69-7.68- 1 17.3-17.8 5.38-1.16 2 6.38-7.5 2 3 8.73-13.33- 15.79 3 4 11.67-16.05- 19.98 4 5 12.78-19.06- 20.13 Mean 7.69 1.03 14.36 1.3 +86 sem p < 0.001 Podocin 1 18.32-14.8- 1 23.4-49.06 4.6-4.85 2 13.3-19.07 2 11.9-14.38- 3 16.81-18.63- 12.23 21.42 3 14.15-16.57- 4 18.78-23.84- 21 36.51 4 19.63-22.76- 5 21.23-23.65- 20.3 27.62 Mean 15.03 1.58 24.13 2.6 +60 sem p < 0.01

(13) TABLE-US-00002 TABLE 1B Podocyte Sample NB Sample NB + % Marker number Controls number Rostafuroxin vs. Ctrl. -Adducin 1 4.42-2.10 1 22.23 2 6.06 2 4.38-6.02 3 5.70-6.77 3 5.17-3.91 4 8.06-7.53 4 4.47-6.75 5 9.33-7.07 Mean 4.65 0.49 9.09 1.9 +95 sem p < 0.001 Synaptopodin 1 1.1 1 2.7 2 2.51 2 1.61 3 4.29 3 0.912 4 1.43 4 0.79 5 2.56 Mean 1.10 0.18 2.70 0.45 +143 sem p < 0.02 Actin 1 64.3-95-90- 1 130.5 88.5 2 115-87.5- 140.4 2 97.3-94.8- 3 111-98- 100-97 98.2-81.2 3 120.0-90.4- 4 86.2-101.2- 94-109.9 101-144.2 4 99.4-92.4- 5 87.7-108- 85.4-160.9 103-91.6 Mean 100.5 4.53 105.3 4.75 ns sem

(14) The results obtained indicate that the compound of the invention is able to antagonize the podocyte protein loss induced bybeta adducin mutation thus favouring the correct function of the glomerular filtration barrier and reducing proteinuria in a normotensive experimental model.

EXAMPLE 2

(15) To test the activity of the compound of the invention for the prevention of proteinuria and loss of renal glomerular proteins, rats chronically infused with ouabain (OS rats) or saline (Control rats) were utilized.

(16) Two groups of 2-month-old OS rats (n=8 each) were orally treated by gavage with vehicle (Methocel 0.5%) or Rostafuroxin (100 g/kg) for 8 weeks. One group of saline infused rats was used as control. After this period, systolic blood pressure and urinary protein excretion was measured in the three groups. The animals of the three groups were then sacrificed for nephrin quantification from renal cortex microsomes by Western blotting.

Ouabain Infusion

(17) Three week-old male Sprague-Dawley rats (Harlan, Ind.), weighing 100-110 g, were subcutaneously implanted with osmotic mini-pumps, releasing either 15 g/kg/day of ouabain (OS rats, n=16) for 14 weeks or sterile saline (CS rats, n=8) (Ferrari P. et al. J. Pharmacol Exp. Ther. 1998; 285: 83-94). At the 6th week of ouabain infusion, OS rats were randomly assigned to two groups (n=8 each): the first (OS treated) received Rostafuroxin orally at 100 g/kg/day, suspended in 0.5% w/v Methocel, and the second group (controls) only vehicle. Systolic blood pressure (SBP) and heart rate (HR) were measured weekly in conscious rats by tail-cuff plethysmography (BP recorder, U. Basile, Italy).

Biochemical Assays for the Measurement of Urinary Parameters

(18) Urinary parameters were measured in conscious OS and control rats at the 12th week of treatment. Rats were housed in individual metabolic cages and acclimated for one day. 24-hours urines collection started at 9 a.m. During urine collection, rats had free access to water and food. After centrifugation (4500 rpm for 20 min; Varifuge 3.2 RS, Haereus Instruments, AHSI, Milan, Italy), rat urines were analyzed for the urinary volume (ml), quantified by weighing the urinary reservoir on a precision Mettler balance; urinary pH (pHM83, Radiometer, Copenhagen) and total urinary protein excretion (mg/24 h), measured with a standard total protein Kit (Sentinel Diagnostics, Milan, Italy). The animals of the three groups were then sacrificed, renal cortical microsomes were prepared from each rat and nephrin, the key protein of the slit diaphragm membrane, was quantified by Western blotting. Samples were separated by SDS-polyacrylamide gel electrophoresis, blotted and overnight incubated at 4 C. with specific primary antibodies (anti-nephrin from Santa Cruz; anti-actin from Sigma-Aldrich), followed by 1 h incubation with fluorescent secondary antibodies (Alexa Fluor), then analyzed and quantified by Odyssey Infrared Imaging detection system (LI-COR Biosciences). Nephrin quantification is expressed as optical density, arbitrary units.

(19) The results obtained are reported in the following Tables 2A; 2B; 2C; and FIG. 6.

(20) TABLE-US-00003 TABLE 2A Saline control Systolic blood Nephrin Saline pressure Proteinuria optical density control mmHg mg/24 h arbitrary units 1 140 31.63 64.51 2 150 45.06 64.3 3 150 28.2 79.92 4 145 51.6 52.88 5 135 29.16 66.06 6 145 21.95 58.92 7 150 25.44 45.91 8 140 45.44 mean sem 144 1.2, n = 8 34.9 3.8, n = 8 61.8 4, n = 7

(21) TABLE-US-00004 TABLE 2B Ouabain-infused rats (OS): effect of treatment with methocel (vehicle) Systolic blood Nephrin Pressure Proteinuria optical density OS rats mmHg mg/24 h arbitrary units 1 170 35.41 51.4 2 180 79.3 49.7 3 170 42.3 35.9 4 170 37.8 41.9 5 165 57.07 47.0 6 160 45.2 52.3 7 175 47.19 44.3 8 170 60.3 52.2 mean sem 170 2.1, n = 8 50.6 5.1, n = 8 46.7 2.1, n = 8 p < 0.01 vs. p < 0.05 vs. p < 0.02 vs. Saline Saline Saline

(22) TABLE-US-00005 TABLE 2C Ouabain-infused rats (OS): effect of treatment with 100 g/kg os Rostafuroxin OS treated Systolic blood Nephrin with Pressure Proteinuria optical density Rostafuroxin mmHg mg/24 h arbitrary units 1 135 53.07 64.7 2 150 20.14 54.8 3 155 55.34 51.4 4 145 29.02 50.8 5 155 39.7 76.1 6 145 40.68 57.4 7 155 20.85 48.8 8 150 48.88 53.8 mean sem 148.8 2.5, n = 8 38.5 4.9, n = 8 57.3 3.1, n = 8 ns vs. Saline ns vs. Saline ns vs. Saline

(23) The results obtained indicate that the compound of the invention is able to antagonize the pathological effects of ouabain on blood pressure, urinary protein excretion and glomerular protein loss thus lowering blood pressure, re-establishing the glomerular nephrin expression and reducing proteinuria.