Patent Application
20130203668
The present invention is related to a composition of antiplatelet agents and erythropoiesis stimulating agents suitable for chronic kidney disorders.
The present invention is related to a composition of suitable erythropoiesis stimulating agents (ESAs) and antiplatelet agents for the reduction of progression of kidney damage due to the use of erythropoiesis stimulating agent in Chronic Kidney Disease (CKD). Further, the present invention is related to a combination of ESAs and antiplatelet agents to reduce the platelet reactivity induced by erythropoiesis stimulating agent in CKD and to reduce the bleeding problem associated with antiplatelet agent, which eventually reduces the cardiovascular risk associated with ESAs in CKD.
Chronic kidney disorder is related to progressive and irreversible decline in the kidney function and considered to be a risk factor for cardiovascular disease. Reduction in kidney function leads to maladaptive changes in fluid retention (extracellular fluid overload), anemia, disturbances of bone and mineral metabolism and dyslipidemia. Indeed, patients with reduced kidney function represent a population not only at risk for progression of kidney disease and for end-stage renal disease (ESRD), but also at even greater risk for cardiovascular diseases. At present, there are no specific cures for reduction of the progression of chronic kidney diseases, and renal transplantation is limited by organ shortage; therefore present composition is useful for the prevention of progression of renal diseases. t is found that treatment with recombinant human erythropoietin (rHuEPO) accelerates the progression of renal diseases (Garcia D L et al, PNAS, 6142-6146, 1988). Moreover rHuEPO also augments the platelet reactivity that makes the patient susceptible to cardiovascular disease and thrombotic events. Therefore it appears that when the chronic kidney patient is treated with rHuEPO it increases both the impairment of kidney functions and the risk of cardiovascular coronary thrombosis.
Cardiovascular disease is very common health problem, which is the major cause of death. Antiplatelet agents are one among the known treatment agents for cardiovascular disorders. There are several antiplatelet agents known like clopidogrel, prasugrel etc. The structure and their functions are known from U.S. Pat. No. 4,847,265, U.S. Pat. No. 6,429,210 and U.S. Pat. No. 6,693,115 respectively.
Clopidogrel is marketed as its bisulfate salt under the brand name Plavix, which is in solid form. Several solid formulations of clopidogrel are known but because of low solubility of clopidogrel bisulfate, it is difficult to prepare its injectable formulation. In fact, there is no injectable formulation available in the market for clopidogrel bisulfate.
These antiplatelet agents are associated with some side effects like it may take longer for bleeding to stop if one experiences a cut or injury, while taking these agents. Clopidogrel (Plavix) and prasugrel both are associated with this problem (Sanofi-Aventis, BMS, www.plavix.com and NEJM Volume 361:940-942)
Combination of clopidogrel and aspirin has better effect in treatment of acute coronary syndrome, but it has been found that there is high risk of increase in bleeding time on administration of this combination. This problem has been observed in hemodialysis population also, when the combination of clopidogrel and aspirin is administered. (NEJM vol 345 No 7, 2001. 494-502 and J Am Soc Nephrol 14: 2313-2321, 2003)
Patients on hemodialysis or chronic kidney disorders face other complications, which mainly include anemia and cardiovascular disorders. For the treatment of anemia, it is necessary to administer erythropoietin to patients of chronic kidney disorders.
Erythropoietin (EPO), mainly produced by the peritubular cells of the kidney is the primary regulator of red blood cell production and is responsible for the terminal differentiation of erythroid progenitor cells.
In bone marrow, EPO acts on a specific receptor (EPO-R), with subsequent activation of various signaling pathways (STATS, MAPK, PI3/Akt). EPO acts primarily as a survival factors for erythroid progenitor cells, and in this manner increases the number of mature red blood cells in the circulation.
At present, Recombinant Human Erythropoietin (rHuEPO) is approved for treatment of anemia caused by conditions, including anemia associated with renal failure, chemotherapy, and HIV antiviral treatment or to reduce the need for transfusion in preoperative surgical patients. It is also one of the products used for the treatment of aplastic anemia. Recombinant Human Erythropoietin (rHuEPO) should be started if patients Hb remains below 11 gm % despite correcting the nutritional and iron deficiencies. The intravenous route is recommended in patients on hemodialysis while in others it can be used subcutaneously two to three times a week.
A study in nephrectomized rat showed that correction of anemia with rHuEPO may have adverse renal hemodynamic and structural consequences (Garcia D L et al, PNAS, 6142-6146, 1988).
A study in dogs suggested that rHuEPO not only promotes the synthesis of increased numbers of reticulated platelets but these newly produced platelets are hyper reactive compared with controls. (Thromb haemost 1997; 78:1505-09, Blood 2000; 95:2983-89). These newly produced platelets increase platelet reactivity and are linked to an increased risk of thrombotic events. EPO seems to affect various cardiovascular and haemostatic parameters unrelated to increase in haematocrit (Blood 2000; 95:2983-89). EPO may also be prothrombotic in humans both by increasing platelet reactivity and by increasing systemic BP through vasoconstriction. It also increased calcium uptake and store in platelets. (Curr Opin Nephrol Hypertens 2001; 10:633-77).
EPO increases the red blood cells significantly in patients which may cause serious risk of venous thrombosis (Red clot) and therefore heparin and low molecular weight heparin (LMWH) are preferred medicament for the treatment of red clot. In addition, another drawback of rHuEPO is that it increases the platelet count and make them hyper-reactive which may lead to formation of platelet rich clot (white clot) in coronary artery that causes myocardial infarction in patient. However, the rHuEPO does not show any suitable therapeutic and preventive effect over the white clot. (Curr Opin Nephrol Hypertens 2001; 10:633-77, Blood 2000; 95:2983-89, BMJ 1990; 300:573-8, N Engl J Med 1998; 339:584-90, Cancer 2003; 9:1514-20, Clin Invest 1994; 72:S36-S43, Lancet 2007; 369:381-8, Clin J Am Soc Nephrol 2007; 2:1274-82, Am J Hypertension 2006; 19:1286-1292, PNAS USA 1988; 85, 6142-6146).
Thus, in a nutshell the problem associated with the administration of rHuEPO is that it not only increases the impairment of kidney function but also make the patient vulnerable to cardiovascular disease. These drawbacks are very severe and complicated which lead to high mortality rate in patient suffering from kidney diseases. Unfortunately, no therapy is available yet to prevent the rHuEPO-induced kidney damages and/or to improve in kidney function.
Hence, it is desirable to develop a composition which significantly improve the kidney function and also prevent further damage caused by rHuEPO and/or lower down the thrombotic events and bleeding problems in patients and also improve the renal function in patient.
The present invention provides a composition comprising of a suitable antiplatelet agent/agents or its pharmaceutical acceptable salts and a suitable erythropoiesis stimulating-agent for the improvement in renal function. Specifically, such a composition provides therapeutic benefit by prevention and treatment of kidney damage and cardiovascular diseases in chronic kidney diseases patient. Furthermore, when administrated simultaneously, sequentially or separately, the suitable antiplatelet agent/agents and an erythropoiesis stimulating agent interact in a synergistic manner to control the problems associated with rHuEPO and improves the renal function.
In another embodiment, there is provided a kit for the treatment of cardiovascular disorders and chronic kidney disorders, which comprises suitable antiplatelet agent/agents and an erythropoietin stimulating agent.
In a preferred feature, the present invention provides a liquid formulation of clopidogrel for intravenous administration so that it can be administered simultaneously with erythropoietin-stimulating agent.
In an embodiment, the present invention provides a composition comprising suitable anti-platelet agent or its pharmaceutical acceptable salts combined with suitable erythropoiesis stimulating agent for the treatment and improvement of impaired kidney function. Preferably the compounds are provided in a suitable ratio depending on the need of the patient.
In another embodiment, the invention provides composition of suitable antiplatelet agent or its pharmaceutical acceptable salts combined with suitable erythropoiesis stimulating agent for the improvement in kidney damage caused by administration of rHuEPO.
In a preferred embodiment, the present invention provides a composition comprising clopidogrel or its pharmaceutical acceptable salts in combination with recombinant human erythropoietin for the improvement in kidney damage caused by administration of rHuEPO and.
In a further embodiment, the present invention provides a composition of clopidogrel or its pharmaceutical acceptable salts in combination with recombinant human erythropoietin for the treatment of cardiovascular disorders wherein the bleeding problem associated with clopidogrel or its pharmaceutical acceptable salt and the risk of thrombotic events associated with recombinant human erythropoietin are reduced.
In yet another embodiment, the present invention provides a combination of more than one antiplatelet agent and a suitable erythropoiesis stimulating agent for the treatment of one or more conditions described above.
In another embodiment, any of the composition according to present invention is further used for the curative, prophylactic treatment of chronic kidney disorders as well as conditions associated with chronic kidney disorders where the risk of thrombotic events associated with recombinant human erythropoietin is reduced.
In a further embodiment, the combination of the present invention comprising of a suitable antiplatelet agent or its pharmaceutical acceptable salts and erythropoiesis stimulating agents may be administrated by oral, intravenous or subcutaneous route of administration.
In another embodiment of the present invention is provided a kit comprising antiplatelet agent/agents with erythropoiesis stimulating agent.
a—Effect of rHuEPO on platelet reactivity in ADP-induced ex vivo platelet aggregation method using male Wistar rats.
b—Effect of rHuEPO on platelet reactivity in arachidonic acid-induced ex vivo platelet aggregation method using male Wistar rats.
c—Effect of rHuEPO on platelet reactivity in thrombin-induced ex vivo platelet aggregation method using male Wistar rats.
d—Effect of rHuEPO on platelet reactivity in collagen-induced ex vivo platelet aggregation method using male Wistar rats.
a—Effect of clopidogrel or aspirin or their combination on rHuEPO-induced platelet reactivity in ADP-induced ex vivo platelet aggregation method using male Wistar rats.
b—Effect of clopidogrel or, aspirin or their combination on rHuEPO-induced platelet reactivity in arachidonic acid-induced ex vivo platelet aggregation method using male Wistar rats.
c—Effect of clopidogrel or aspirin or their combination on rHuEPO-induced platelet reactivity in thrombin-induced ex vivo platelet aggregation method using male Wistar rats.
d—Effect of clopidogrel or aspirin or their combination on rHuEPO-induced platelet reactivity in collagen-induced ex vivo platelet aggregation method using male Wistar rats.
FIG. 4—Effects of various treatment on ex vivo % platelet aggregation using 2.5 μM ADP as aggregating agent in chronic kidney disease model in rat.
a—Effects of various treatment on RBC count (106/4) in chronic kidney disease model in rat.
b—Effects of various treatment on Hemoglobin (Hb) (g/dL) in chronic kidney disease model in rat.
c—Effects of various treatment on % Hematocrit (HCT) in chronic kidney disease model in rat.
a—Effects of various treatment on Serum Creatinine (mg/dL) in chronic kidney disease model in rat.
b—Effects of various treatment on Serum Urea (mg/dL) in chronic kidney disease model in rat.
c—Effects of various treatment on Serum Uric acid (mg/dL) in chronic kidney disease model in rat.
d—Effects of various treatment on Blood Urea Nitrogen (BUN) (mg/dL) in chronic kidney disease model in rat.
e—Effects of various treatment on Total Protein (g/dL) in chronic kidney disease model in rat.
According to the present invention, a composition is provided comprising suitable antiplatelet agent/agents or their suitable pharmaceutically acceptable salts and an erythropoietin-stimulating agent for the curative, prophylactic treatment of chronic kidney diseases and its associated conditions. In addition, the present invention also provides a composition comprising suitable anti-platelet agent/agents and an erythropoietin-stimulating agent for the curative, prophylactic treatment of cardiovascular diseases.
Thus the present invention provides a composition comprising suitable anti-platelet agent or its pharmaceutical acceptable salts combined with suitable erythropoiesis stimulating agent for the treatment and improvement of impaired kidney function. Preferably the compounds are provided in a suitable ratio depending on the need of the patient.
In a preferred embodiment the invention provides composition comprising a suitable antiplatelet agent or its pharmaceutical acceptable salts combined with suitable erythropoiesis stimulating agent for the improvement in kidney damage caused by administration of rHuEPO.
The “antiplatelet agent” according to present invention is an agent which decreases the platelet aggregation. It may be selected from, but not limited to, clopidogrel and its pharmaceutically acceptable salt, prasugrel and its pharmaceutically acceptable salt and aspirin.
“Erythropoiesis stimulating agent” according to present invention is an agent, which stimulate the erythropoiesis. Erythropoiesis stimulating agent according to present invention may include recombinant human erythropoietin, darbepoetin, PEG-recombinant human erythropoietin, PEG-darbepoetin and other short peptides as well as small molecules etc. which stimulates erythropoiesis.
“Cardiovascular disorders” is well known in the art and can be described as the disorders associated with arteries and veins, which may include angina, atherosclerosis, cerebrovascular accidents, cerebrovascular diseases, congestive heart failure, coronary artery diseases, myocardial infarction, peripheral vascular diseases and cardiovascular diseases associated with chronic kidney diseases.
“Chronic kidney diseases” includes the diseases related to renal function and disorders associated with that and the diseases associated with chronic kidney disorders as given in for e.g. NEJM Volume 351:1296-1305. Chronic kidney disease in the general population is generally characterized by the existence of increased serum creatinine, serum urea, uric acid, blood urea nitrogen and decreased serum total protein and these can be used as kidney biomarkers. Worsening of renal function is defined as increase in about 25% of serum creatinine which roughly corresponds to a decrease in glomerular filtration rate (GFR)≧9.0 ml/min/1.73 m2. (Goldstein S L. Kidney International, 2010: 78, 433-435; A Leelahavanichkul et al. Kidney International, 2010: doi:10.1038/ki.2010.287; Damman K et al., Eur J Heart Fail, 2007:6, 17-18; Khan N A et al., J Am Soc Nephrol, 2006:17, 244-253; Krumholz H M et al., Am J Cardiol, 2000:85, 1110-1113).
In an embodiment, the present invention provides improved therapeutic benefits and safety when a combination comprising anti-platelet agents and rHuEPO is used. In addition, the present combination of antiplatelet agents or their pharmaceutically acceptable salts with rHuEPO reduces the occurrence of problems associated with each other and acts complimentary to each other with better safety benefits. For example, rHuEPO has been linked with increased risk of thrombotic events because of hyper-reactivity of the platelets. On the other hand antiplatelet agent, for example, clopidogrel or its pharmaceutically acceptable salt, specifically those which are ADP-receptor antagonist, has been associated with increased risk of bleeding and causing aplastic anemia as well as bone marrow depression.
Pharmaceutically acceptable salts of clopidogrel which can be used are selected from bisulfate, besylate, tosylate, hydrochloride, hydrobromide and mesylate. In a preferred embodiment the pharmaceutically acceptable salts used may be clopidogrel besylate, clopidogrel bisulfate, clopidogrel hydrochloride and clopidogrel hydrobromide. Surprisingly, when used in combination, rHuEPO takes care of bleeding complications of anti-platelet agents such as clopidogrel; while anti-platelet agent/agents improves kidney functions which are impaired by the administration of rHuEPO as well as rHuEPO-induced hyper-reactivity of platelet therefore reducing the risk of thrombotic events.
Unless otherwise mentioned, in the specification whenever the term antiplatelet agent/agents or clopidogrel, prasugrel etc as well as Erythropoiesis stimulating agents selected from recombinant human erythropoietin, darbepoetin, PEG-recombinant human erythropoietin, PEG-darbepoetin are used, the term where applicable, also includes their suitable pharmaceutical composition.
In another embodiment a mechanistically different anti-platelet agent such as aspirin is used in combination with rHuEPO which also shows synergistic interaction and improves the kidney function caused by administration of rHuEPO in varying degrees. In another embodiment aspirin is used in combination with rHuEPO which shows synergistic interaction and normalizes the platelet reactivity-induced by rHuEPO with better safety in terms of bleeding. In another embodiment is provided a combination of rHuEPO with one or two mechanistically different anti-platelet agents for the curative and prophylactic treatment of chronic kidney disease patients. In another embodiment is provided a combination of one or more suitable antiplatelet agents with suitable erythropoiesis stimulating agent, for the treatment of cardiovascular disorders and chronic kidney disorders.
In an embodiment, the combination of one or more antiplatelet agent/agents and erythropoietin stimulating agent used in said combination may be formulated for oral, parenteral or injectable administration. Injectable administration includes intravenous, intramuscular or subcutaneous injection.
This combination of antiplatelet agent/agents and recombinant human erythropoietin may be given either simultaneously or sequentially or at different time intervals as per requirement of dosing. The combination of the invention can be administered alone but one or both elements will generally be administrated with suitable pharmaceutical excipients, diluents or carrier selected with regard to intended route of administration as are known in the art.
In a preferred embodiment, the anti-platelet agents such as clopidogrel or its pharmaceutically acceptable salt such as clopidogrel bisulfate, clopidogrel besylate, clopidogrel hydrochloride and clopidogrel hydrobromide is administrated either orally or through parenteral route.
In an embodiment, the Clopidogrel salts can be given twice a day or once a day preferably, once a day. The dosage amount of clopidogrel or its pharmaceutically acceptable salt such as clopidogrel besylate is in the range from 5 mg to 600 mg, more preferably less than or equal to 75 mg in patients either given in a single dose or divided in multiple doses depending on the need of the patient, it will be appreciated that the amount of dose will also depend on the severity of disease condition in patient.
In an embodiment the antiplatelet agent aspirin administrated orally or through parenteral route. Aspirin can be given twice a day or once a day, preferably once a day. The dosage amount of aspirin is in the range from 25 to 100 mg in the patient. However, the amount of dose depends over the severity of disease condition in patient.
In another embodiment the erythropoiesis stimulating agent such as rHuEPO is administrated through injection. The rHuEPO can be given once a month or thrice a week or once a week, preferably thrice a week. However, the dosage administration frequency depends over the pharmacokinetic profile of erythropoiesis stimulating agent, the erythropoiesis stimulating agent used, severity of disease, patient profile etc. Preferably, the dosage amount of rHuEPO which may be used is in the range from 0.5 μg/kg to 50 μg/kg more preferably 0.5 μg/kg to 2 μg/kg. The dosage may be varied depending upon the requirements of the patient, the severity of the condition being treated.
In another embodiment, the suitable therapeutic amount of antiplatelet agent used in the combination is at least sufficient to reduce blood clotting. The suitable therapeutic amount of erythropoiesis stimulating agent used in the combination is at least sufficient to start erythropoiesis.
Further in another embodiment is provided a kit for the treatment of one or more of disease conditions referred elsewhere in the specification. The Kit comprises as active ingredients a pharmaceutically effective amount of suitable antiplatelet agent/agents and an erythropoiesis stimulating agent. The active ingredients are formulated in same or different unit dosage form for different or same route of administration. The Kit comprises suitable anti-platelet agent/agents, which are formulated as oral, parenteral or injectable form and an erythropoiesis stimulating agent, which is formulated in an injectable form. Preferably, the antiplatelet agent/agents used in the kit are formulated to be administered by injection.
The present invention can be further illustrated with the help of following examples which disclose some of the embodiments of carrying out the invention and should not be considered as restriction on the scope of the invention.
Six to eight week old male Wistar rats used and the methods and procedures described here have been reviewed and approved by the Institutional Animal Ethics Committee (IAEC). Wistar male rats were divided in groups [each group containing 6 animals (n=6)]. Control animal received vehicle only (0.9% w/v NaCl normal saline), treatment groups received three consecutive doses of Recombinant Human Erythropoietin (25 and 50 μg/kg, s.c.) at an interval of 24 hour. Blood collection was performed at 48 hour after the last injection of Erythropoietin in citrated tubes. Platelet rich plasma (PRP) was separated. Determination of ex-vivo platelet aggregation was performed by using 1) ADP as aggregation inducing agent 2) Arachidonic acid as aggregation inducing agent 3) Thrombin as aggregation inducing agent and 4) Collagen as aggregation inducing agent. Platelet aggregation was performed by turbidometric method using high through put 96-well plate. Optical density was measured at 405 nM by Spectra Max plate reader. We found that rHuEPO causes increase in platelet aggregation. Increase in platelet aggregatory response meaning increase in platelet reactivity when same concentration of aggregating agent is used. When constant concentration of aggregating agents like ADP (1.25 μM), arachidonic acid (150 μM), thrombin (0.0125 IU/ml) and collagen (0.25 and 0.5 μg/ml) are used, platelet aggregation increases on increasing concentration of rHuEPO (25 μg/kg, s.c and 50 μg/kg, s.c (
Six to eight week old male Wistar rats were divided in groups [each group containing 6 animals (n=6)]. Control animal received vehicle only (0.9% w/v NaCl normal saline), treatment groups received three consecutive doses of Recombinant Human Erythropoietin (25 μg/kg, s.c.) at an interval of 24 hour. Aspirin (75 mg/kg p.o.), and Clopidogrel (2.5 mg/kg, p.o.) were dosed individually and in combination with each other to respective groups at 48 hour after the last injection of erythropoietin. Blood collection was performed after 2 hours of antiplatelet treatment. Platelet rich plasma (PRP) was separated. Determination of ex-vivo platelet aggregation was performed by using 1) ADP as aggregation inducing agent 2) Arachidonic acid as aggregation inducing agent 3) Thrombin as aggregation inducing agent and 4) Collagen as aggregation inducing agent. When constant concentration of aggregating agents like ADP (1.25 μM), arachidonic acid (150 μM), thrombin (0.0125 IU/ml) and collagen (0.25 and 0.5 μg/ml) are used, on administration of clopidogrel (2.5 mg/kg p.o.) to rHuEPO (25 μg/kg, S.C.) treated rats, platelet aggregatory response is normalized (
Six to eight week old male Wistar rats were divided in groups [each group containing 6 animals (n=6)]. Control animal received vehicle only (0.9% w/v NaCl normal saline), treatment groups received three consecutive doses of Recombinant Human Erythropoietin (25 μg/kg, S.C.) at an interval of 24 hour. Aspirin (75 mg/kg p.o.), and Clopidogrel (2.5 mg/kg, p.o.) individually and in combination with each other were dosed to respective groups at 48 hour after the last injection of Erythropoietin. Bleeding time experiment using tail transaction test was performed. When clopidogrel and aspirin are used in combination, it increases the bleeding time as compared to normal. When rHuEPO is administered with combination of clopidogrel and aspirin, it significantly reduces this increased bleeding time in Wistar rats (
Six to eight week old male Wistar rats used and the methods and procedures described here have been reviewed and approved by the Institutional Animal Ethics Committee (IAEC). We used human therapeutic dose of rHuEPO in rats and same frequency of administration as in humans (Kirkeby A et al., Thromb Haemost, 2008; 99:720-728). A 5/6 nephrectomy has been performed on male wistar rats (Toba H et al., European Journal of Pharmacology 656, 2011, 81-87) and treatment has been started after one month of nephrectomy.
The rats were divided into 5 groups (n=6):
1) a control group, which received a sham operation (SHAM control);
2) a 5/6 nephrectomized group (NPX), removed the upper and lower poles of the left kidney and after one week the entire right kidney under anesthesia (NPX vehicle);
3) a 5/6 nephrectomy+rHuEPO group, nephrectomized and injected with rHuEPO at 500 IU/kg 3 times a week intravenously (i.v.) for 4 weeks (NPX+EPO);
4) a 5/6 nephrectomized+rHuEPO+clopidogrel group, nephrectomized and administered with rHuEPO (500 IU/kg)+clopidogrel (10 mg/kg) every 3 times a week intravenously (i.v.) for 4 weeks.
5) a 5/6 nephrectomized+clopidogrel group, nephrectomized and administered with rHuEPO (500 IU/kg)+clopidogrel (10 mg/kg) every 3 times a week intravenously (i.v.) for 4 weeks.
After 4 weeks of treatment, blood samples were collected via retro-orbital route in tubes containing 3.8% tri-sodium citrate for the collection of platelet rich plasma (PRP) for ADP-induced platelet aggregation studies. Blood samples were also collected for hematological analysis in tubes containing 5% EDTA. Serum samples were used for measurement of various kidney biomarkers such as creatinine, urea, uric acid, blood urea nitrogen and total protein.
Increase in platelet aggregatory response meaning increase in platelet reactivity when same concentration of aggregating agent is used. When constant concentration of aggregating agents like ADP (2.5 μM) used, platelet aggregation increases with rHuEPO When constant concentration of aggregating agents like. ADP (2.25 μM) is used, on administration rHuEPO (500 IU/kg)+clopidogrel (10 mg/kg) every 3 times a week intravenously (i.v.) for 4 weeks in nephrectomized rats, platelet aggregatory response is reversed (
There was increase in RBC count, hemoglobin and hematocrit when rHuEPO is used alone and in combination with antiplatelet agent every 3 times a week intravenously (i.v.) for 4 weeks in nephrectomized rats (
There was increase in the serum creatinine, serum urea, serum uric acid, blood urea nitrogen and decrease in serum total protein in nephrectomized animals. Surprisingly, there was further increase in the serum creatinine, serum urea, serum uric acid, blood urea nitrogen in nephretomized+rHuEPO treated rats which is the indicative of worsening the kidney function upon rHuEPO treatment. There was improvement in the kidney function when combination of rHuEPO and antiplatelet agent has been given every 3 times a week intravenously (i.v.) for 4 weeks in nephrectomized rats (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1281/MUM/2010 | Apr 2010 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IN2011/000259 | 4/18/2011 | WO | 00 | 12/17/2012 |
