HK1172555B - Use of amides of mono- and dicarboxylic acids in the treatment of renal diseases - Google Patents

Description

Use of amides of monocarboxylic and dicarboxylic acids in the treatment of renal diseases

Technical Field

The present invention relates to the treatment of renal disease and the consequent alteration of renal function, and in particular, though not exclusively, to the treatment of renal disease occurring in diabetic patients or patients who have undergone anti-tumour chemotherapy with platinum derivatives.

Background

Chronic kidney disease and renal failure derived therefrom are extremely common, if not diagnostic, diseases; in fact, it is estimated that 17% of the adult population is affected by this disease.

The most common renal diseases are characterized by glomerular damage.

Renal disease may be congenital or acquired; in particular, acquired kidney disease may have various etiologies:

● immune, such as Goodpasture's syndrome, lupus nephritis and immunoglobulin A nephropathy. In the case of immune-mediated renal disease, the etiology consists in the presence of a strong antigenic stimulus that triggers an immune response;

● abnormal in metabolism, particularly diabetic nephropathy, which is one of the most common causes of chronic kidney disease. Its incidence is 20-30% in patients with type 1 diabetes and about 10% in patients with type 2 diabetes. This is an insidious disease, as it is characterized by a particularly slow onset (maximum of 20-30 years from the onset of diabetes) and by the fact that it is asymptomatic for a long period of time; it initially occurs by microalbuminuria (amount of albumin in urine between 30 and 300 mg/l), which slowly progresses to macroalbuminuria (amount of albumin in urine exceeding 300mg/l up to a value of 3g within 24 hours) showing marked nephropathy;

● is hemodynamic and is caused by arterial hypertension. Over time, changes in the pressure mechanisms of renal blood flow cause a decrease in renal filtration capacity;

● ischemic, renal ischemia being the most common pathological event associated with acute kidney disease and consequent tubular necrosis, in both autologous and transplanted kidneys;

● toxic, most clinically important drugs (cytotoxic agents, chemotherapeutic agents, non-steroidal anti-inflammatory drugs, corticosteroid therapy, etc.) and various chemical products (e.g., radiographic media, solvents, etc.) produce nephrotoxicity, which can very frequently cause inflammation at the renal parenchymal level and short-and long-term insufficiency.

Even in veterinary medicine, renal disease, which must progress to chronic kidney disease, constitutes an important clinical category, second only to the death from tumors in dogs and first in aging cats. From an etiological point of view, the causes that determine the progressive and irreversible loss of nephron function in small animals are precisely classified in (Squires et al, 1998) as:

-degenerative: chronic interstitial nephritis; renal infarction

-autoimmunity: anti-GBM glomerulonephritis

-metabolic: diabetes mellitus; hyperthyroidism (cat); hypercalcemia

-tumorigenicity: renal lymphoma and renal carcinoma

-idiopathic: amyloidosis; idiopathic glomerulonephritis

-infectivity: bacterial pyelonephritis; lyme disease (borreliosis)

-immune-mediated: immune complex glomerulonephritis

-toxicity: nephrotoxic drugs (e.g. cisplatin, aminoglycosides, non-steroidal anti-inflammatory drugs)

-traumatic: the bladder and urethra rupture.

In any case of all acquired nephropathies of whatever etiology, both in humans and animals, there is activation of inflammatory processes whose primary purpose is to combat adverse events, but which may become the cause of glomerulosclerosis and tubulointerstitial fibrosis, the latter being able to determine the progression of chronic kidney disease to the prophase of the end stage (prophase of end stage nephropathies) in which the majority of the nephrons are destroyed. One of the two main goals of nephrology, first and foremost, is to understand the regulatory mechanisms of the process from acute kidney injury to chronic fibrotic nephropathy, as it can be very difficult at present to intervene in the fibrotic process once fibrogenesis has begun; in any case, it is still of paramount importance to prevent or at least slow down the progression of chronic kidney disease, considering that it also constitutes an important risk factor for cardiovascular diseases. In this connection, there are currently several studies aimed at accurately understanding the most important pathogenesis, with the aim of preventing the phenomena that determine the irreversibility of the disease. Among these phenomena, the most important is the induction of the phenomenon of tubulointerstitial fibrosis, which is considered to be a major cause of chronic kidney disease; fibrosis causes excessive accumulation of extracellular types composed primarily of collagen, and when normal tissue is replaced by scar tissue, it is often accompanied by progressive loss of renal function. One of the most studied phenomena at present is the control of the processes that take place by myofibroblasts and the role these cells play in the formation of fibrotic scar tissue. In particular, these studies seek to understand the repair phenomena usually provided by tissues that continuously undergo a large number of etiologies, such as kidney tissues, why it is possible to determine at one time an excessive increase in the extracellular matrix and, therefore, the tubulointerstitial fibrosis. Currently, special attention is given to the occurrence of myofibroblasts starting from both tubular epithelial cells and endothelial cells through a phenotypic transformation process from epithelial to mesenchymal, strongly stimulated by TGF-1 β (transforming growth factor). Indeed, expression of TGF-1 β in the tubular epithelium is constantly increasing during the active process of fibrogenesis. In animal models of renal injury, the dose of TGF-1 β in the renal tubular epithelium is taken as an important indicator of the activation state of fibrogenesis and thus also of the state of functional alterations induced by renal disease.

Despite the large amount of new information obtained regarding the pathogenic mechanisms involved in the development of renal disease, satisfactory therapeutic solutions for controlling these conditions remain to be discovered.

Palmitoylethanolamide (PEA) is a parent compound of the family of N-acyl amides known as aliamides, a class of endogenous lipid molecules capable of normalizing the activity of immune cells by local antagonistic mechanisms. In contrast, analgesic effects are associated with the normalization of the controlled release of trophic factors such as NGF, which, if present in excess in tissues, over-sensitize and over-excite neuronal structures, producing hyperalgesia and allodynia. From a clinical point of view, oral ingestion of a product containing PEA can improve the symptomatology of neuropathy associated with peripheral neuropathy (neuropathicsymptomatology), as well as promote functional recovery of motor conduction velocity. At the experimental level, PEA is also effective in neuropathies with metabolic abnormalities, in particular when it is administered to animals made diabetic with streptozotocin (streptozotocin), eliminating the allodynia and inducing a partial recovery of body weight and an increase in blood insulin levels. These animals also showed low overproduction of blood free radicals and low NGF levels in the sciatic nerve.

Similar to PEA, although the N-acyl amides formed generally from monoethanolamine and saturated or unsaturated fatty dicarboxylic acids are themselves non-physiological, they are also capable of forming substances that are physiologically present in the mammalian organism during catabolic processes, and therefore do not produce any sort of accumulation and/or toxicity, and have been shown to produce pharmacological effects similar to that of the parent PEA.

Disclosure of Invention

We have now surprisingly found that certain molecules belonging to the class of amides between amino alcohols and mono-or dicarboxylic acids are active in the treatment of renal diseases. In particular, it was observed that Palmitoylethanolamide (PEA), and diethanolamide of fumaric acid, a monounsaturated dicarboxylic acid normally present in mammalian organisms, show a rather high activity on said diseases.

A first object of the present invention is therefore a mono-or di-amide or a mixture thereof of a saturated or monounsaturated C12-C20 monocarboxylic acid or a saturated or monounsaturated C4-C14 dicarboxylic acid, respectively, with an amine selected from monoethanolamine and serine, for use in the treatment of renal diseases, in particular but not exclusively those caused by metabolic disorders or toxic agents.

Another object of the invention is Palmitoylethanolamide (PEA) for use in the treatment of renal diseases, wherein the PEA is preferably in micronized or ultra-micronized form.

Another object of the present invention is PEA for use in the treatment of renal disease, wherein said PEA is administered orally.

Another object of the invention is the diethanolamide of fumaric acid in aqueous solution for the treatment of renal diseases.

Detailed Description

The present invention is based on the surprising finding that exogenous administration of a mono-or di-amide of a saturated or mono-unsaturated C12-C20 mono-or saturated or mono-unsaturated C4-C14 dicarboxylic acid, respectively, with an amine selected from monoethanolamine and serine, in particular oral administration of palmitoylethanolamide, preferably in micronized form (PEAm) or in ultra-micronized form (PEAum), and/or diethanolamide of fumaric acid, preferably in dissolved form in a suitable aqueous medium, enables a significant improvement of renal function in mammals affected by renal disorders, in particular diabetic renal disorders and renal disorders originating from antitumor agents. The inventors have also found that an improvement in renal function is associated with a lower expression of TGF-1 β which is considered to be an important indicator of ongoing fibrogenesis. Improvement in renal function has also been demonstrated in patients affected by inflammatory renal disease and diabetic nephropathy.

In an embodiment of the invention, the saturated or monounsaturated C12-C20 monocarboxylic acid is selected from palmitic acid, stearic acid and oleic acid.

In an embodiment of the invention, the saturated or monounsaturated C4-C14 dicarboxylic acid is selected from fumaric acid, azelaic acid and trans-callus acid.

Palmitoyl ethanolamide is a commercial product that can be prepared by conventional methods well known to those skilled in the art, such as a method that provides for the reaction between ethanolamine or serine, possibly in protected form, and the mono-or dicarboxylic acid under suitable condensation conditions, which can also be provided using a condensing agent.

The term "micronized form of PEA" or "PEAm" is used to refer to palmitoyl ethanolamide in which at least 94% or at least 95% or about 96% of the particles have a size of less than 10 microns, and preferably at least 77% or at least 78% or about 80% of the particles have a size of less than 6 microns. PEAm can be prepared according to the disclosure of European patent EP 1207870B 1.

The term "ultra micronized form of PEA" or "PEAum" is used to refer to palmitoylethanolamide in which at least 97% or at least 98% or at least 99% or about 99.9% of the particles have a size of less than 6 microns, and preferably at least 57% or at least 58% or at least 59% or about 59.6% of the particles have a size of less than 2 microns. PEAum may be prepared according to the disclosure of patent application PCT/IT 2009/000399.

The diethanolamide of fumaric acid can be prepared by synthesis according to the disclosure of example 10 of patent No. US 5,618,842.

The present invention therefore relates to monoamides or diamides or mixtures thereof formed from saturated or monounsaturated C12-C20 monocarboxylic acids or saturated or monounsaturated C4-C14 dicarboxylic acids, respectively, and amines selected from monoethanolamine and serine, for use in the treatment of renal disease, particularly but not exclusively renal disease caused by metabolic abnormalities or toxic agents.

In embodiments, the mono-or di-amide of a saturated or monounsaturated C12-C20 monocarboxylic acid or a saturated or monounsaturated C4-C14 dicarboxylic acid is PEA, or diethanolamide of fumaric acid.

In embodiments, the PEA is used in micronized form (PEAm).

In various embodiments, PEA is used alone in ultra-micronized form (PEAum), or in admixture with PEAm.

In embodiments, the diethanolamide of fumaric acid is used in dissolved form in a suitable aqueous solvent.

Pharmacological Activity of the Compounds of the invention

Occurrence of renal injury following streptozotocin administration to mice

The streptozotocin model in mice represents a classical known hyperglycemic model that is capable of inducing progressive renal injury in animals, leading to renal disease with markedly altered characteristic parameters.

The model used is as follows: male C57BL6/J mice were maintained under standard care conditions. Diabetes was induced in mice 8 weeks old and having an average body weight of about 22g by intraperitoneal injection of streptozotocin (55mg/Kg body weight/day) in citrate buffer for 5 consecutive days. Control mice were treated under the same conditions with citrate buffer alone.

Using both micronized palmitoylethanolamide-PEAm (10.0mg/Kg) suspended in a carrier and micronized palmitoylethanolamide PEAum (10.0mg/Kg) suspended in a carrier, the treatment was administered orally using a tube; the results were compared to control mice treated with vehicle alone. As a carrier, 0.5% carboxymethyl cellulose was used.

Diethanolamide of fumaric acid in sterile saline solution administered by intraperitoneal injection (10.0 mg/Kg); the results were compared to animals treated with sterile saline solution only.

Administration of the vehicle and two different suspensions containing palmitoylethanolamide or injections containing diethanolamide of fumaric acid was performed once daily from the day of the last administration of streptozotocin. Prior to sacrifice, blood was collected from the saphenous vein using a microsyringe and blood glucose, glycated hemoglobin, and serum creatinine levels were determined by conventional methods.

Assessment of TGF-1 β on renal tissue was performed by the following method:

carefully isolated and weighed pieces of renal cortex were homogenized in 10mM Tris-HCl buffer pH7.4 containing 2M NaCl, 1mM PMSF (phenylmethylsulfonyl fluoride as a protease inhibitor), 1mM EDTA and 0.01% Tween 80. The samples were centrifuged at 19,000rpm for 30 minutes and the supernatants were collected, measured and stored at-80 ℃. The evaluation of TGF-1 β was performed using a commercial ELISA kit (Quantikine kit, Res & Diagn Systems, Minneapolis, USA), with values expressed in pg/mg total protein. Total protein concentrations were measured using Bio-Rad commercial detection reagents (Hercules, Ca, USA).

The results obtained are summarized in table 1.

TABLE 1

Occurrence of renal injury following administration of cisplatin to mice

Cisplatin is a known and widely used chemotherapeutic agent that is known to produce severe renal injury in 50% of patients undergoing treatment. A mouse animal model in which cisplatin induces severe nephrotoxicity and consequent renal disease was used in the experiments. The model used is as follows: male C57BL6/J mice were maintained under standard care conditions. Nephrotoxicity was induced in mice 8 weeks old and having an average body weight of about 23g by intraperitoneal injection of cisplatin dihydrochloride in saline solution (20 mg/Kg per administration). Control animals were treated under the same conditions with saline solution alone. Animals were sacrificed 72 hours after cisplatin treatment.

6 oral administrations were carried out using a tube using both micronized palmitoylethanolamide-PEAm (10.0mg/Kg) suspended in a carrier and micronized palmitoylethanolamide PEAum (10.0mg/Kg) suspended in a carrier, once every 12 hours; the first treatment was performed 12 hours prior to cisplatin administration. The results were compared to control animals treated with vehicle alone. A 0.5% carboxymethyl cellulose solution was used as the carrier.

Diethanolamide of fumaric acid in a similar dosage to PEA by intraperitoneal injection (10.0mg/Kg) in a sterile saline solution; the results were compared to animals treated with sterile saline solution only.

Prior to sacrifice, blood was collected from the saphenous vein using a microsyringe and serum creatinine levels were determined by conventional methods.

The level of TGF-1 β on renal tissue is measured by:

carefully isolated and weighed pieces of renal cortex were homogenized in 10mM Tris-HCl buffer pH7.4 containing 2M NaCl, 1mM PMSF (phenylmethylsulfonyl fluoride as a protease inhibitor), 1mM EDTA and 0.01% Tween 80. The samples were centrifuged at 19,000rpm for 30 minutes and the supernatants were collected, measured and stored at-80 ℃. The amount of TGF-1 β was measured using a commercial ELISA kit (Quantikine kit, Res & Diagn Systems, Minneapolis, USA), and the values are expressed in pg/mg total protein. Total protein concentrations were measured using Bio-Rad commercial detection reagents (Hercules, Ca, USA).

The results obtained are summarized in table 2.

TABLE 2

Effect of ultra-micronized palmitoylethanolamide-PEAum in patients with renal disease

Administering palmitoylethanolamide to the patient in the form of tablets containing 600mg of active ingredient per tablet in micronized form; the administration is carried out for 60 days, 2 tablets per day (one tablet every 12 hours, taken after meals).

GRF (glomerular filtration rate) was determined by creatinine endogenous markers using the Cockcroft-Gault equation (Cockcroft D.W. et al, 1976) according to the National Kidney Foundation Standard (US National Renal Foundation criterion, K/DOQI, clinical practice guidelines for chronic kidney disease, 2002).

The results are shown in table 3.

TABLE 3

The results shown above clearly show that PEA, particularly when administered orally in micronized or ultra-micronized form, can be successfully used to treat kidney disease in mammals. In addition, diethanolamide of fumaric acid is revealed to be active when injected intraperitoneally.

Thus, the compounds of the present invention are useful for both human and veterinary purposes in the treatment of renal disease.

Such diseases are preferably selected from:

diabetic nephropathy

Renal arteriosclerosis

Pyelonephritis

Polycystic kidney disease (polycystic kidney)

Alport syndrome

Lei-nie syndrome

-Goodpasture's syndrome

Lupus nephritis

Immunoglobulin A nephropathy

Tubular necrosis of the kidney

-glomerulonephritis

Urethral stricture

Iatrogenic nephropathy (from non-steroidal anti-inflammatory drugs, cytotoxic drugs, lithium, antibiotics, cyclosporine etc.)

Renal disease from therapeutic radiation

Senile nephropathy.

Thus, the compounds of the present invention may be formulated for oral, buccal, parenteral, rectal or transdermal administration.

The PEA is preferably formulated for oral administration.

In view of the high solubility of the synthetic molecule, diethanolamide of fumaric acid, in water, it can preferably be formulated for oral or injectable administration.

For oral administration, the pharmaceutical compositions may be provided, for example, in the form of tablets or capsules, which may be prepared conventionally using pharmaceutically acceptable excipients, for example, admixtures (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethyl cellulose), extenders (e.g., lactose, microcrystalline cellulose or dibasic calcium phosphate), lubricants (e.g., magnesium stearate, talc or silicon dioxide), disintegrants (e.g., potato starch or sodium starch glycolate) or inhibitors (e.g., sodium lauryl sulfate). Tablets may be coated by means well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a lyophilized product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (for example sorbitol syrup, cellulose derivatives or edible hydrogenated fats), emulsifying agents (for example lecithin or acacia), non-aqueous vehicles (for example almond oil, oily esters, ethanol or fractionated vegetable oils) and preservatives (for example methyl or propyl p-hydroxybenzoates or sorbic acid). The formulation may also suitably contain flavouring, colouring and sweetening agents.

Formulations for oral administration may be suitably formulated to allow controlled release of the active ingredient.

For buccal administration, the composition may take the form of a tablet which is conventionally formulated and is suitable for absorption at the buccal mucosal level. A typical buccal dosage form is a tablet for sublingual administration.

The compounds of the present invention may be formulated for parenteral administration by injection. The injectable dosage form may take the form of, for example, a single dose in a vial, and contain an added preservative. Compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and they may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may take the form of a powder for constitution with a suitable vehicle, e.g., sterile water, before use.

The diethanolamide of fumaric acid can be easily formulated in sterile pyrogen-free aqueous solutions according to the conventional literature of the pharmaceutical industry.

According to the invention, the compounds of the invention can also be formulated in rectal compositions such as suppositories or retention enemas, containing for example the essential components of conventional suppositories such as cocoa butter or other glycerides.

In addition to the compositions described above, the compounds of the present invention may also be formulated as depot preparations. Such long-term dosage forms may be administered by implantation (e.g., subcutaneously, transdermally or intramuscularly) or by intramuscular injection. Thus, for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (e.g. in the form of an emulsion in a suitable oil) or ion exchange resins.

According to the invention, the doses of the compounds of the invention or their mixtures, which are suggested for administration to humans (body weight approximately 70Kg), are in the range of 1mg to 2g, preferably 100mg to 1g, of active ingredient per dosage unit. The dosage unit may be administered, for example, 1 to 4 times per day. The dosage should be determined by the method of administration chosen. It is contemplated that frequent changes in dosage may be required depending on the age and weight of the patient and the severity of the clinical condition to be treated. Finally, the specific dose and method of administration should be determined at the discretion of the particular physician or veterinarian.

The Pharmaceutical compositions of the invention may be prepared using conventional methods, for example as described in the Remington's Pharmaceutical Sciences Handbook, Mack pub.Co., N.Y., USA, 17 th edition, 1985.

Detailed description of the invention

The following are non-exhaustive examples of pharmaceutical compositions of the present invention.

Formulation examples

Example 1

Each tablet contains:

example 2

Each tablet contains:

example 3

Each tablet contains:

example 4

Each tablet contains:

example 5

A 5g dose of an orally-dissolvable microgranule for paediatric use, comprising:

-PEAum mg 50.00

-non-cariogenic sugar mg 200,00

A suitable amount of pharmaceutically acceptable excipients up to g 5.00

Example 6

A 5ml dose sterile suspension for pediatric use comprising:

-PEAum mg 80,00

-carboxymethyl cellulose mg 25.00

Proper amount of double distilled water, up to ml 5.00

Example 7

A 5g dose of an orally-dissolvable microgranule comprising:

-PEAum mg 600.00

-non-cariogenic sugar mg 200.00

A suitable amount of pharmaceutically acceptable excipients up to g 5.00

Example 8

5ml of bi-layer containers per sterile single dose containing:

in aqueous gels：

Hyaluronic acid sodium salt mg 80.00

Proper amount of double distilled water, up to ml 2.50

In oily gels：

-PEAum mg 600.00

-Glycerol monostearate (gelenol) mg 40.00

Proper amount of vegetable oil, up to ml 2,50

Example 9

A veterinary (dog and cat) soft gelatin capsule, each comprising:

-PEAum mg 100.00

pharmaceutically acceptable oily excipient mg 300.00

Example 10

A 2ml glass vial containing:

diethanolamide mg of fumaric acid 100.00

An appropriate amount of sterile saline solution up to ml 2.0

Example 11

A 4ml freeze-dried glass vial containing:

diethanolamide mg of fumaric acid 200.00

-glycine mg 85.00

A 4ml solvent vial containing:

sterile saline solution ml 4.0ml

Claims (14)

1. Use of palmitoylethanolamide in a wholly or partially micronized form alone for the preparation of a medicament for the treatment of renal diseases.

2. Use according to claim 1, wherein at least 94% of the particles of the micronized palmitoyl ethanolamide have a size of less than 10 microns.

3. Use according to claim 1, wherein at least 77% of the particles of the micronized palmitoylethanolamide have a size less than 6 microns.

4. Use according to claim 1, wherein the palmitoylethanolamide is in all or part in a micronized form.

5. Use according to claim 4, wherein at least 97% of the particles of the micronized palmitoylethanolamide have a size less than 6 microns.

6. Use according to claim 4, wherein at least 57% of the particles of the micronized palmitoylethanolamide have a size less than 2 microns.

7. The use according to any one of claims 1 to 6 for the treatment of renal diseases caused by metabolic disorders or toxic agents.

8. The use of any one of claims 1 to 6, wherein the renal disease is selected from:

diabetic nephropathy

Renal arteriosclerosis

Pyelonephritis

Polycystic kidney disease

Alport syndrome

Lei-nie syndrome

-Goodpasture's syndrome

Lupus nephritis

Immunoglobulin A nephropathy

Tubular necrosis of the kidney

-glomerulonephritis

Urethral stricture

Iatrogenic nephropathy from non-steroidal anti-inflammatory drugs, cytotoxic drugs, lithium, antibiotics or cyclosporines

Renal disease from therapeutic radiation

Senile nephropathy.

9. Use according to any one of claims 1 to 6, wherein the treatment is carried out orally.

10. Use according to any one of claims 1 to 6, wherein the palmitoylethanolamide is comprised in a pharmaceutical composition in an amount ranging from 1mg to 2g of active ingredient per dosage unit.

11. Use according to claim 10, wherein the palmitoylethanolamide is comprised in a pharmaceutical composition in an amount ranging from 100mg to 1g of active ingredient per dosage unit.

12. Use according to any one of claims 1 to 6, wherein the palmitoylethanolamide is contained in a pharmaceutical composition selected from the group consisting of: tablets, capsules, solutions, syrups, suspensions of the controlled release type or of the non-controlled release type for oral administration; tablets for buccal or sublingual administration; suspensions, solutions or emulsions in oily or aqueous vehicles for injectable administration; suppositories or retention enemas for rectal administration; depot formulations for subcutaneous, transdermal or intramuscular administration.

13. Use according to any one of claims 1 to 6 for human therapy.

14. The use according to any one of claims 1 to 6 for veterinary treatment.