LIPOSOME COMPOSITION FOR USE IN PERITONEAL DIALYSIS

Abstract

The present invention is directed to a liposome composition for use in the peritoneal dialysis of patients suffering from endogenous or exogenous toxicopathies, wherein the pH within the liposomes differs from the pH in the intraperitoneal cavity and wherein the pH within the liposome results in a liposome-encapsulated charged toxin. The invention also relates to a pharmaceutical composition comprising said liposomes. A further aspect of the present invention relates to a method of treating patients suffering from endogenous or exogenous toxicopathies, preferably selected from drug, metabolite, pesticide, insecticide, toxin, and chemical warfare toxicopathies, more preferably hyperammonemia, comprising the step of administering liposomes of the invention in a therapeutically effective amount into the peritoneal space of a patient in need thereof. Next to human, the present invention is particularly suitable to veterinary aspects.

Claims

1.-12. (canceled)

13. A method for treating a metabolite toxicopathy in a patient in need thereof by peritoneal dialysis, comprising: the step of administering a therapeutically effective amount of a liposome composition into the peritoneal cavity of said patient, wherein the pH within the liposomes differs from the pH in the peritoneal cavity, wherein the pH within the liposomes results in liposomes-encapsulated charged metabolite, wherein the metabolite toxicopathy is selected from the group consisting of hyperammonemia, argininosuccinic acidemia, hyperuricemia, isovaleric acidemia and propionic acidemia.

14. The method according to claim 13, wherein said metabolite toxicopathy is hyperammonemia, isovaleric acidemia or propionic acidemia.

15. The method according to claim 14, wherein said metabolite toxicopathy is hyperammonemia.

16. The method according to claim 13, wherein the liposome composition comprises liposomes having a diameter size larger than 600 nm.

17. The method according to claim 16, wherein the liposome composition comprises liposomes having a diameter size of 600 nm to 10 m, 700 nm to 10 m, or 800 nm to 5 m.

18. The method according to claim 13, wherein the pH within the liposomes is 1 to 6.5.

19. The method according to claim 18, wherein the pH within the liposomes is 1.5 to 5.

20. The method according to claim 19, wherein the pH within the liposomes is 1.5 to 4.

21. The method according to claim 13, wherein the pH within the liposomes is 8.5 to 12.

22. The method according to claim 21, wherein the pH within the liposomes is 9 to 11.

23. The method according to claim 22, wherein the pH within the liposomes is 9 to 10.

24. The method according to claim 13, wherein the liposomes in the liposome composition are uni- and/or multilamellar, and comprise at least one of: (i) 1 to 100 mol % physiologically acceptable phospholipids; (ii) 1 to 100 mol % sphingolipids; (iii) 1 to 100 mol % surfactants; (iv) 5 to 100 mol % amphiphilic polymers and/or copolymers; (v) 0 to 60 mol % toxin retention-enhancing compounds; or (vi) 0 to 30 mol % steric stabilizers.

25. The method according to claim 24, wherein the physiologically acceptable phospholipids are selected from the group consisting of DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DSPE, DOPE, MPPC, PMPC, SPPC, PSPC, DMPG, DPPG, DSPG, DOPG, DMPA, DPPA, DPPS, EPC, and SPC.

26. The method according to claim 24, wherein the sphingolipids comprise sphingomyelin.

27. The method according to claim 24, wherein the surfactants are selected from the group consisting of hydrophobic alkyl ethers, alkyl esters, polysorbates, spans, and alkyl amides.

28. The method according to claim 24, wherein the amphiphilic polymers and/or copolymers are selected from the group consisting of block copolymers comprising at least one block of a hydrophilic polymer or copolymer, and at least one block of a hydrophobic polymer or copolymer.

29. The method according to claim 28, wherein the at least one block of a hydrophilic polymer or copolymer comprises polyethylene glycol (PEG).

30. The method according to claim 24, wherein the toxin retention-enhancing compounds are selected from the group consisting of cholesterol and sterol derivatives.

31. The method according to claim 24, wherein the steric stabilizers are selected from the group consisting of PEGylated compounds, PEGylated lipids, and DSPE-PEG.

32. The method according to claim 24, wherein the liposome composition comprises liposomes having a diameter of 800 nm or larger.

33. The method according to claim 32, wherein the liposome composition comprises liposomes having a diameter size of 900 nm or larger.

34. The method according to claim 32, wherein the liposome composition comprises liposomes having a diameter size of 1000 nm or larger.

35. The method according to claim 13, wherein the bilayer of the liposomes comprises: (i) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (ii) cholesterol (CHOL), and (iii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), wherein the liposome composition comprises liposomes having a diameter of 800 nm or larger.

36. The method according to claim 35, wherein the aqueous solution within the liposomes comprises a citrate solution buffered at pH 1.5 to 3.

37. The method according to claim 35, wherein the bilayer of the liposomes comprises 0.5 to 2 mol % of DSPE-PEG.

38. The method according to claim 37, wherein the liposome composition comprises liposomes having a diameter size of 900 nm or larger.

39. The method according to claim 38, wherein the liposome composition comprises liposomes having a diameter size of 1000 nm or larger.

40. The method according to claim 24, wherein the physiologically acceptable phospholipid is DPPC, the toxin retention-enhancing compound is cholesterol, and the steric stabilizer is DSPE-PEG.

41. The method according to claim 13, wherein the bilayer of the liposomes comprises: (i) 1 to 100 mol % of DPPC; (ii) 0 to 60 mol % of cholesterol; and (iii) 0 to 30 mol % of DSPE-PEG.

42. The method according to claim 41, wherein the bilayer of the liposomes comprises: (i) 10 to 100 mol % of DPPC; (ii) 0 to 60 mol % of cholesterol; and (iii) 0.5 to 2% mol % of DSPE-PEG.

43. The method according to claim 13, wherein the patient in need thereof is a human.

44. The method according to claim 13, wherein the patient in need thereof is a mammal or a bird.

45. The method according to claim 44, wherein the mammal is selected from the group consisting of swine, cattle, dog, cat, sheep, goat and horse.

Description

FIGURES

[0080] FIG. 1 illustrates the sequestration of toxic substances (e.g. drugs or ammonia, NH.sub.3) in the peritoneal space by transmembrane pH-gradient liposomes (case of a weak base). The unionized compound diffuses from the blood capillaries to the peritoneal space, where it gets trapped in an ionized form (NH.sub.4) in the vesicles. Diffusion continues until the liposomes internal buffering capacity is overwhelmed.

[0081] FIG. 2 is a graph illustrating the liposomes drainage from the peritoneal space to the blood after intraperitoneal administration. A non-exchangeable sterol dye (Cholesteryl BODIPY® FL-C12, Invitrogen) was incorporated (0.05 mol %) in the liposomal membrane (during the lipid film production process). After intraperitoneal administration of liposomes, the dye fluorescence (λ.sub.ex=470 nm, λ.sub.em=520 nm) was measured in the plasma aliquots and compared to a calibration curve to obtain the liposomal lipid concentration. Larger liposomes remained longer (8 h) in the peritoneal space whereas small liposomes were found in blood at important concentrations after 4 h. Mean±SD (n=3).

[0082] FIG. 3. is a graph showing the in vitro ammonia uptake by pH-gradient liposomes in 50% fetal bovine serum at 37° C. Liposomes exhibited a rapid and efficient uptake of ammonia. The initial ammonia and liposome concentrations were set at 1.7 and 3.8 mM, potentially tolerating a maximal capture capacity of 0.45 μmol ammonia/μmol lipid. Interestingly, the vesicles sequestered more than the total amount of ammonia loaded in the system (dashed line). The surplus came from the native ammonia present in the serum. The liposome diameter was of 840 nm. Mean±SD (n=6).

[0083] FIG. 4 is a graph showing the concentration of ammonia (NH.sub.3) in peritoneal dialysate in the absence (closed triangles) and presence (open triangles) of liposomes. The dialysis fluid was injected intraperitoneally at t=0 h in healthy rats. The injected liposome dose was 180 mg/kg, and the lipid concentration in the dialysis fluid was of 15 mM. The liposome diameter was of 850 nm.

[0084] FIG. 5 is a graph showing the concentration of verapamil (VP) in peritoneal dialysate in the absence (closed triangles) and presence (open triangles) of liposomes. VP was administered by oral gavage at t=0 h (50 mg/kg, ), followed by the intraperitoneal injection of the dialysis fluid at t=1 h with . The injected liposome dose was 180 mg/kg, and the lipid concentration in the dialysis fluid was of 15 mM. The liposome diameter was of 850 nm.

EXAMPLES

[0085] In the following examples it was demonstrated that an illustrative liposome composition (Example 1) can be retained for a prolonged time period in the peritoneal space after intraperoneal administration depending on the size of the liposomes (Example 2). These liposomes exhibited a rapid and efficient uptake of ammonia in 50% fetal bovine serum (Example 3). Moreover, these liposomes were capable of entrapping and concentrating ammonia (Example 4) and orally administered drug verapamil (Example 5) in the peritoneal space, thus demonstrating the utility of such liposomes for the detoxification of metabolites and drugs by intraperitoneal administration.

Example 1—Liposome Composition and Preparation

[0086] The formulation tested in the following experiments in vivo was composed of DPPC with 45 mol % of CHOL and 5 mol % of DSPE-PEG. The aqueous solution within the liposomes was a 250 mM sodium citrate solution buffered at pH 2. The formulations were prepared by the lipid film hydration/extrusion method (Hope M, Bally M, Webb G, Cullis PR. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim Biophys Acta 1985, 55-65). Lipids, CHOL, and eventually the Cholesteryl BODIPY® FL-C12 dye, from Invitrogen (0.05 mol %) were dissolved in chloroform which was subsequently removed under continuous nitrogen flow and high vacuum for >12 h. The lipid film was hydrated with citrate buffer (250 mM, pH 2). The large vesicles were obtained by extrusion through 2 stacked membranes of 5 m. The transmembrane pH-gradient was established by dialysis in normal saline for >12 h (membrane cut-off: 1000 kDa).

Example 2—Liposomes Drainage from the Peritoneal Space to the Blood after Intraperitoneal Administration

[0087] Sprague-dawley rats (male, 300 g) were lightly anesthetized by isofluran inhalation (2%) and 20 mL of a solution of icodextrin 7.5% containing liposomes (either 250 or 850 nm of diameter) bearing the non-exchangeable sterol dye (Cholesteryl BODIPY® FL-C12, Invitrogen, 0.05 mol %) in their membrane were slowly injected in the peritoneal space through sterile puncture. Then, blood aliquots of 250 μL were sampled through the tail veins at 15 min, 1, 2, 4, 6, 8, 10, 12, 14, 16 h after i.p. injection. Plasma was separated from the blood aliquots by centrifugation (6000 g for 10 min) and the dye fluorescence measured in plasma at λ.sub.em=520 nm (λ.sub.ex=470 nm).

Example 3—In Vitro Ammonia Uptake b pH-Gradient Liposomes in 50% Fetal Bovine Serum

[0088] Ammonia (NH3) uptake kinetics were monitored in 50% FBS in side-by-side diffusion cells (PermGear, Hellertown, Pa.) at 37° C. The liposomes used in this experiment had a diameter of 850 nm and contained 54 mol % DPPC, 45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and an internal citrate solution (250 mM) buffered at pH 2. The donor compartment (liposome-free) was separated from the receiver compartment (containing liposomes) by a polycarbonate membrane with 100 nm pores. The NH.sub.3-to-lipid molar ratio was set to 0.45 with an initial NH.sub.3 concentration of 1.7 mM in both cells to achieve equilibrium. NH.sub.3 uptake by the vesicles in the receiver compartment was directly related to the reduction of toxin concentration in the donor cell. Aliquots of 100 L were sampled from the donor compartment 3, 30 min, 1, 2, 4, 8, and 24 h after injection of pH-gradient liposomes in the receiver compartment. NH.sub.3 was then quantified by a colorimetric assay (Berthelot MPE, Violet d'aniline. Repert Chim Appl 1859, 1:284).

Example 4—Concentration of Ammonia in Peritoneal Dialysate in the Absence and Presence of Liposomes

[0089] Sprague-Dawley rats (300 g) were lightly anesthetized with isoflurane (2.5%, 0.6 L/min O2), kept on a warming blanket, and 20 mL of a solution of icodextrin 7.5% with (or without) liposomes (3 mg/mL) was slowly infused in the peritoneal space through sterile abdominal puncture with a 22G silicon catheter (Venflon; Becton Dickinson). The liposomes used in this experiment had a diameter of 850 nm and contained 54 mol % DPPC, 45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and an internal citrate solution (250 mM) buffered at pH 2. Aliquots of peritoneal dialysate were sampled 0.5, 1, 1.5, 2, 3, and 4 h after dialysis onset. The ammonia content in peritoneal fluid samples was assayed by a colorimetric assay (Berthelot MPE, Violet d'aniline. Repert Chim AppI 1859, 1:284).

Example 5—Concentration of Verapamil in Peritoneal Dialysate in the Absence and Presence of Liposomes

[0090] One hour after administration of verapamil (50 mg/kg, p.o.) to Sprague-Dawley rats (300 g), animals were lightly anesthetized with isoflurane (2.5%, 0.6 L/min O2), kept on a warming blanket, and 20 mL of a solution of icodextrin 7.5% with (or without) liposomes (3 mg/mL) was slowly infused in the peritoneal space through sterile abdominal puncture with a 22G silicon catheter (Venflon; Becton Dickinson). The liposomes used in this experiment had a diameter of 850 nm and contained 54 mol % DPPC, 45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and an internal citrate solution (250 mM) buffered at pH 2. Aliquots of peritoneal dialysate were sampled 2, 4, 6, 8, 10, and 12 h after the oral gavage of verapamil. The drug content peritoneal fluids was assayed by HPLC, as described in, e.g. Forster et al., Biomaterials 33, 3578-3585, 2012).