MXPA06009699A - Methods and compositions for detection of microbial contaminants in peritoneal dialysis solutions - Google Patents

Abstract

Methods and compositions for detection of microbial contaminants in peritoneal dialysis solutions are provided. A novel cause of aseptic peritonitis is provided--aseptic peritonitis associated with gram positive microbial contamination of a dialysis solution. Peptidoglycan is a major component of a gram positive bacterial cell wall and thus can serve as a marker for gram positive bacteria. In this regard, testing for peptidoglycans can be utilized to effectively prevent peritonitis in patients that use the peritoneal dialysis solutions, such as peritoneal dialysis solutions that contain a glucose polymer including an icodextrin and the like.

Description

METHODS AND COMPOSITIONS FOR THE DETECTION OF MICROBIAL CONTAMINANTS IN SOLUTIONS FOR PERITONEAL DIALYSIS Background of the Invention The present invention relates generally to the detection of gram positive microbial contaminants. More specifically, the present invention relates to methods and compositions employing the detection of peptidoglycan in solutions for peritoneal dialysis. Peptidoglycans are major components of the cell wall of gram-positive organisms and therefore serve as good markers for these microbes. Due to illness, trauma or other causes, the renal system may fail. In renal failure due to any cause, there are several physiological disorders. The balance of water, minerals (eg Na, K, Cl, Ca, P, Mg, SO4) and the excretion of a daily metabolic charge of fixed ions is no longer. possible in renal failure. During renal failure, the final toxic products of nitrogen metabolism (eg, urea, creatinine, uric acid, and the like) can accumulate in blood and tissues. Dialysis processes have been designed for the separation of elements in a solution by diffusion through a semipermeable membrane (solute diffusive transport) through a concentration gradient. Examples of dialysis processes include hemodialysis, peritoneal dialysis and hemofiltration.

The hemodialysis treatment uses the patient's blood to eliminate waste, toxins and excess water from the patient. The patient is connected to a hemodialysis machine and the patient's blood is pumped through the machine. Catheters or the like are inserted into the patient's veins and arteries to connect the blood flow to and from the patient's blood and the blood is infused back into the patient. Hemodialysis treatments can last several hours and are usually done in a treatment center approximately three or four times a week. To overcome the disadvantages often associated with classical hemodialysis, other techniques were developed, such as peritoneal dialysis. Peritoneal dialysis uses the patient's peritoneum as a semipermeable membrane. The peritoneum is the membranous covering of the body cavity which, due to the large number of blood vessels and capillaries, is capable of acting as a natural semi-permeable membrane. In peritoneal dialysis, a sterile dialysis solution is introduced into the peritoneal cavity using a catheter or the like. After a sufficient period of time, an exchange of solutes between the dialysate and the blood is achieved. The elimination of the fluids is achieved by providing an adequate osmotic gradient between the blood and the dialysate to allow the efflux of the water from the blood. This allows a proper acid-base balance of electrolytes and fluids to be restored to the blood. The dialysis solution is simply drained from the body cavity through the catheter. Examples of different types of peritoneal dialysis include continuous ambulatory peritoneal dialysis, automated peritoneal dialysis, and continuous flow peritoneal dialysis. Standard solutions for peritoneal dialysis contain dextrose to transport water and metabolic waste products through the peritoneum. Although dextrose has the advantage of being relatively safe and inexpensive, it has a number of disadvantages. Due to its small size, dextrose is transported rapidly through the peritoneum, thus leading to loss of osmotic gradient and loss of ultrafiltration within approximately 2 to 4 hours of infusion. It has been suggested that the ultrafiltration characteristics of peritoneal dialysis solutions could be improved by replacing the dextrose with high molecular weight substances, such as glucose polymers. An example of a novel high molecular weight agent is icodextrin. Dialysis solutions containing icodextrin are available commercially and have been found to be useful in the treatment of patients with end-stage renal disease Peritonitis is a major complication of peritoneal dialysis. The clinical suspicion of peritonitis is generated by the development of a cloudy dialysate in combination with variable clinical manifestations that may include abdominal pain., nausea, vomiting, diarrhea and fever. See, for example, Vas SI: Peritonitis. In: Nolph KD, ed. Peritoneal Dialysis. 3rd ed. Dordrecht, Holland: Kluwer Academic Publishers, 1989: 261 -84. most episodes of peritonitis are caused by intraperitoneal bacterial infections and diagnosis is usually easily established by positive dialysate cultures. However, there are several well-documented causes of noninfectious or sterile peritonitis. Aseptic or sterile peritonitis, which is also described as aseptic, chemical or negative culture peritonitis, is typically caused by a chemical or a foreign irritant to the body. One of the largest outbreaks of sterile peritonitis among patients undergoing peritoneal dialysis occurred in 1977. This was attributed to intrinsic contamination and hidden endotoxins of the dialysis solution. Batches of peritoneal dialysate suspected of causing the event had endotoxin levels in the range of 2 to 2.5 units of endotoxin (EU) / ml. See, for example, Karanicolas S., Oreopoulos D.G., Izatt S.H. , et al. : Epidemic of aseptic peritonitis caused by endotoxin during chronic peritoneal dialysis, N Engl J Med 1977; 296: 1336-7. A similar epidemic of aseptic peritonitis caused by endotoxin contamination in continuous cycle peritoneal dialysis patients was reported in 1998. See, for example, Mangramo AJ, Archbald LK, Hupert M., et al .: Outbreak of sterile peritonitis among continuous cycling peritoneal dialysis patients, Kidney Int 1988; 54: 1367-71. Other reported causes of aseptic peritonitis include vancomycin administered intraperitoneally (See, for example, Smith T., Baile G., Eisele G .: Chemical peritonitis associated with intraperitoneal vancomycin, Ann Pharm 1991; 25: 602-3, and Chancy DI, Gouse SF: Chenmical peritonitis secondary to intraperitoneal vancomycin, Am J Kidney Dis 1991; 17: 76-9), amphotericin B (See, for example, Benevent D., The Akoun N., Lagarde C :: Dangers of administration of intraperitoneal amphotericin B in continuous ambulatory peritoneal dialysis, Press Med 1984; 13: 1844), and acetaldehyde (See, for example, Tuncer M., Sarikaya M., Sezer T., et al .: Chemical peritonitis associated with high dialysate acetaldehyde concentrations , Nephrol Dial Transplant 2000; 15: 2037-40). A unique form of aseptic peritonitis, eosinophilic peritonitis, is a much more common entity that can occur shortly after the onset of peritoneal dialysis. See, for example, Gokal, R., Ramos J.M., Ward M.K., et al .: "Eosinophilic peritonitis" in CAPD, Clin Nephrol 1981; 15: 328-330. As discussed above, glucose polymers, such as icodextrin, can be used in place of dextrose in solutions for peritoneal dialysis. Icodextrin is a glucose polymer derived from the hydrolysis of corn starch. It has a molecular weight of 12-20,000 Daltons. Solutions for peritoneal dialysis containing codextrin as an osmotic agent in general are used for long-term exchanges (> 4 hours). Most of the glucose molecules of icodextrin are linearly linked with (1-4) glycosidic bonds (> 90%), while a small fraction is linked by (1-6) bonds. Icodextrin was introduced into clinical practice in the United Kingdom in 1994 and in other European countries started in 1996. The clinical advantages of icodextrin for long residence times, especially in patients with high or high average transport status and loss of ultrafiltration, are well accepted and contributed to its global popularity. See, for example, Wilkie M.E., Plant M.J. , Edwards L., et al. : Icodextrin 7.5% dialysate solution (glucose polymer) in patients with ultrafiltration failure: extension of technique survival, Perit Dial Int 1997; 17: 84-7; Wolfson M., Piraino B., Hamburger R.J. , Morton A.R. , for the Icodextrin Study Group: A randomized controlled trial to evaluate the efficacy of icodextrin in peritoneal dialysis, Am J Kidney Dis 2002; 40: 1055-65; and Mujáis S., Nolph K., Gokal R., et al .: Evaluation and management of ultrafiltration problems in peritoneal dialysis, Perit Dial Int 2000; 20 (Suppl 4): S5-S21. Since the introduction of icodextrin for use in peritoneal dialysis solutions, sporadic cases of aseptic peritonitis have been reported. See, for example, Pinerolo M.C. , Porri M.T. , D'Amico G.; Recurrent sterile peritonitis at onset of treatment with icodextrin, Perit Dial Int 1999; 19: 491 -2; Williams P.F .: Timely initiation of dialysis. Am J Kidney Dis 34: 594-595, 1999; Williams P.F., Foggensteiner L.: Sterile / allergic peritonitis with icodextrin in CAPD patients, Perit Dial Int 2002; 22: 89-90; Foggensteiner L., Bayliss J., Moss H., et al .: Timely initiation of dialysis - single-exchange experiences in 39 patients starting peritoneal dialysis, Perit Dial Int 2002; 22; 471 -6; Heering P., Brause M., Plum. , et al. : Peritoneal reaction to icodextrin in a female patient on CAPD. Perit Dial Int 2001; 21: 321-2; Del Rosso G., Di Liberato L., Pirilli A., et al. : A new form of acute adverse reaction to icodextrin in peritoneal dialysis patient, Nephrol Dial Transplant 2000; 15: 927-8; Goffin E., Scheiff J.M .: Transient sterile chemical peritonitis in a CAPD patient usibg icodextrin, Perit Dial Int 2002; 22: 90-1; Tintillier M., Pochet J.M., Christophe J.L., Scheiff J.M., et al .: Transient sterile chemical peritonitis with icodextrin: clinical presentation, prevalence, andliterature review, Perit Dial Int 2002; 22: 534-7; and Gokal R.: Icodextrin-associated sterile peritonitis, Perit Dial Int 2002; 22: 445-8. These patients typically presented with a cloudy dialysate, without abdominal pain, and dialysis cell counts ranging from 300 to 3500 / mm3, with varying percentages of neutrophils, lymphocytes, and macrophages. In general, there are no changes in the ultrafiltration profile or the peritoneal permeability to the solutes. The cultures were invariably negative, with no evidence of peritoneal eosinophilia or peripheral blood. What's more, all the components and endotoxin levels of the solution were within the product specifications, and the solutions for icodextrin-based peritoneal dialysis met the standards of the current Pharmacopoeia. Driven by these reports, in 2001 the manufacturer of the solution containing icodextrin (BAXTER HEALTHCARE CORPORATION) modified the Summary of Product Characteristics (SPC) to include the cloudy effluent as an "undesirable side effect" of icodextrin. Parenteral pharmaceutical products are required to be free of contaminating substances, such as substances that can cause fever. Because endotoxins derived from gram-negative bacteria are the most common contaminant in products for parenteral use, the historical pyrogens of care are LPS. The current Pharmacopoeia standards establish that one of two pyrogen contamination tests be applied to products for parenteral use. These tests are the rabbit pyrogen test and the LAL test. Although generally reliable, both trials have deficiencies. The rabbit test is based on a febrile response that in turn depends on the preparation of pyrogenic cytokines. The pyrogen test of the rabbit can be falsely negative if the pyrogen is at too low a concentration to induce a systemic response, but of sufficient magnitude to produce a local inflammatory reaction. In turn, the more sensitive LAL assay does not detect pyrogens other than LPS. Pyrogens such as viruses, fungi, DNA, gram-positive exotoxins or bacterial cell wall components from gram-positive bacteria, such as peptidoglycans and the like, are not detected by the LAL assay. See, for example, Dinarello C.A. , O 'Conner J.V., LoPreste G .: Human leukocyte pyrogen test formula detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli, J Clin Microbiol 1984; 20: 323-9; Poole, S., Thorpe R., Meager A., et al .: Detection of pyrogen by cytokine relay, Lancet 1988; 1 (8577): 130; Ray A., Redhead K., Selkirk S., et al .; Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis, FEMS Microbiol Lett 1991; 63: 21 1-7; Taktak Y.S., Selkirk S., Bristow A.F., et al .: Assay of pyrogens by interleukin-6 relase from monocytic cell lines, J Pharm Pharmacol 1991; 43: 578-82; and Fennrich S., Fischer M., Hartung T., et al .: Detection of endotoxins and other pyrogens using human whole blood, Dev Biol Stand 1999; 101: 131 -9. The global outbreak of aseptic peritonitis observed with solutions for peritoneal dialysis discussed above serves as a sentinel example of how contemporary products for parenteral use with microbial contaminants other than endotoxins can be considered safe according to the standards of the Pharmacopoeia but cause adverse clinical effects. Therefore, there is a need to provide improved standards for parenteral products that employ screening procedures to better ensure that parenteral products are effectively free of contaminants.

Brief Description of the Invention The present invention relates in general to the detection of gram positive bacterial contaminants. In particular, the present invention relates to methods and compositions employing the detection of peptidoglycan in solutions for peritoneal dialysis. The inventors have surprisingly discovered a novel cause of aseptic peritonitis: aseptic peritonitis associated with contamination by positive gram microbes of a dialysis solution. Peptidoglycan is a major component of the cell wall of gram-positive bacteria and can thus serve as a marker for gram-positive bacteria. Thus, the determination of peptidoglycans can be used to effectively prevent peritonitis in patients using solutions for peritoneal dialysis, such as peritoneal dialysis solutions containing a glucose polymer, including an icodextrin and the like. Icodextrin is derived from corn starch, a natural product. It is well known that products of natural origin are contaminated with a wide variety of microorganisms. The inventors have discovered that some natural products, such as corn starch, contain a thermophilic acidophilic bacterium, such as Alicyclobacillus acidocaldarius. This last organism is ubiquitous in the food industry, particularly in acidic beverages. It is the Alicyclobacillus that produces guaiacol, which is the substance that causes the unpleasant smell of orange juice. See, for example, Matsubara H., Goto K., Matsubara T., et al. Alicyclobacillus acidiphilus sp. Nov., a novel thermo-acidophilic, omega-alicyclic fatty acid-containing bacterium isulated from acidic beverages. Int J Syst Evol Microbiol 2002; 52: 1681 -5. It has not been generally recognized that solutions for peritoneal dialysis and solutions for parenteral use are contaminated by this organism or its degradation products. This is mainly because current testing procedures for microbial contamination of peritoneal dialysis solutions and solutions for parenteral use in general are not capable of detecting this organism or its degradation products. To this end, in one embodiment, the present invention provides a method for manufacturing a solution for peritoneal dialysis. The method includes providing a solution based on a glucose polymer; add a reagent to the solution based on a glucose polymer, where the reagent is capable of reacting with a peptidoglycan; determine a proportion of the peptidoglycan using the reagent; and using the solution based on a glucose polymer to provide the solution for peritoneal dialysis if it is determined that a sufficiently low level of the peptidoglycan is present. In one embodiment, the reaction initiates a serine protease cascade. In one reaction, the serine protease cascade includes a profenol oxidase cascade. In one embodiment, the reagent is derived from a plasma of silkworm larvae. In one embodiment, the proportion of peptidoglycan is further determined by a colorimetric measurement in response to the reaction between the peptidoglycan and the reagent. In one embodiment, the sufficiently low level of peptidoglycan is about 10 ng / ml or less. In one embodiment, the solution based on a glucose polymer includes an icodextrin. In one embodiment, the reagent is added to the icodextrin as a raw material that is used to prepare the solution based on a glucose polymer. In another embodiment, the present invention provides a method for delivering peritoneal dialysis to a patient. The method includes preparing a solution for peritoneal dialysis using a reagent to ensure that the solution for peritoneal dialysis has a sufficiently low level of a peptidoglycan as to prevent peritonitis in the patient; and supplying the peritoneal dialysis solution to the patient. In a modality, the solution for peritoneal dialysis includes a solution based on icodextrin. In one modality, peritoneal dialysis includes automated peritoneal dialysis, continuous ambulatory peritoneal dialysis, and the like. In one modality, the patient is monitored to detect peritonitis during peritoneal dialysis. For example, a dialysis effluent may be collected from the patient to determine an IL-6 response that correlates with an incidence of peritonitis. In one embodiment, the reagent is used to determine whether the proportion of the peptidoglycan exceeds about 10 ng / ml in the peritoneal dialysis solution before use during peritoneal dialysis. In yet another embodiment, the present invention provides a method for determining in a solution for peritoneal dialysis a proportion of peptidoglycan that exceeds a level sufficient to cause peritonitis. The method includes adding a reagent to the solution for peritoneal dialysis, where the reagent is able to react with peptidoglycan to initiate a serine protease cascade; and determine the proportion of the peptidoglycan. For example: the icodextrin-based solution can be tested to determine if the proportion of peptidoglycan exceeds about 10 ng / ml. In yet another embodiment, the present invention provides an icodextrin composition that includes a reagent capable of reacting with a peptidoglycan. In one embodiment, a level of peptidoglycan can be established above which the product would produce sterile peritonitis. In one embodiment, the use of affinity columns with resins that bind to peptidoglycans can be exploited to minimize contamination by peptidoglycans in glucose polymers. An advantage of the present invention is to provide improved solutions for peritoneal dialysis. Another advantage of the present invention is to provide improved methods for manufacturing and using solutions for peritoneal dialysis employing a detection protocol for determining the presence of peptidoglycans in the solution for peritoneal dialysis. Yet another advantage of the present invention is to provide improved assay procedures that can be employed to prevent peritonitis in patients receiving peritoneal dialysis therapy. Yet another advantage of the present invention is to provide an improved composition of icodextrin which utilizes a detection method in the manufacture thereof to determine the presence of peptidoglycans. Additional features and advantages of the present invention are described in, and will be apparent from, the following Detailed Description of the Invention and the Figures.

Brief Description of the Figures Figure 1 illustrates a correlation between the response of I L-6 in a PBMC assay and the concentration of peptidoglycan in icodextrin. The IL-6 response was measured in freshly isolated monocytes from healthy volunteers. Each symbol and line represents data from a single donor. Figure 2 illustrates an effect of peptidoglycan on neutrophil infiltration in rat peritoneal fluid. The peritoneai fluid was collected six hours after a single injection (35 ml / kg) of icodextrin containing peptidoglycan. Figure 3 illustrates an effect of peptidoglycan on TN F-a in rat peritoneal fluid. Figure 4 illustrates an effect of peptidoglycan on IL-6 in rat peritoneal fluid. Figure 5 illustrates the frequency (%) of the aseptic peritonitis reported with the use of icodextrin.

Detailed Description of the Invention The present invention relates generally to the detection of gram positive organisms and fragments thereof. In particular, the present invention relates to methods and compositions employing the detection of peptidoglycans in solutions for peritoneal dialysis. The inventors have surprisingly discovered a novel cause of aseptic peritonitis: aseptic peritonitis associated with contamination by positive gram microbes of a dialysis solution. Peptidoglycan is a major component of the cell wall of gram-positive bacteria and can thus serve as a marker for gram-positive bacteria. In this regard, the determination of peptidoglycans can be used to effectively prevent peritonitis in patients using solutions for peritoneal dialysis, such as peritoneal dialysis solutions containing a glucose polymer, including an icodextrin and the like.

Aseptic peritonitis associated with solutions for peritoneal dialysis based on icodextrin is considered to be the largest adverse event reported for a solution for peritoneal dialysis due to a contaminant of microbial origin. Based on the experimental investigations described in detail below, this suggests that the peptidoglycans present in the solution for peritoneal dialysis based on icodextrin was the causative agent of aseptic peritonitis. In addition, the pharmacovigilance data detailed below supports the effectiveness of the corrective action and the manufacturing procedure with selection to prevent the onset of peritonitis. These findings illustrate that although endotoxins are deservedly one of the most worrisome bacterial products that can cause adverse effects in patients, they are not the only ones. In this regard, pyrogens other than endotoxins, such as peptidoglycans, which have not been previously identified, are capable of producing clinically significant inflammation. Thus, this demonstrates that pharmaceutical products for parenteral use that approve the summarized trials and thereby comply with Pharmacopoeia standards, may require an additional level of testing to effectively determine the efficacy and safe use of such products to better ensure the quality of life issues associated with their use. In the present invention, contamination by pyrogens other than LPS was recognized as a problem since exchanges of peritoneal dialysis allowed direct observation of the inflammation induced by peptidoglycan in situ. A peptidoglycan is a heteropolymer formed from N-acetylmuramic acid linked by β-bonds (1-4) and N-acetyl-D-glucosamine residues cross-linked by peptide bridges. See, for example, Royce C.L., Pardy R.L.; Endotoxin-like properties of an extract from a symbiotic, eukaryotic cholerella-like green algae, J Endotoxin Res 1996; 3: 437-44. The skeleton of glycan is chemically homogeneous, while the peptides that cross sugars vary. The peptidoglycan occupies approximately 40% by weight of the positive gram cell wall, but approximately 1-10% of the total weight of the gram negative cell walls. Royce C. L., Pardy R.L.; Endotoxin-like properties of an extract from a symbiotic, eukaryotic cholerella-like green algae, J Endotoxin Res 1996; 3: 437-44. the peptidoglycan and another constituent of the cell wall, lipoteichoic acid, include virtually all the main inflammatory inducing components of the gram-positive cell walls. See, for example, Sriskandan S., Cohen J: Gram-positive sepsis, in: Opal S.M., Cross A.S. , eds. Bacterial Sepsis and Septic Shock, Philadelphia: W. B Saunders Company, 1999: 397-412. Like endotoxins, peptidoglycans can induce the production of cytokines in a wide variety of cells and it has long been recognized that they have immunomodulatory actions. See, for example, Garner R.E. , Hudson J.A .: Intravenous injection of candida-derived mannan results in elevated tumor necrosis factor alpha levéis in serum, Infect Immun 1996; 64: 4561 -6; and Schwab J .: Phlogistic properties of peptidoglycan- polysaccharide polymers from cell walls of pathogenic and normal flora bacteria which colonize humans, Infect Immun 1993; 61: 4535-9. however, peptidoglycans are several orders of magnitude less potent than endotoxins as triggers of these biological effects. See, for example, Henderson B., Poole S., Wilson M; Bacterial modulins: a novel class of virulence factors which cause host tissue pathology by induction cytokine synthesis, Microbial Rev 1996; 60: 316-41; and Nakagawa y., Maeda H., Murai T.: Evaluation of the in vitro pyrogen test system based on proinflammatory cytokine relase from human monocytes: Comparison with a human whole blood culture test with the rabbit pyrogen test, Clin Diag Lab Immunol 2002; 9: 588-97. For example: the minimum pyrogenic dose of peptidoglycans in rabbits is 7.3 μg / kg, while that of endotoxins is 0.0027 μg / kg. See, for example, Henderson B., Poole S., Wilson M; Bacterial modulins: a novel class of virulence factors which cause host tissue pathology by inducing cytokine synthesis, Microbial Rev 1996; 60: 316-41. In addition to the absence of substances containing pyrogenic, the safety of products for parenteral use in general is defined by Pharmacopoeia tests to determine their sterility. Bacterial cultures are generally carried out at neutral pH using an incubation temperature between 20-35 ° C. These are suboptimal conditions for the growth of thermophilic and acidophilic microorganisms such as Alicyclobacillus acidocaldarius, which require an acid medium and high temperature for their growth. Therefore, the "definitions of sterility" routinely used and the trials that support them may fail to detect microorganisms that do not grow under conventional conditions. In one embodiment of the present application, acid hydrolysis is used at elevated temperature for hydrolysis of the starch to produce icodextrin. These manufacturing conditions are suitable for the growth of Allcyclobacillus acidocaldarius, but differing from those used to determine sterility e? based on bioburden. As mentioned above, a detailed description of the findings arising from the investigation is given below according to one embodiment of the present invention by way of example and without limitation: Chemical and Physical Investigations Most of the glucose molecules of the icodextrin are linearly linked with glycosidic bonds to (1-4) (> 90%), while a small fraction is linked by (1-6) bonds. The molecular weight distribution of icodextrin was performed by gel permeation chromatography. The distribution of glycosidic linkages to (1? 4) and a (1 -> 6) in icodextrin was evaluated by nuclear magnetic resonance spectroscopy. Volatile and semi-volatile impurities were examined by high performance liquid chromatography and mass spectroscopy. Dialyzed effluent analysis Samples of dialysate effluent from patients were analyzed to determine metabolites of icodextrin, triglycerides, total proteins and selective pyrogenic cytokines (IL-6, IL-1β and TNF-a). The metabolites of icodextrin were analyzed using high resolution anion exchange chromatography with pulsatile amperometric detection. See, for example, Burke R.A., Hvizd M.G. , Shockley T.R .: Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection, J Chromatogr B 1997; 693: 353-7. The analysis of triglycerides and proteins was carried out with a Boehringher Mannheim / Hitachi 91 1 Chemistry analyzer. Measurements of cytokines were made using ELISA kits (R & amp; amp;; D Systems, Minneapolis, MN). Pyrogen measurement The endotoxin concentration of the icodextrin solution was determined by a fixed-point chromogenic LAL assay. See, for example, Weary M., Dubczak J., Wiggins J. Et al: Validating an LAL chromogenic substrate pyrogen test for large volume parenterals, in: Watson S.W. , Levin J., Novítsky T.J., eds., Detection of bacterial endotoxin with limulus amebocyte lysate test. New York: Alan R. Liss, 1987: 307-22. The rabbit pyrogen test was conducted in accordance with the instructions of the European Pharmacopoeia. European Pharmacopoeia, 4th. Ed. Strasbourg, France: Council of Europe, 2002; 131 -2. An ex vivo pyrogen test was used to measure the response of IL-6 in mononuclear cells isolated from peripheral blood (PBMC) after exposure to a test substance, to quantify non-endotoxin pyrogens. See, for example, Dinarello C.A., O 'Conner J.V. , Le Preste G .: Human leukocyte pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli, J Clin Microbiol 1984; 20: 323-9; and Poole S., Thorpe R., Meager A., et al .: Detection of pyrogen by cytokine relase, Lancet 1988; 1 (8577): 130. Measurement of peptidoglycans The quantification of peptidoglycans (PG) was carried out with the plasma test of silkworm larvae (SLP) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). See, for example, Tsuchiya M., Asabi N., Suzouki F.: Detection of peptidoglycan and B-glucan with silkworm larvae plasma test, FEMS Immunol Medical Microbiol 1996; 15: 129-34; and U.S. Patent No. 4,970,152. The SLP contains all the factors of the profenol oxidase (PPO) cascade, a self-defense mechanism for insects. The PPO cascade is initiated by the peptidoglycan, where the PPO is finally activated to phenol oxidase. The activity of phenol oxidase is detected colorimetrically with 3,4-dihydroxyphenylalanine as substrate. It was found that the detection limit of peptidoglycan in icodextrin solution is 7.4 ng / ml. The SLP assay does not detect endotoxins.

As fully described in U.S. Patent No. 4,970,152, for example, the detection of peptidoglycan (or β-G) can be carried out in the following manner. A mixture containing the peptidoglycan is mixed well with a reagent that includes a fraction that specifically reacts with the peptidoglycan ("PG") to prepare the reaction solution. After a certain period of time, an enzymatic activity, for example BAEEase activity, PPAE, PO, etc., can be measured in the reaction solution by a conventional method and compared with previously obtained calibration curves using standard solutions of PG with known concentrations to determine the proportion of PG. Alternatively, it is possible to apply the phenomenon consisting in that the time required for the activation of PO depends on the concentration of PG in the sample. That is, after mixing the PG reagent with a sample in the presence of the PO substrate, the time required to reach a certain value of the amount of reaction product generated by the PO is measured. By way of example and not limitation, an experimental procedure using the SLP assay according to one embodiment of the present invention may be carried out as follows. Peptidoglycan (PG) and (1,3) -D-glucan (BG) are components of the cell walls of gram positive bacteria and fungi, respectively. PG and BG are measured with the Plasma of the Silkworm Larvae (SLP) test. The SLP assay contains all the factors of the profenol oxidase (PPO) cascade, an important self-defense mechanism for insects. The PPO cascade is initiated by peptidoglycan, where PPO converts 3,4-dihydroxyphenylalanine (DOPA) into melanin. The resulting melanin formation is detected colorimetrically at 650 nm using a standard plate reader. The icodextrin raw material is tested undiluted and has a detection limit (LD) of 0.74 ng / ml (the lowest detectable standard point). The finished product, such as EXTRANEAL, is tested after diluting ten times in order to mitigate the inhibition of the matrix caused by the presence of electrolytes. The dilution step of the sample results in an LD of 7.4 ng / ml after correction by the 1: 10 dilution. Animal studies The No Observed Effect Level (NOEL) was determined in a rat model. in English) for peptidoglycan in icodextrin. A total of 45 male Sprague-Dawley rats (Harían Inc, Indianapolis, IN, USA), weighing 255-280 g, were divided into 9 equal groups. Each group received a single intraperitoneal injection of icodextrin inoculated with 0, 1, 5, 10, 50, 100, 500, 1000 or 5000 ng / ml of peptidoglycan at a dose of 35 ml / kg. Peptidoglycan derived from Staphylococcus aureus (Toxin Technology Inc., Sarasota, FL, USA) was used in these experiments. Six hours after the injection, the rats were sacrificed and the fluid of the peritoneal cavity was quantitatively collected determining the weight and the volume. The peritoneal fluid was analyzed to determine the nucleated cell count and the differential count, the total proteins and the concentration of IL-6 and TNF-α. Statistical analysis Data on post-sale surveillance in several hundred batches of icodextrin manufactured between July 2000 and September 2002 were analyzed to determine an association between the level of peptidoglycan and the incidence of aseptic peritonitis. To accumulate enough claim frequencies to effectively show claims per million (CPM), the data was sorted by peptidoglycan level in logarithmic scale, in particular < 7.4, > 7.4-15, > 15-30, > 30-60 and > 60 ng / ml. For each range of peptidoglycan levels, total claims and total units sold were calculated. The CPM units sold were calculated as the total claims divided by the total units sold, multiplied by one million. Negative binomial regression was used to evaluate the association between CPM and peptidoglycan level. Statistical analysis was performed using the SAS GENMOD procedure (SAS Institute, Cary, NC, USA). Clinical cases According to reports received through the global pharmacovigilance system of BAXTER HEALTHCARE CORPORATION in 2002, the frequency [(number of complaints - * - number of patients treated) x 100] of aseptic peritonitis was 0.095%. There was a sustained increase in the cases reported in 2001 up to a maximum frequency of 1.04% in March 2002. The patients were rarely feverish or with the appearance of intoxicated; the abdominal pain was between discrete and absent; the dialysate was turbid and contained cells, with cell counts in the dialysate ranging from 300 to 3500 / mm3 (variable percentages of neutrophils, lymphocytes and macrophages without eosinophils). Unlike bacterial peritonitis, there were no changes in ultrafiltration or peritoneal permeability for small solutes. The cultures of blood and dialysate were invariably negative. Antibiotics were instituted in varying ways by local doctors. All clinical signs were resolved by suspending icodextrin. In some cases, icodextrin was resumed and aseptic peritonitis recurred. BAXTER HEALTHCARE CORPORATION initiated a worldwide voluntary withdrawal of several hundred batches of icodextrin in May 2002. Chemical and physical investigations of icodextrin Chemical and physical investigations of the recovered icodextrin batches were conducted. These analyzes revealed no difference in the molecular weight distribution of icodextrin, in the percentage of branching of the glycosidic linkages or in the volatile and semi-volatile trace level organic impurities, between the lots associated with aseptic peritonitis and the not associated with adverse clinical events. All the components of the questioned lots of dialysate containing icodextrin were within the specifications of the product and complied with the standards of the Pharmacopoeia. Dialysate effluent analysis A marked elevation was observed in the concentration of I L-6 in the dialysate effluent of a patient with aseptic peritonitis, compared to a control effluent (> 5000 versus 59 pg / ml, respectively). An increase in protein concentration was observed with the sample of the claim compared with the control (236 mg / dl versus 125 mg / dl, respectively). There was no difference in the icodextrin and its metabolites (glucose polymers with degree of polymerization from 2 to 7) between the samples of the claim and the control. These results indicated that neither icodextrin nor its metabolites were the probable cause of aseptic peritonitis. Pyrogenic Analysis in Codextrin The endotoxin levels in all samples associated with aseptic peritonitis were found to be within the product limit (<0.25 EU / ml), as determined, by the LAL assay. In the pyrogen tests in rabbit, there was no increase in temperature neither with the lots of the claim nor with those not questioned. However, an increase in the IL-6 response was observed in the PBMC test in vitro both with the dialysate batches containing icodextrin and with the icodextrin raw material used to manufacture the questioned batches of peritoneal dialysate, as illustrated in FIG. Table 1 below: Table 1: Response of IL-6 from questioned and unquestioned batches of icodextrin in the PBMC assay. In this assay, an IL-6 response greater than 500 pg / ml is considered a positive pyrogenic response. a Minimum essential eagle Eagle with supplementary components, Baxter's experimental product, solution for standard peritoneal dialysis containing glucose The IL-6 provocative substance from the questioned samples was not affected in the presence of polymyxin B, suggesting that it was not LPS. See, for example, Pool E.J. , Johaar G., James S., et al .: Differentiation between endotoxin and non-endotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay, J Immunoassay 1999; 20: 79-89. In a filtration experiment using a 30 kD molecular weight cutoff filter, the contaminant that produced the inflammatory response in the PBMC assay was found in the retentate, suggesting that the molecular weight of the substance was > 30kD, that is to say superior to that of icodextrin. Negative tests for LPS, but positive response of IL-6 in PMBC indicated that the probable cause of aseptic peritonitis was a pyrogenic contaminant distinct from the endotoxins of the icodextrin raw material used to make the final solution for peritoneal dialysis. Peptidoglycan and microbiological analysis Analysis of several hundred returned batches of icodextrin indicated that 41% of the batches were contaminated with peptidoglycan, which was detected by the SLP assay. The concentrations ranged from the detection limit of 7.4 ng / ml to 303 ng / ml. Because the manufacture of icodextrin from maltodextrin requires heat and acidification, a microbiological investigation was carried out to determine the presence of unruly organisms. It was found that the first steps in the elaboration of icodextrin were contaminated with an acid-fast thermophilic gram-positive organism, Alicyclobacillus acidocaldarius. Alicyclobacillus was the source of contaminating peptidoglycan. Sterile heating and filtration procedures applied to the nearly finished product eliminated the bacteria, but not the contaminating peptidoglycans. A positive correlation was found between peptidoglycan levels in the icodextrin and the IL-6 response observed in the PBMC assay (See Fig. 1). A substantial variability in the IL-6 response between donors was observed, suggesting a range of peptidoglycan sensitivities. Animal studies The effects of intraperitoneal administration of peptidoglycan in rats were investigated in order to establish a NOEL. This can be used to establish a regulatory specification for the manufacture of icodextrin. Total proteins, white blood cells and neutrophils were elevated in the peritoneal fluid of rats treated with icodextrin + peptidoglycan, compared to control rats that received icodextrin without peptidoglycan. The infiltration of neutrophils in the peritoneal fluid showed a dose-dependent increase with a NOEL at 100 ng / ml. (See Fig. 2). Both inflammatory cytokines showed dose-dependent increases with NOEL values at 10 ng / ml for TNF-a (See Fig. 3) and 100 ng / ml for IL-6 (See Fig. 4). The lowest NOEL for peptidoglycan was for the TNF-α response, which was 10 ng / ml. Correlation between peptidoglycan and aseptic peritonitis A positive correlation was found between the concentration of peptidoglycan in icodextrin and CPM, as illustrated in Table 2 below: Table 2: Correlation between Claims Per Million (CPM) of units sold and concentration of peptidoglycan in icodextrin solution aClaims Per Million (CPM) of units sold were calculated as the total of claims divided by the total units sold, multiplied by one million. The threshold CPM value was 8.2, when the peptidoglycan levels were below the detection limit of 7.4 ng / ml. In contrast, the CPM value was 252.9 when the peptidoglycan levels were equal to or greater than 60 ng / ml. The association between CPM and peptidoglycan was highly significant (p <; 0.0001). Incidence of aseptic peritonitis after corrective action In May of 2002, BAXTER HEALTHCARE CORPORATION rescued all batches of icodextrin contaminated with peptidoglycan at a concentration> 0.05. 10 ng / ml, as well as all batches in which peptidoglycan was not analyzed at the time of rescue. In addition, an internal regulatory specification for peptidoglycan was implemented below the detection limit (<7.4 ng / ml) of the assay for product release. A routine and serial monitoring of peptidoglycan and thermophilic bacteria was implemented. Peptidoglycan concentrations in all recently manufactured lots have been below the detection limit of SLP and are free of IL-6 generating agent according to the PBMC assay. Fig. 5 shows the rate of incidence of aseptic peritonitis per month, starting in September 2001 until January 2003. The frequency of claims has decreased from a maximum value of 1, 04% in March of 2002 to 0.013% in January of 2003, after the implementation of corrective actions. The number of patients who used a solution for commercial peritoneal dialysis known as EXTRANEAL, of BAXTER HEALTHCARE CORPORATION, during this period remained at approximately 7,000. These results suggest that the corrective actions have been effective in the prevention of excessive claims of aseptic peritonitis due to contamination of peptidoglycan in the dialysis solution containing icodextrin. As previously discussed, the present invention relates to methods and compositions employing the detection of peptidoglycan in solutions for peritoneal dialysis. This allows analyzing the presence of bacterial fragments since peptidoglycans are the main cell wall component of gram positive organisms. In this regard, detection of peptidoglycan can be effectively used to prevent peritonitis in patients using solutions for peritoneal dialysis, such as peritoneal dialysis solutions containing a glucose polymer, including icodextrin and the like. It should be appreciated that the compositions, solutions for peritoneal dialysis and methods of manufacturing thereof may be provided in any suitable manner in accordance with a modality of the present invention. In one embodiment, the present invention provides bioburden assays, such as bioburden assays for determining thermophilic acidophilic organisms according to standard procedures described in various Pharmacopoeias. This type of assay can be used as an aggregate level of assay in addition to assays for determining bacterial fragments by peptidoglycan detection, as discussed previously. In one embodiment, the present invention provides methods for making a solution for peritoneal dialysis. The method can include any number and type of processing steps that are suitable. For example: the process includes providing a glucose polymer; add a reagent to the glucose polymer where the reagent is capable of reacting with a peptidoglycan; determine a proportion of the peptidoglycan; and using the glucose polymer to prepare the solution for peritoneal dialysis if it is determined that a sufficiently low level of the peptidoglycan is present in the glucose polymer. If the proportion of peptidoglycan or the like exceeds this level, such as about 10 ng / ml or less, the glucose polymer can be further processed to remove the peptidoglycan or the like in order to reach a sufficiently low level thereof. The glucose polymer can be further processed in any suitable manner. In one embodiment, the glucose polymer can be processed with any number and type of separation devices, such as an affinity device and / or the like. In one embodiment, the present invention provides compositions, such as glucose polymer compositions, which can be used to prepare solutions for peritoneal dialysis. A variety of different types of compositions and solutions containing them can be used. For example, the type of compositions and solutions can be found in U.S. Patent No. 4,761,237, entitled "PERITONEAL DIALYSIS SOLUTION CONTAINING CARBOHYDRATE POLYMERS"; U.S. Patent No. 4,886,789, entitled "PERITONEAL DIALYSIS AND COMPOSITIONS FOR USE THEREIN"; U.S. Patent No. 6,077,836, entitled "PERITONEAL DIALYSIS AND COMPOSITIONS FOR USE THEREIN"; and U.S. Patent No. 6,248,726 B1, entitled "METHOD OF PERITONEAL DIALYSIS USING GLUCOSE POLYMER SOLUTIONS", the entire descriptions of which are incorporated herein by reference. Additional examples of compositions and solutions containing the same can be found in U.S. Patent Application No. 10 / 327,264, entitled BIOCOMPATIBLE DIALYSIS SOLUTION CONTAINING MODIFIED ICODEXTRINS, filed December 4, 1998, the entire descriptions of which are incorporated herein. as a reference. In one modality, peritoneal dialysis solutions may include BAXTER HEALTHCARE CORPORATION's EXTRANEAL or appropriate modifications thereof. In one embodiment, the present invention includes methods for delivering peritoneal dialysis, such as continuous ambulatory peritoneal dialysis and automated peritoneal dialysis. In continuous ambulatory peritoneal dialysis, the patient performs several cycles of drainage, filling and residence during the day, for example approximately four times per day. Each treatment cycle, which includes drainage, filling and residence, takes approximately four hours. Automated peritoneal dialysis is similar to continuous ambulatory peritoneal dialysis in the fact that the dialysis treatment includes a drain, fill and residence cycle. However, a dialysis machine automatically performs three or more cycles of peritoneal dialysis treatment, typically at night while the patient is asleep. With automated peritoneal dialysis, an automated dialysis machine is connected in fluid form to an implanted catheter or the like. The automated dialysis machine also connects fluidly to a source or bag of fresh dialysis solution and fluid drainage. The dialysis machine pumps the spent dialysis solution from the peritoneal cavity, through the catheter, to the drain. The dialysis machine then pumps fresh dialysis solution from the source, through the catheter, and into the patient's peritoneal cavity. The automated machine allows the dialysis solution to reside within the cavity so that the transfer of waste, toxins and excess water from the patient's bloodstream to the dialysis solution can take place. A computer controls the automated dialysis machine so that the dialysis treatment occurs automatically when the patient is connected to the dialysis machine, for example when the patient sleeps. In this regard, the dialysis system automatically and sequentially pumps fluid into the peritoneal cavity, allows residence, pumps fluid out of the peritoneal cavity and repeats the procedure. Several cycles of drainage, filling and residence occur during the treatment. Also, a final "final filling" volume is typically used at the end of the automated dialysis treatment, which remains in the peritoneal cavity of the patient when the patient is disconnected from the machine for the day. Automated peritoneal dialysis relieves the patient of manually performing the steps of drainage, residence and loading during the day. In one embodiment, automated peritoneal dialysis can be performed using a mixing device, such as the ADMIX HOMECHOICE, of BAXTER HEALTHCARE CORPORATION, or suitable modifications thereof. In one embodiment, the present invention provides an assay for determining whether a solution for peritoneal dialysis, such as a solution based on icodextrin, includes peptidoglycan that exceeds a sufficiently low level to cause peritonitis in the patient who uses it. The present invention provides a detection protocol that uses a reagent, such compound a reagent derived from a plasma of silkworm larvae, for that purpose. It should be appreciated that the detection method can be carried out at any suitable stage prior to the use of the solution for peritoneal dialysis. For example, the reagent can be added to a glucose polymer composition, such as a icodextrin composition, as a raw material, to determine the presence of peptidoglycan. If the level of peptidoglycan is at a sufficiently low level, the composition can then be used to prepare the solution for peritoneal dialysis. In another modality. The reagent can be added to the solution for peritoneal dialysis, such as after the sterilization process in the form of a finished product. For example, the reagent can be added to the solution for peritoneal dialysis that is contained in any bag for suitable solution, such as a bag for solution of a camera or a bag for multi-chamber solution. An example of a bag or container for multi-chamber solution is provided in U.S. Patent No. 5,431,496, the entire disclosure of which is incorporated herein by reference. As described herein, all patent applications and publications, including patent publications, are hereby incorporated by reference in their entirety. It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its relevant advantages. Therefore, such changes and modifications should be covered by the appended claims.

Claims (31)

1. CLAIMS 1. A method for manufacturing a solution for peritoneal dialysis, characterized in that it comprises: providing a glucose polymer; add a reagent to the glucose polymer, where the reagent is capable of reacting with a peptidoglycan; determine a proportion of the peptidoglycan; using the glucose polymer to prepare the solution for peritoneal dialysis if it is determined that a sufficiently low level of the peptidoglycan is present.
2. 2. The method of claim 1, characterized in that the reaction initiates a serine protease cascade.
3. 3. The method of claim 2, characterized in that the serine protease cascade includes a profenol oxidase cascade.
4. 4. The method of claim 1, characterized in that the reagent is derived from a plasma of silkworm larvae.
5. The method of claim 1, characterized in that the proportion of peptidoglycan is further determined by a colorimetric measurement in response to the reaction between the peptidoglycan and the reagent.
6. 6. The method of claim 1, characterized in that the sufficiently low level of the peptidoglycan is about 10 ng / ml or less.
7. The method of claim 1, characterized in that the solution based on a glucose polymer includes an icodextrin.
8. 8. The method of claim 7, characterized in that the reagent is added to the solution based on the glucose polymer.
9. The method of claim 1, further comprising the step of removing the peptidoglycan to provide the sufficiently low level thereof if it is determined that the sufficiently low level of the peptidoglycan is not present.
10. The method of claim 1, characterized in that the glucose polymer includes an icodextrin. eleven .
11. A method for delivering peritoneal dialysis to a patient, the method comprising the steps of: preparing a solution for peritoneal dialysis using a reagent to ensure that the solution for peritoneal dialysis has a sufficiently low level of peptidoglycan as to prevent peritonitis in the patient; and provide the solution for peritoneal dialysis to the patient.
12. The method of claim 1, wherein the sufficiently low level of the peptidoglycan includes about 10 ng / ml or less.
13. The method of claim 1, wherein the solution for peritoneal dialysis includes a solution based on a glucose polymer.
14. The method of claim 13, wherein the solution based on the glucose polymer includes an icodextrin.
15. 15. The method of claim 1, wherein the peritoneal dialysis is selected from the group consisting of automated peritoneal dialysis and continuous ambulatory peritoneal dialysis.
16. 16. The method of claim 1, wherein the patient is monitored to detect peritonitis during peritoneal dialysis.
17. The method of claim 16, wherein a dialysis effluent is collected from the patient to determine an IL-6 response that correlates with an incidence of peritonitis.
18. The method of claim 11, wherein the reagent is used to determine if the proportion of peptidoglycan exceeds about 10 ng / ml in the solution for peritoneal dialysis before use during peritoneal dialysis.
19. The method of claim 18, wherein the reagent is derived from a plasma of silkworm larvae.
20. 20. A method for testing in a solution for peritoneal dialysis the presence of a positive gram organism that exceeds a level sufficient to cause peritonitis, characterized in that it comprises: adding a reagent to the solution for peritoneal dialysis, where the reagent is capable of reacting with peptidoglycan to initiate a serine protease cascade; and determine the proportion of the peptidoglycan.
21. The method of claim 20, characterized in that the serine protease cascade includes a profenol oxidase cascade.
22. 22. The method of claim 21, characterized in that the reagent is derived from a plasma of silkworm larvae.
23. 23. The method of claim 20, characterized in that the solution for peritoneal dialysis includes a solution based on a glucose polymer.
24. The method of claim 23, characterized in that the solution based on a glucose polymer includes an icodextrin.
25. 25. The method of claim 23, characterized in that the reagent is added to a glucose polymer as raw material that is used to prepare the solution based on a glucose polymer.
26. 26. The method of claim 23, characterized in that the solution based on a glucose polymer is assayed to determine the proportion of peptidoglycan that exceeds about 10 ng / ml.
27. 27. A composition of a glucose polymer characterized in that it comprises a reagent that is capable of reacting with a peptidoglycan.
28. 28. The composition of a glucose polymer of claim 27, characterized in that the reagent is capable of reacting with the peptidoglycan to initiate a serine protease cascade.
29. 29. The composition of a glucose polymer of claim 28, characterized in that the serine protease cascade includes a profenol oxidase cascade.
30. 30. The composition of a glucose polymer of claim 27, characterized in that the reagent is derived from a plasma of silkworm larvae.
31. 31. The composition of a glucose polymer of claim 27, characterized in that the composition of a glucose polymer is an icodextrin.