MXPA97009003A - Cojugados de polimix - Google Patents

Abstract

Water-soluble conjugates of polymyxin B / dextran (or salts thereof), which have an activity against endotoxin greater than unconjugated polymyxin B. These may be prepared by reacting polymyxin B or a salt thereof with dextran in an aqueous medium, at a pH of about 9.3 to about 10, and at a temperature of about 30 ° C to about 35.

Description

CONJUGATES OF POLYMIXINE SUMMARY OF THE INVENTION The present invention relates to a process for producing water-soluble conjugates (or salts thereof) of polymyxin B (PMB) or salts thereof and dextran. which comprises reacting polymyxin B or a salt thereof with dextran in an aqueous medium, at a pH of about 9.3 to about 10, preferably about 9.5 to about 9.7, and at a temperature of about 30 ° to about 35. ° C, preferably of about 32 ° C. A variant of the preferred process involves reacting polymyxin B sulfate with oxidized dextran at a temperature of about 32 ° C, to covalently bind the polymyxin B molecule with the dextran to through an amine link. The improved conjugates (or salts thereof) prepared by the process of the present invention, can be reproduced in an easy and consistent manner, and exhibit significantly improved pharmacological properties, such as higher potency and higher endotoxin activity than the activity against the endotoxin of unconjugated polymixipa B. The invention also includes water soluble conjugates (or salts thereof) comprising polymyxin B or a salt thereof and dextran, the conjugates (or salts thereof) having an activity against endotoxin greater than the activity against endotoxin. of unconjugated polymyxin B Background of the Invention The medical necessity of an effective therapy for systemic inflammatory response syndrome (SIRS) and septic shock is widely recognized, and the patient base is large, requiring more than 100,000 critical cases of SIRS / sepsis in developed nations. , an acute therapy, and almost 1 million patients at high risk of sepsis who could benefit from prophylaxis. Endotoxin is recognized as the primary primary initiator / mediator in the pathogenesis of SIRS / sepsis, based on a large body of studies in humans and animals, thus providing a clear scientific rationalization for an approach against endotoxin for sepsis. Endotoxins or lipopolysaccharides are structural molecules derived from the cell walls of Gram-negative bacteria. When they enter the bloodstream, they can interfere with the regulation of body temperature and cause fever. They also have a toxic effect, which leads to heart, lung, and kidney failure. Endotoxin-related diseases are a leading cause of death among patients in intensive care units.

The ability of PMB to neutralize endotoxin is unique among antibiotics. which is accomplished by linking to the lipid A region of the endotoxin molecule. The PMB of Bacillus polymixa (B. aerosporus) is a highly charged cyclic amphiphilic peptidol. It is also useful to fight different fungal infections, especially those that occur in immunocompromised individuals. However, PMB has some properties that make it less than an ideal antibiotic. First, it has a short half-life in the body, which requires repeated dosages in order to be effective. Second, as it passes through the kidney, it can cause extensive damage. Third, in high doses, it has neurotoxic properties that cause respiratory paralysis. Previously, researchers have conjugated PMB with immobile or fixed molecules, see, for example, Issekutz, J. Immunol. Methods 61 (1983) 275-281, which describes the binding of PMB with Sepharose. These conjugates, although useful in purification techniques, are not suitable for therapeutic use in vivo. An approach to achieve pharmacological activity, a longer duration, or a reduced toxicity of the organ, has involved the conjugation of drugs with macromolecules of great molecular weight, such as dextran, polyethylene glycol, or polyvinyl pyrrolidine. However, attempts in this area of polymer conjugation have met with only limited success. For example, the conjugated form of procainamide (an antiarrhythmic drug) was less active and exhibited a shorter half-life than native procainamide (Schact et al., Ann N.Y. Acad Sci. 416 [1985] 199-211). In a similar manner, the prostaglandin analogue B245, bound to a carrier, was less effective (by several orders of magnitude) than the native molecule (Bamford et al., Bioch. Biophys. Acta 886 [1986] 109-118). Reductions in biological potency have also been described for the conjugated forms of kallikrein, aprotinin, bradykinin (Odya et al, Biochem Pharmacol 27 [1978] 173-179), daunorubicin antitumor drugs (Hurwitz et al., J. Appl. Biochem 2 [1980] 25-35), and mitomycin C (Taka ura et al., Cancer Res. 44 [1984] 2505-2510). The conjugated enzymes also suffer a reduction in biological activity due to steric hindrance and reduced accessibility of the substrate (Blomhoff et al., Biochem. Biophys. Acta 757 [1983] 202-208; Marshall et al., J. Biol. Chem. 251 (4) [1976] 1081-1087; R.L. Foster Experimentia 11 (7) [1975] 772-773; Wileman et al., J. Pharm. Pharmacol. 35 [1983] 762-765). However, there are some examples of improvements in the circulatory half-life after conjugation (Wileman, supra, Kaneo, Chem. Pharm, Bull. 22 (1) [1989] 218-220). It would be desirable to develop a form of PMB that stays in the bloodstream for longer, and / or that has no neuro- or nephro-toxicity at the therapeutic doses. Patent of the United States USP 5,177,059 Number and its equivalents in other countries, such as European Patent EP 428 486 Number, describe conjugates of polymyxin B with polysaccharides such as dextran or hydroxyethyl starch, proteins such as albumin, and polymers such such as polyvinyl pyrrolidone, polyethylene glycol, and polyvinyl alcohol. That patent specifically describes a process for the chemical conjugation of polymyxin B with dextran, by reacting the materials at room temperature (ie, at approximately 25 ° C or less), and at a pH of 8.5 to 9.0. The conjugates described in that patent are less toxic than the unconjugated polimixipa B, and have an activity against endotoxipa that is not clearly defined but appears to be less than or equal to that of unconjugated polymyxin B. The process of the present invention is an improvement over the process described in that patent, and provides conjugates (or salts thereof) of polymyxin B or a salt thereof, and dextran, which have, in a surprising manner, better characteristics comparing with the known PMB-dextran conjugates, in particular a higher activity against endotoxin than unconjugated polymyxin B, a higher potency, a better reproducibility, and better pharmacological properties. It is particularly surprising that the novel PMB-dextran conjugates (or salts thereof) of the present invention have, in a consistent and reproducible manner, a higher activity against endotoxin, than unconjugated PMB. Although the molecular basis for these unexpected novel features is not clearly understood, it is possible that, under the conditions of the reaction used with the present invention in the preparation of the conjugates, the pH change alters the distribution of the protons over the basic side chains. At a higher pH, one or more side chain amino groups may be available to bind to the dextran by means of reductive amination. Since each side chain amino group will have a characteristic pKa, the higher pH will release the same amine each time. If that amine provides a particularly favorable binding (from the point of view of conjugate potency), then a better conjugate form arises, because that particular binding path contributes more to the overall structure. This structure confers to the conjugates, in a consistently reproducible manner, a level of activity that is above the level of activity of the native MBP, eg, 125 percent or more, of the activity of the native MBP.

Detailed Description The present invention involves an improved process for producing a water-soluble conjugate (or a salt thereof) of polymyxin B and dextran, which is useful in the treatment of fungal and bacterial infections, and in the prevention of induced disease. by bacterial endotoxin. In a specific manner, the present invention relates to a process for producing a water-soluble conjugate of polymyxipa B / dextran (or a salt thereof), which comprises the reaction of polymyxin B or a salt thereof with dextran in an aqueous medium, at a pH of about 9.3 to about 10, and at a temperature of about 30 ° C to about 35 ° C. PMB is an antibiotic peptide and an approved pharmaceutical agent, with modest antibiotic activity. It has been in clinical use for more than 40 years for local and parenteral applications. The development of bacterial resistance to PMB is very rare. The PMB binds the endotoxin with an affinity of about 10%. M, and can neutralize the biological effects of endotoxin against all clinically important Gram-negative bacteria in a large number of models in vitro and in vivo. In vitro, it has been used as a standard to neutralize the endotoxipa. Furthermore, in vivo, PMB protects against the pathology of bacterial sepsis in many animal models, such as acidosis and hypotension in enterobacterial infections, and lethality due to gram-negative sepsis in dogs, rabbits, rats, and mice. It also has a potential to prevent sepsis in humans, such as burn patients. The PMB used for the preparation of the conjugate of the present invention is commercially available. The PMB can be prepared by fermentation of Bacillus polymixa (Prazmowski) Migula. PMB consists of a mixture of several related decapeptides. The PMB, or a pharmaceutically acceptable salt thereof, is useful in this invention (such as polymyxin B sulfate and the like). The dextran used in the process of the present invention can be any of the conventional pharmaceutically acceptable dextrans. Preferably, the dextran is chemically modified to bind in a covalent manner with peptides, such as to be oxidized by an oxidizing agent, for example, sodium periodate. The dextran preferably should have a weight average molecular weight of from about 25,000 to about 500,000; more preferably, from about 50,000 to about 200,000; and most preferably from about 63,000 to about 76,000. The molecular weight can be determined, for example, by high performance liquid chromatography with gel permeation. The most preferred dextran is prepared by fermentation of Leuconostoc mesenteroides (NRRL B-512). This is composed of glucose units that are a [1, 6] linked in a long linear chain with about 5 percent branching to [l-3]. Of the branched chains, approximately 85 percent have one to two glucose units, and the remaining 15 percent an average of 33 units.

General procedures for the preparation of conjugates of PMB and dextran in aqueous media are described in U.S. Patent No. USP 5,177,059, and are applicable to the present invention. In a preferred embodiment of the present invention, partially oxidized dextran is prepared by reaction with an oxidizing agent, such as sodium periodate (NaT.04). This treatment creates aldehydes by means of oxidative dissociation of the vicinal diols on the glucose monomers. By exposing the partially oxidized dextran to the PMB or to a salt thereof, Schiff bases are formed. The production of conjugates within the scope of the present invention requires careful control of the pH in from about 9.3 to about 10., preferably from about 9.5 to about 10, and most preferably from about 9.5 to about 9.7, during the reaction of PMB and dextran, especially during the Schiff base formation step. The pH is preferably maintained using a borate regulator, especially one comprising tetrabora or sodium. Also, the temperature of the aqueous medium is maintained at from about 30 ° C to about 35 ° C, more preferably at about 32 ° C, while the PMB or the salt thereof and the dextran are reacted. The subsequent introduction of sodium borohydride (NaBH) reduces the Schiff bases and the remaining aldehydes to provide stable covalent bonds, preferably through one or more amine bonds, between the PMB and the dextran. The conjugate is preferably purified by ultrafiltration, such as using a 10,000 molecular weight cut-off membrane, to remove residual PMB, inorganic byproducts (eg, borates), and potential low molecular weight degradation products. The pH of the aqueous medium is preferably adjusted to about 5 to 7, before purification. Accordingly, a preferred embodiment of the process of the present invention comprises: a) preparing partially oxidized dextran by the reaction of dextran with sodium periodate.; b) reacting polymyxin B or a salt thereof with the resulting partially oxidized dextran in an aqueous release, at a pH of about 9.3 to about 1C, and at a temperature of about 30 ° C to about 35 ° C; c) adding sodium borohydride to the resulting aqueous medium; and d) purifying the resulting conjugate of polymyxin B-dextran. The invention also includes water soluble conjugates (or pharmaceutically acceptable salts of the same polymyxin B or a pharmaceutically acceptable salt of the same and dextran, the conjugates having an activity against the endotoxin greater than the activity against the endotoxin of the polymyxin B not conjugated, for use in the prophylactic or curative treatment of the systemic inflammatory response syndrome and septic shock Also includes conjugates (or pharmaceutically acceptable salts thereof) such as can be obtained by the process defined above, and those conjugates (and pharmaceutically salts) acceptable thereof) provided that they are obtained by the process described above, pharmaceutical compositions comprising a water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a pharmaceutically acceptable salt thereof and dextran as defined above , along with less a pharmaceutically acceptable vehicle or diluent; and the use of these conjugates (or pharmaceutically acceptable salts thereof) in the preparation of a medicament for use in the prophylactic or curative treatment of the systemic inflammatory response syndrome and septic shock. In the process of the present invention, as well as in the conjugates produced therefrom, the molecular ratio of PMB or a salt thereof to the dextran, is from about 1:15 to about 200: 1; more preferably from about 1: 2 to about 1: 5; most preferably of about 1.5: 5. A salt of polymyxin B is preferably a pharmaceutically acceptable salt of PMB. A conjugate should be seen as soluble in water if it has a solubility in water of approximately 25 milligrams / milliliter or greater at 20 ° C; preferably about 50 milligrams / milliliter or greater: more preferably about 60 milligrams / milliliter or greater; and most preferably about 65 milligrams / milliliter or greater, at 20 ° C. The conjugates of PMB-dextran (and salts thereof) prepared according to the present invention, can be used in a manner consistent with the use of PMB itself, that is, they can be used alone, for example, as an antibiotic for bacterial or fungal infections, or combined with other bactericidal and / or anti-inflammatory agents. These can be administered in any of the ways by which the native PMB is conventionally administered, for example, intramuscularly, intravenously, intrathecally, subcopjuptivamente, or local epte. Accordingly, formulations for intramuscular injections usually comprise an effective amount of PMB-dextran conjugate (or a pharmaceutically acceptable salt thereof) in sterile water, physiological saline, or procaine.HCl at about 1 percent. Intravenous formulations usually comprise an effective amount of the conjugate in 5 percent dextrose and sterile water. Intrathecal formulations usually comprise an effective amount of the conjugate in physiological saline. For local ophthalmic use, an effective amount can be mixed with water or saline, and optionally glycerin, and cupric sulfate in eye drops, or it can be made in an ointment or suspension. Creams for local applications, especially for burned areas, typically comprise an effective amount of PMB-dextran conjugate (or a pharmaceutically acceptable salt thereof) on a base of inactive ingredients such as liquid petrolatum, propylene, polyoxyethylene, polyoxypropylene, and emulsifying wax. The purified PMB-dextran conjugates (or the pharmaceutically acceptable salts thereof) of the present invention are essentially non-toxic. The amount of conjugate of the present invention to be used can be determined based on the amount of native PMB that would normally be prescribed for a particular patient, taking into account such factors as the condition being treated and the age and patient's weight, and the activity of the particular conjugate used. A reduction in the dosage of half or more can be made, compared to the native PMB, due to the greater effective activity of the conjugate, its reduced toxicity, and its longer duration of activity. The molecular weight of the conjugates of the present invention can be determined, for example, using high performance liquid chromatography by size exclusion (CLAR-CET, TSK-GEL G4000 PWxl eluted with 40 mM phosphate buffer, at a pH of 4.4, detected with low angle laser light scattering (with reference to a dextran standard having a weight average molecular weight (MW) of 79,800.) The conjugates of the present invention preferably have a weight average molecular weight ( MW) from approximately 55,000 to approximately 80,000; more preferably from about 60,000 to about 80,000; and most preferably from about 63,000 to about 76,000. The improved conjugates of the present invention have a better reproducibility over the conjugates of the prior art, and have an activity against the endotoxin greater than the activity against the endotoxin of the unconjugated polymyxin B. The conjugates of the present invention have a better activity against endotoxin, that is, the ratio of the IC 50 of the polymyxin B unconjugated to the IC 50 of the conjugates of the invention is greater than 1.0; preferably greater than about 1.25 or 1.3; and most preferably greater than about 1.4. As used herein, IC50 means the concentration of polymyxin B measured by the carbocyanine dye test which abrogates 50 percent of the absorbance change of the carbocyanin dye at 450 nanometers in the presence of 0.05 milligrams of E. endotoxin. coli As used herein, the activity (%) is defined as follows: IC50 of unconjugated polymyxin B × 100 IC50 of polymyxin B conjugate / dex rano The carbocyanine dye test is an in vitro biochemical assay used to measure the endotoxin properties of polymyxin B and the conjugates of the present invention. This assay is modified from previous reports (Oga and Kanoh, Microbiol, Immunol, 28 [1984] 1313-1323, Zey and Jackson, Applied Microbiol, 26. [1973] 129-133), and optimized for use in plates. of microtitration. In this assay, the unconjugated conjugate or PMB (positive control) competes with the carbocyanin dye (a small cationic compound that changes in spectrophotometric absorbency when bound to endotoxin) by the endotoxin. The preparation of reagents for the carbocyanin dye test is as follows: 1) a solution of 0.15 M sodium acetate and 0.15 M acetic acid solution is prepared. The two are combined (40.2 milliliters of 0.15 M sodium acetate and 159.8 milliliters of 0.15 M acetic acid) to make a 0.03 M acetate buffer; 2) 8.0 milligrams of carbocyanine dye are dissolved in 12.5 milliliters of ethyl alcohol. This is diluted with 37.5 milliliters of acetate buffer. This is the final dye solution at 160 micrograms / milliliter in 25 percent ethanol. It is kept on ice, in the dark, and mixed for at least 30 minutes; 3) serial dilutions of PMB or of each conjugate are prepared in a 40 mM phosphate buffer (pH = 7.4). Each dilution is 6 times greater than its final test concentration; 4) After sonification, 0.111: B of E. coli endotoxin is diluted to 0.5 milligrams / milliliter of 0.9 percent serum of a concentration of a pre-prepared frozen supply of 1.0 milligram / milliliter of 0.9 percent serum . Plates are placed, and the test is carried out as follows, with the addition of samples or regulator to the microtiter cavities first: a) control: 100 microliters of serum and 50 microliters of 40 mM phosphate buffer (pH = 7.4) in triplicate; b) positive control: 100 microliters of endotoxin and 50 microliters of 40 mM phosphate buffer (pH = 7.4) in triplicate; c) samples of PMB: 100 microliters of endotoxin and 50 microliters of samples of PMB, in triplicate; d) Negative controls: 100 microliters of serum and 50 microliters of conjugate samples, in triplicate; e) experimental: 100 microliters of endotoxin and 50 microliters of conjugate samples, in triplicate. The plates are centrifuged for several minutes. 150 microliters of dye are added to each cavity, and the plate is wrapped in foil and kept on ice and in the dark for 60 minutes. The cavities are mixed with a multi-channel pipette, and read immediately at 450 nanometers in a microtiter plate reader. Reed-Muench calculations are performed for the estimation of the IC50. The invention is illustrated by the following non-limiting examples. The temperatures are in degrees Celsius.

Example 1 A) Oxidation of dextran A 5-liter four-necked round bottom flask is charged with 2.31 liters of water for injection and 150 grams of dextran. The mixture is stirred at 21 ° C to 24 ° C, at about 140 rpm, to effect dissolution. A solution of 3.9 grams of sodium periodate in 60 milliliters of water for injection is added (moderate exotherm, approximately Io C). The addition funnel is rinsed with 30 milliliters of water for injection. The resulting mixture is stored at 21 ° C-24 ° C for 1 hour. The solution is vacuum filtered through a 90 millimeter Corning cellulose acetate membrane filter of 0.22 microns, and heated to 30 ° C-32 ° C, and maintained at that temperature.

B) Preparation of polymyxin B sulphate solution / borate regulator This preparation is started 45 minutes before the oxidation of dextran, filtration, and heating of A) above is finished. A four-neck round bottom flask, of 12 liters, is charged with 3.00 liters of borate regulator at a pH of 9.7, and regulator temperature is adjusted to 32 ° C. Next, 45 grams of polymyxin B sulfate are added, and the resulting suspension is stirred at 32 ° C for 45 minutes. The pH of the mixture is measured (initially 9.4) and adjusted to a pH of 9.7 at 32 ° C by the addition of 30 milliliters of 5N NaOH. The borate regulator is prepared as follows: a 5-liter four-necked round bottom flask, heat treated, is charged with 114.3 grams of sodium tetraborate decahydrate and 2,906 liters (2.90 kilograms) of water for injection. The solution is heated to 32 ° C, and the pH is adjusted to 9.7 (initially 9.3) by the addition of 37 milliliters of 5N NaOH. The solution is vacuum filtered through a 90 millimeter Corning cellulose acetate sterile membrane filter and stored.

C) Reaction of polymyxin B sulfate with oxidized dextran The oxidized dextran solution previously heated (30 ° C-32 ° C) (from part A) is added as quickly as possible to the stirred mixture of polymyxin sulfate B in borate regulator at 32 ° C (from part B). After the addition is complete, the pH of the mixture is measured (initially 9.5), and adjusted to a pH of 9.7 by the addition of 18 milliliters of 5 N NaOH. The mixture is stirred at 32 ° C for 1 hour . To the heterogeneous mixture, a solution of 3.6 grams of sodium borohydride in 50 milliliters of water for injection is added. The mixture is stirred at 32 ° C for 2 hours. Another fresh solution of 3.6 grams of sodium borohydride in 50 milliliters of water for injection is added to the mixture. The mixture is stirred for 2 hours at 32 ° C. To the mixture is added a solution of 3.6 grams of sodium borohydride in 50 milliliters of water. The heating is removed, and the reaction mixture is allowed to cool to room temperature with stirring for 14 hours. The pH of the reaction mixture is measured (10.0), and adjusted to a pH of 5.7 by the addition of 1.27 liters of IN HCl. The resulting mixture is stirred for 10 minutes after acidification, and filtered under vacuum through a sterile 0.22 micron Corning cellulose acetate membrane filter to yield 6.81 liters of a conjugate solution of polymyxin B-dextran. , which is saved for purification.

D) Purification by ultrafiltration The solution of the polymyxin B-dextran conjugate (from part C) is purified using a previously treated Amicon CH2PRS Ultrafiltration Unit, with S1Y10 membrane cartridges. The purified product is removed from the ultrafiltration unit, and stored frozen at -25 ° C. The analysis of the conjugate obtained showed that the product has the following characteristics: IC50 = 16.8 micrograms / milliliter activity = 124.7 percent dextran content = 65.37 milligrams / milliliter total content of polymyxin B = 2.47 milligrams / milliliter content of free polymyxin B = 0.65 percent ratio of PMB: dextran = 37.71 milligrams of PMB / gram of dextran It appears that, at a reaction temperature of 32 ° C and a pH of 9.5, the product obtained has an activity that is approximately 125 percent of the activity of the unconjugated PMB.

Example 2: The process of Example 1 is repeated, and produces a polymyxin B-dextran conjugate having the following characteristics: IC50 = 16.4 micrograms / milliliter activity = 125.8 percent dextran content = 65.30 milligrams / milliliter total content of polymyxin B = 2.49 milligrams / milliliter content of free polymyxin B = 0.67 percent ratio of PMB: dextran = 38.06 milligrams of PMB / gram of dextran.

Again, at a reaction temperature of 32 ° C and a pH of 9.5, the conjugate obtained has approximately 126 percent activity, compared to unconjugated PMB.

Example 3; Polymyxin B / dextran conjugates are prepared according to the process of Example 1 (except as indicated), to determine the effects of the reaction temperature between PMB and dextran at a pH of 9.7. The payload is determined (milligrams of PMB / gram of dextran), the average IC50 (micrograms / milliliter), and the activity (%), and the results are presented in Table 1: Table 1; Effect of temperature at a pH of 9.7 ¡10 grams of PMB; 0.65 grams of NaI04; 50 grams of dextran) From Example 3, it can be seen that, when the reaction temperature is lowered to 10 ° C, at a pH of 9.7, the product obtained has a reduced activity. Example 4; Polymyxin B / dexane conjugates are prepared according to the process of Example 1 (except as indicated), to determine the effect of the pH of the reaction between PMB and dextran. The payload (milligrams of PMB / gram of dextran), the average IC50 (micrograms / milliliter), and activity (%) are determined as in Example 3, and the results are presented in Table 2: Table 2; Effect of PH at a temperature of 32 ° C (15 grams of PMB, 1.3 grams of NaI04, 50 grams of dextran) a) acetate regulator; b) phosphate regulator; c) borate regulator; ND: not determined (the samples are too diluted to determine these parameters).

The data in Table 2 show that decreasing the temperature of the reaction to 32 ° C while using a pH between 9.5 and 9.7, still slows down the production of the conjugate having an activity that is higher than that of the Unbound PMB (Examples 41, 4n, and 4o), while increasing the pH further, to a pH of 10, is not particularly beneficial (Example 4p). Example 5: Polymyxin B / dextran conjugates are prepared as in Example 1 (except as indicated) and their activity against endotoxin is measured. The results are presented in the following Table 3: Table 3: Effect of the amount of PMB (1.3 grams of NaI04, pH of 9.7, 32 ° C) The results of Table 3 further confirm that the optimum conditions reside at a reaction temperature of about 32 ° C, and at a pH of about 9.5 to about 9.7. After all, the data of Examples 1 to 5 thus show that the use of a reaction temperature of about 30 ° C to about 35 ° C, preferably about 32 ° C, and a pH of about 9.3 to about 10, preferably from about 9.5 to about 9.7, results in the improved conjugates of the present invention.

Claims (36)

1. A water-soluble conjugate (or a salt thereof) comprising polymyxin B or a salt thereof and dextran, the conjugate having an activity against endotoxin greater than the activity against endotoxin of unconjugated polymyxin B, ie, where the ratio of the IC 50 of polymyxin B unconjugated to the IC 50 of the conjugate is greater than 1.0.

The conjugate (or a salt thereof) of claim 1, wherein the ratio is greater than about 1.25 or 1.3.

3. The conjugate (or a salt thereof) of claim 1, wherein the ratio is greater than about 1.4.

4. The conjugate (or a salt thereof) of claim 1, wherein the activity against endotoxin is measured by a carbocyanine dye test, wherein the IC50 of the conjugate is measured as the concentration of the conjugate that abrogates the conjugate. percent of the absorbance change of the carbocyanin dye at 450 nanometers in the presence of 0.05 milligrams of E. coli endotoxin per milliliter of 0.9 percent serum.

The conjugate (or a salt thereof) of any of claims 1 to 4, wherein the molecular ratio of the polymyxin B or a salt thereof to the dextran is from about 1:15 to about 200: 1.

6. The conjugate (or a salt thereof) of any of claims 1 to 4, wherein the molecular ratio of the polymyxin B or a salt thereof to the dextran is from about 1: 2 to about 1: 5.

The conjugate (or a salt thereof) of any of claims 1 to 4, wherein the dextran has a weight average molecular weight of from about 25,000 to about 500,000.

8. The conjugate (or a salt thereof) ) of any of claims 1 to 4, wherein the dextran has a weight average molecular weight of from about 50,000 to about 200,000.

The conjugate (or salt thereof) of any of claims 1 to 4, wherein the dextran has a weight average molecular weight of about 63,000 to about 76,000.

10. The conjugate (or a salt thereof) ) of any of claims 1 to 4, which has a weight average molecular weight of from about 55,000 to about 80,000.

11. The conjugate (or a salt thereof) of any of claims 1 to 4, which has a weight average molecular weight of about 60,000 to about 80,000.

12. The conjugate (or a salt thereof) of any of claims 1 to 4, which has a weight average molecular weight of from about 63,000 to about 76,000.

13. A process for producing a water-soluble conjugate (or a salt thereof) of polymyxin B or a salt thereof and dextran, which comprises reacting polymyxin B or a salt thereof with dextran in an aqueous medium, at a pH of about 9.3 to about 10, and at a temperature of about 30 ° C to about 35 ° C.

The process of claim 13, wherein the pH is from about 9.5 to about 9.7.

15. The process of claim 13, wherein the temperature is about 32 ° C.

The process of claim 13, wherein the reaction of polymyxin B or a salt thereof with dextran comprises covalently linking polymyxin B or a salt thereof with dextran through one or more amine bonds.

The process of claim 13, wherein the polymyxin B salt is polymyxin B sulfate.

18. The process of claim 13, wherein the molecular ratio of the polymyxin B or a salt thereof to the dextran is about 1:15 to about 200: 1.

19. The process of claim 13, wherein the molecular ratio of the polymyxin B or a salt thereof to the dextran is from about 1: 2 to about 1: 5.

The process of claim 13, wherein the molecular ratio of the polymyxin B or a salt thereof to the dextran is about 1.5: 5.

21. The process of claim 13, wherein the dextran has a weight average molecular weight of from about 25,000 to about 500,000.

22. The process of claim 13, wherein the dextran has a weight average molecular weight of from about 50,000 to about 200,000.

The process of claim 13, wherein the dextran has a weight average molecular weight of from about 63,000 to about 76,000.

24. The process of claim 13, wherein the dextran has been partially oxidized.

25. The process of claim 24, wherein the dextran has been partially oxidized using sodium periodate.

26. The process of claim 13 wherein the pH is maintained using a borate regulator.

The process of claim 26, wherein the borate regulator comprises sodium tetraborate.

28. The process of claim 13, which comprises the subsequent reaction with sodium borohydride.

29. The process of claim 13, which further comprises adjusting subsequently the pH of the aqueous medium to about 5 to 7, and purifying the resulting conjugate.

30. The process of claim 29, wherein the conjugate is purified by ultrafiltration.

31. A process according to claim 13, for producing a water-soluble conjugate (or a salt thereof) of polymyxin B or a salt thereof and dextran, which comprises: a) preparing partially oxidized dextran by the reaction of dextran with sodium periodate; b) reacting polymyxin B or a salt thereof with the resulting partially oxidized dextran in an aqueous medium, at a pH of about 9.3 to about 10, and at a temperature of about 30 ° C to about 35 ° C; c) adding sodium borohydride to the resulting aqueous medium; and d) purifying the resulting conjugate.

32. A pharmaceutical composition comprising a water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a pharmaceutically acceptable salt thereof, and dextran, the conjugate having an activity against endotoxin as defined in claim 1 , together with at least one pharmaceutically acceptable carrier or diluent.

33. A water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a pharmaceutically acceptable salt thereof and dextran, the conjugate having an activity against endotoxin as defined in claim 1, for use in the treatment prophylactic or curative of systemic inflammatory response syndrome or septic shock.

34. A water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a salt thereof and dextran as defined in claim 1, obtainable by the process of claim 13 or 31.

35 A water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a salt thereof and dextran as defined in claim 1, provided that it is obtained by the process of claim 13 or 31.

36. The use of a water-soluble conjugate (or a pharmaceutically acceptable salt thereof) of polymyxin B or a pharmaceutically acceptable salt thereof and dextran, the conjugate (or salt thereof) having an activity against endotoxin as defined in claim 1, in the preparation of a medicament for use in the prophylactic or curative treatment of systemic inflammatory response syndrome or septic shock. pRgj r Tew Water-soluble conjugates of polymyxin B / dextran (or salts thereof), which have an activity against endotoxin greater than unconjugated polymyxin B. These can be prepared by reacting polymyxin B or a salt thereof with dextran in an aqueous medium, at a pH of about 9.3 to about 10, and at a temperature of about 30 ° C to about 35 ° C. * * * * *