WO1996007420A1

WO1996007420A1 - Macrophage stimulating composition comprising a non-ionic surfactant

Info

Publication number: WO1996007420A1
Application number: PCT/GB1995/002077
Authority: WO
Inventors: Seyed Moein Moghimi; Stanley Stewart Davis
Original assignee: University of Nottingham
Current assignee: University of Nottingham
Priority date: 1994-09-03
Filing date: 1995-09-04
Publication date: 1996-03-14
Anticipated expiration: 1997-03-03
Also published as: GB9417742D0; AU3394095A

Abstract

A non-ionic surfactant, such as a poloxamer, poloxamine, or polyethylene glycol, is used to stimulate the phagocytic activity of macrophages and thereby enhance their anti-infective role in the immune system.

Description

MACROPHAGE STIMULATING COMPOSITION COMPRISING A NON-IONIC SURFACTANT

Mononuclear phagocytes (macrophages) are widely distributed in the body, being present in blood, bone marrow, connective tissue, liver, lungs, lymphoid tissue, nervous tissue and serous cavities. These cells constitute an important part of the defence mechanism of the body (the reticuloendothelial system, RES) by clearing the blood, the lymph and other tissues of particulate pollutants, for instance microorganisms and damaged body constituents.

The RES also participates in a myriad of other host functions such as secretion of numerous biochemical products that affect a wide range of functions (complement components, coagulation factors, colony stimulating factors, enzymes like lysozyme and elastase, enzyme inhibitors such as plasmin, serum amyloid A and P factors, etc.), determining the host response to he orrhagic, traumatic, septic and endotoxin shock, burns, surgery, X-irradiation, prolonged tissue ischemia, response to drugs, bacteria and viral infections, tumour growth, atherosclerosis and immunological depression (reviewed by Altura, B. M. , 1980, Adv. Microcirc lation 9, 252).

The hepatic Kupffer cells manifest 80-90% of total reticuloendothelial activity, and thus a direct or an indirect depression of Kupffer cell function will have a significant effect on the defence mechanisms of the body. Factors that depress Kupffer cell function, particularly phagocytic function, include age, traumatic shock, sepsis, neoplastic diseases, X- irradiation. aflatoxin B, alcohol, diabetes, obstructive jaundice, etc. [reviewed by Jones, E. A. and Summerfield. J. A. In: The Liver - Biology and Pathobiology, (Arias, I. M., Jakoby, W. B. , Popper, H., Schachter. D. and Shafritz, D. A., eds.) Raven Press, New York, pp 683- 704].

Efforts have been directed to develop systems that can stimulate the phagocytic function of the RES, particularly the Kupffer cells. Factors or signals that activate Kupffer cells, particularly increased phagocytic function, respiratory burst activity and release of lysosomal enzymes, include zymosan, glucan (soluble and paniculate forms), estrogen, colony- stimulating factor, lymphokines, somatostatin, Corynebacterium parvum with or without phorbol myristate, endotoxin, bacillus Calmette-Guerin, hydroxyethyl starch, partial hepatectomy and 3-palmitoyl-(-)-)-catechin. Some of these agents cause Kupffer cell proliferation and some will cause enhanced expression of certain plasma membrane receptors [reviewed by Jones, E. A. and Summerfield, J.A. In: The Liver - Biology and Pathobiology, (Arias, I. M., Jakoby, W. B., Popper, H., Schachter, D. and Shafritz, D. A., eds). Raven Press, New York, pp 683-704; de Villiers, W. J. S. et al. 1994, J. Exp. Med. 180, 705]. Agents that stimulate Kupffer cells increase their ability to mediate cytotoxicity against tumour cells and their antibacterial properties (Nomoto, K. et al. 1985, J. Gin. Lab. Immunol. 17, 91 ; Xu, Z. and Fidler, 1985, Cancer Immunol. 18, 118). Increased respiratory burst activity induced by agents that stimulate Kupffer cells is usually characterized by glucose oxidation via the hexose monophosphate shunt, luminol-dependent chemiluminescence and production of superoxide oxygen and hydrogen peroxide.

Some factors which stimulate Kupffer cell phagocytic function also exert radioprotective activity (usually via production of cytokines). These include a genetically engineered (atypical) β-glucan from a variant of S. cerevisiae (Alpha Beta Technology, WO9103248-A. 1991), polysaccharides from plant sources, for example extracts of ginseng root (Takeda, A. et al. 1981 , J. Radiat. Res., 22, 323), Thuja occidemale (Deutsche Wissenschaft, EP-315182-A, 1989) and the Compositae family (Wagner, H., DE3042491 , 1982), various monosaccharides such as 6-thio- D-fructose (Purdue Research Foundation, US4420489, 1983), N-decanoyl- D-glucosamine (GIRPI, DE2708667, 1978) and acylated monosaccharides (Sandoz, DE3941078, 1991) and endotoxin-free bacterial extracts such as, Picibanil (Chugai Pharmaceuticals), Biostim (Roussel), Broncho- vaxom (OM Labs) and trehalose dimycolate (RIBI Immunochem Research) (Patchen, M.L. et al. 1988, Comments Toxicol. 2, 217; Fedorocko, P. et al., 1992, Int. J. Radiat. Bioi , 61, 511 ; Madonna. G. S. , et al. , 1989, Infect. Immunity 57. 2495).

We have now surprisingly found that administration of a substantially endotoxin-free solution of non-ionic surfactants can stimulate the activity of the reticuloendothelial system. The present invention therefore provides the use of a substantially endotoxin-free non-ionic surfactant having a molecular weight of 1200 or greater in the manufacture of a medicament for stimulating the phagocytic function of macrophages of the reticuloendothelial system.

By "endotoxin-free" we mean that the composition is not toxic when administered in beneficial doses to humans. Preferably the endotoxin level is of OJng/ml or below (for example when measured using the Endotec Kit (ICN Biomedical, UK)).

The non-ionic surfactant is preferably a polymeric material containing polyethylene oxide or polypropylene oxide or a combination of both subunits at any molar ratio, or contains polyethylene glycol.

The non-ionic surfactant may be provided as a conjugate. It is preferably coupled to another biologically active material, such as a protein, peptide, sugar or phospholipid. However, the non-surfactant part of the conjugate need not itself be immunologically active and, in particular, need not be an immunoglobulin. If the conjugate is itself a non-ionic surfactant then clearly it can be used as it is. However, the invention also encompasses conjugates or derivatives which are not themselves non-ionic surfactants but which are, for example, metabolized in the body to form or release a non-ionic surfactant following administration. In such a conjugate, it is preferably the surfactant part which has a m.w. of 1200 or greater.

Preferred non-ionic surfactants are poloxamers and poloxamines with a molecular weight of 1200 or more, the Pluronic and Tetronic block co- polymers (Registered Trademarks, BASF, USA). Especially preferred materials are Pluronic L10, L35 (poloxamer 105), F38 (poloxamer 108), L42, L43, L44 (poloxamer 124), L61 (poloxamer 181), L62 (poloxamer 182), L63 (poloxamer 183), L64 (poloxamer 184), P65, L72, P75, F77, L81, L101, L121 (poloxamer 401), L122 (poloxamer 402), F127 (poloxamer 407); poloxamines 304, 504; 701, 702, 704, 901 , 904, 908, 1304, 1307. Poloxamines are preferred, especially poloxamine-908. Also preferred are polyethylene glycols of molecular weight greater than 1200. Preferred polyoxyethylene-comprising conjugates of surfactants include Tweens 40, 60 and 80 (poloxyethylene sorbitan mono-palmitate, -stearate and -oleate, respectively). The molecular weight of the non-ionic surfactant is preferably at least 3000, more preferably at least 5000 or 10000.

Preferred non-ionic surfactants having a molecular weight of 1200 or greater are Pluronics L35, L42, L43, L44, L61, L62, L63, L64, Tetronic 304 and Tween 80. Preferred surfactants having a molecular weight of 3000 or greater are Pluronics F68, F38, F65, P84, P85, F87 and Tetronics 904, 908, 1304, 1307 and 1504. Activation can be achieved by a single administration of 0.3 g/kg body weight or higher of the non-ionic surfactant. The dose will generally be chosen to cause stimulation of the phagocytic function of macrophages within 4 days after administration. The effect can last up to 10 days after administration. Preferably the surfactant is administered at 3 mg/kg body weight, more preferably 6 or 18.5 mg/kg body weight. The activation of the RES has been found to be obtained three days after intravenous administration of the polymer solution. The activation is defined as enhanced phagocytosis by macrophages of the RES, particularly the Kupffer cells. The activation is achieved either directly on cells or via cytokine release (hence the polymer may exert radioprotective activity) and may cause proliferation of Kupffer cells and up-regulate certain Kupffer cell plasma membrane antigens and receptors such as ED2 and scavenger receptor respectively. Similar effects may occur on other macrophages (lymph nodes, spleen, etc).

The surfactant is preferably administered by intravenous administration, but may also be administered by other parenteral routes such as subcutaneous, intramuscular and intraperitoneal, or non parenteral routes such as orally.

For parenteral routes of administration, the surfactant can be administered in water, saline, or any other non-toxic solution containing electrolytes, for example dextrose or other sugars, as will be appreciated by those skilled in the art. For oral administration the surfactant may be administered in a solid or liquid dosage form made with standard components known to those skilled in the art.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules. -cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the additior of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The enhanced phagocytic activity of macrophages following administration of the surfactant can be demonstrated by measuring clearance of "phagocyte-resistant" substrates from the blood. Such substrates have been previously described. They include certain poloxamer and poloxamine coated polystyrene and gold particles (Ilium, L., US4904479, Davis, S. S. et al. WO9402122,; Moghimi, S. M. et al. 1983, Biochem. Biophys. Acta 1179, 157) and other sterically-stabilized colloids such as polyethyleneglycol-coated liposomes, etc. The size of the test-substrate is below 100 nm in diameter which under normal conditions is not cleared effectively from the blood by organs of the RES. By "not cleared", we mean that there is a total liver uptake of test-substrate of not more than 10-15 % of the administered dose within 3 h after intravenous administration or 2 h after subcutaneous administration by the liver in normal control subjects. In test subjects the following should preferably be achieved:

1. Administration of the endotoxin-free polymer should stimulate the RES within 3 to 4 days;

2. At day 3 or 4 "phagocyte-resistant" substrate is injected intravenously, within 3 h at least 25% of the administered dose is cleared by the liver (compared to less than 10-15% of the administered dose in control subjects) and 4-5 % of the dose by the spleen (compared to less than 3 % of the administered dose in the control subjects);

3. Both liver and spleen should retain their ability to efficiently remove "phagocyte-resistant" particles up to 10 days after administration of the polymer.

It has been found that injection of poloxamer- 188 may have undesirable effects on complement hemolytic activity. The non-ionic surfactant used in the invention should therefore preferably have minimal effect on the activation of human serum complement activity and preferably should give a residual total complement hemolytic activity of surfactant-treated human serum of 50% or less than that of the residual total complement activity of poloxamer- 188 treated human serum when measured by the method described by Devine, C.V. et al. in 1994 Biochem. Biophys. Acta. 1191, 43.

The compositions of the invention may be used to enhance the phagocytic activity of macrophages of the RES in any patient who requires a strengthened immune system, for example elderly, malnourished or diabetic patients or patients with compromised immunity because of other illnesses, for example AIDS, and those given immuno-suppressive agents such as cyclosporin. The compositions may also be administered 3-10 days before surgery, especially abdominal surgery, to reduce the risk of post-operative infection. The compositions can also be used to prevent or treat infections, whether viral, bacterial, fungal, protozoal or parasitic, as an alternative to, or as well as, treatment with other anti-infective agents.

Other expected pharmacological effects of the compositions of the invention include: interfering with the clotting mechanism or reducing the blood viscosity; decreasing the incidence of tumour metastases; reducing the endothelial adherence of tumour cells and improving the rheology of the sickle erythrocytes; and radioprotective activity.

Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawing, Figure 1 , which shows the effect of poloxamer-188 and poloxamine-908 on residual total human complement hemolytic activity. Example 1. Removal of any endotoxin associated with polymer solutions was achieved using polymyxin-B-agarose as described in Moghimi. S. M. et al.. 1993 Biochim. Biophys. Acta 1157, 233). Briefly, solutions of non- ionic surfactants (2.0% w/v) were passed ten times through a column of polymyxin-B-agarose. The procedure was repeated to ensure complete removal of endotoxins employing a new column. The systems were then tested for the presence of endotoxin using Endotec Kit (ICN Biomedical. UK). The kit is based on the Limulus lysate reaction with the endotoxin (the sensitivity is 0.06 - 0J ng/ml). The residual total complement activity of poloxamine-908 was compared to that of poloxamer- 188 treated human serum according to established methods, using sheep erythrocyte stroma sensitized with rabbit antibody as described in detail elsewhere (Devine, D. V. et al., 1994, Biochim. Biophys. Acta 1191, 43). The results are presented in Figure 1. The human serum must have a CH^, (complement half-life of 50%) of 200-220 units (1 in 30 dilution). Serum was serially diluted to obtain an optimal concentration for the detection of changes in complement levels. The residual complement activity is expressed as a percentage of the hemolytic activity of human serum diluted with the standard DGNB²⁺ buffer (isotonic veronal buffered saline). The results show minimum activation of complement by poloxamine-908 when compared to that of poloxamer- 188 (50% or less than that of poloxamer- 188 up to 0J2 μmole of polymer).

Example 2. Groups of three male Wistar rats (body weight 150+ 10 g) were injected intravenously with 18.5 mg/kg of body weight of endotoxin- free poloxamine-908 1.0% (w/v) via the lateral tail vein. Control animals received intravenous administration of sterile saline. At 3 h and then at daily intervals, all animals received an intravenous dose of poloxamine- 908 coated [^I25I]-radiolabelled polystyrene nanospheres of 60 nm in diameter (Moghimi, S. M. et al., 1993, Biochim. Biophys Acta 1157, 233). The circulatory activity of nanospheres was monitored and animals were killed 3 h post administration of nanospheres for the analysis of radioactivity associated with organs of the RES. Only a small fraction of sterically-stabilized nanospheres was sequestered by the liver in animals predosed with saline; the majority of nanosphere remained in the blood at the time of sacrifice. The results are shown in Table 1. In contrast, the majority of uncoated nanospheres (60.0 ± 4.2% of the dose) were cleared by the liver.

Prior administration of the free poloxamine-908 up to 3 days before the injection of poloxamine-coated spheres had no influence on blood clearance and organ localization and the results were comparable to those of saline-treated rats (Table 1). Remarkably, from day 3 onward test- nanospheres were rapidly cleared from blood by both the liver and spleen despite the presence of the steric-barrier on the nanosphere surface (Table 1). The electron micrographs further confirmed these observations and show that only Kupffer cells are loaded with test nanospheres in polymer- treated animals. Kupffer cells had ingested few or none of the poloxamine-coated nanospheres throughout the organ in saline-treated rats. Both liver and spleen retained their ability to efficiently remove poloxamine-coated particles up to 10 days after administration of the polymer.

Table 1 Distribution of poloxamine-908 coated nanospheres in control (saline treated) and polymer-treated animals at different days after polymer administration. Time after % of administered dose/organ total polymer radioactivity administration Liver Spleen Blood collected*

Control:

Day 4 ** 8.8 ± 1.9 1.5 ± 0J 53.0 ± 4.9 •54.2 ± 6.8

Test: 3 h 5.9 ± 0.4 1.8 ± 0.1 61.6 ± 3.3 60.3 ± 3.1 Day 1 9.2 ± 2.0 1.4 ± 0.1 48.3 ± 4.6 58.9 ± 5.7 Day 2 12.4 ± 2.7 2.8 ± 0.6 67.8 ± 3.0 82.7 ± 6J Day 3 48.1 ± 6.2 5.9 ± 0.3 25.5 ± 4.9 81.4 ± 1.7 Day 4 42.6 ± 7.9 5J ± 0.6 25.2 ± 10.5 73.0 ± 2.4 Day 7 43.3 ± 9.3 5.9 ± 1.7 15.4 ± 8.9 64.6 ± 1.8 Day 10 19.7 ± 3.5 3.5 ± 0.8 43.3 ± 3.6 67.7 ± 3.6

* Excluding carcass

Organ distribution of nanospheres were the same in saline-treated rats 3 h and 10 days prior to administration of test-nanospheres.

The enhanced hepatic sequestration of test-nanospheres in polymer-treated rats cannot be solely explained on the basis of opsonization processes, since preincubation of poloxamine-coated spheres in serum (50% v/v) derived from both the control and polymer-treated animals had no effect on blood clearance and hepatic uptake of nanospheres after intravenous administration to rats that had not previously been exposed to poloxamine- 908 (data not shown). These observations further support the fact that the serum of polymer-treated animals cannot displace the adsorbed layer of poloxamine. and hence the steric barrier, from the surface of nanospheres which could make them prone to phagocytosis. Furthermore, no significant changes in the level of specific antibodies against the poloxamine were detected by ELISA in serum of polymer- treated rats when compared to control animals during the course of the experiment. Poloxamine-908 coated nanospheres, which are resistant to phagocytosis by lymph node macrophages (Moghimi. S. M. et al. 1994, FEBS Lett. 344, 25), were also sequestered by the phagocytic cells of the subcapsular sinus of the cortex and the classical macrophages of the medulla in both the popliteal and iliac nodes 2 h after subcutaneous administration into the footpad of polymer-treated rats when compared to control animals (data not shown). A large fraction of the nanospheres reached the systemic circulation of both animal groups but only in polymer-treated rats were particles rapidly cleared from the body by Kupffer cells.

Example 3. Groups of three male or female Wistar rats (body weight 150 ± 10 g) were injected intravenously with 17 mg/kg body weight of endotoxin-free L31 , PEG-1000. L121 , F68. PEG-20000 and tetronic 908 via the lateral tail-vein. Control animals received i.v. administration of sterile saline. 4 days later all animals received an intravenous dose of poloxamine-908 control (^l2iI)-radiolabelled polystyrene nanospheres of 60nm in diameter. The circulatory activity of nanospheres was monitored and animals were killed 3 hours post administration for the analysis of radioactivity associated with organs of the RES. The results (shown in Table 2) demonstrate that polymers with m.w. of less than 1200 are not effective in stimulating nanosphere clearance by liver and spleen. The results also demonstrate that the most effective polymers in stimulating nanosphere clearance by liver and spleen have m.w. of greater than 3000 (eg. L121 , F68, PEG-20000 and 908).

Table 2

Distribution of phagocyte-resistant poloxamine-908 coated nanospheres in saline treated or animals treated iv with 17 mg/kg (in saline) of designated polymers 3-4 days before the administration of particles. Treatment (m.wt) % uptake per organ

Liver Spleen

Control 7.7 + 2.5 1.6 ± 0.3

Pluronic L31 (1 100) 6.0 ± 0.3 1.3 ± 0.3

PEG- 1000 (1000) 9.0 ± 2.5 3.1 ± 0.9

Pluronic L121 (4400) 34.7 ± 2.7 9.8 ± 0.2

F68 (8400) 39.6 ± 3.9 5.6 ± 1.1

PEG-20000 (20000) 42.8 ± 1 , 1 7.8 ± 0.3

908 (25000) 32.4 ± 10.8 7.0 ± 0.4

Example 4: The method of Example 3 was repeated using endotoxin-free Tween-80 (sorbitan onooleate (ethylene oxide 20)). The liver and spleen uptake of nanospheres was found to be 31.9 ± 3.6 and 8.9 ± 1.2% of the administered dose. This is compared to values of 7.7 ± 2.5 and 1.6 ± 8.3 for liver and spleen in saline (control) treated animals.

The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of any of the above structures.

Example A: Tablet

Poloxamer 181 100 mg Lactose 200 mg Starch 50 mg

Polyvinylpyrrolidone 5 mg Magnesium stearate 4 mg

359 mg

Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.

Example B: Ophthalmic Solution

Tween 80 0.5 g

Sodium chloride, analytical grade 0.9 g

Thiomersal 0.001 g

Purified water to 100 ml pH adjusted to 7.5

Example C: Tablet Formulations

The following formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

Formulation A mg/tablet mg/tablet

(a) Poloxamine 908 250 250

(b) Lactose B.P. 210 26

(c) Povidone B.P. 15 9

(d) Sodium Starch Glycolate 20 12

(e) Magnesium Stearate 5 3

500 300 Formulation B mg/tablet mg/tablet

(a) PEG 10000 250 250

(b) Lactose 150 -

(c) Avicel PH 101^* 60 26

(d) Povidone B.P. 15 9

(e) Sodium Starch Glycolate 20 12

(f) Magnesium Stearate 5 3

500 300

Formulation C mg/tablet

PEG 20000 100

Lactose 200

Starch 50

Povidone 5

Magnesium stearate 4

359

The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direction compression type. Formulation D mg/capsule

Poloxamer 401 250

Pregelatinised Starch NF15 150

400

Formulation E mg/capsule

Poloxamine 904 250

Lactose 150

Avicel " 100

500

Formulation F ^Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. mg/tablet

(a) Poloxamer 105 500

(b) Hydroxypropylmethylcellulose 112

(Methocel K4M Premium)^*

(c) Lactose B.P. 53

(d) Povidone B.P.C. 28

(e) Magnesium Stearate 7

700 Example D: Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule

(a) Pluronic L10 250

Ob) Lactose B.P. 143

(c) Sodium Starch Glycolate 25

(d) Magnesium Stearate 2

420

Formulation C mg/capsule

(a) Poloxamer 402 250

(b) Macrogol 4000 BP 350

600

Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule. Formulation D mg/capsule

Poloxamine 408 250

Lecithin 100

Arachis Oil 100

450

Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. mg/capsule (a) Poloxamine 908 250

(b) Microcrystalline Cellulose 125

(c) Lactose BP 125

(d) Ethyl Cellulose 13

513 Example E: Injectable Formulation

Poloxamine 908 0.200 g

Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35- 40°C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Example F: Intramuscular injection

Poloxamine 908 0.20 g

Benzyl Alcohol 0J0 g Glucofurol 75' 1.45 g

Water for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Claims

1. The use of a substantially endotoxin-free non-ionic surfactant having a molecular weight of 1200 or greater in the manufacture of a medicament for stimulating the phagocytic function of macrophages of the reticuloendothelial system.

2. The use of claim 1 wherein the non-ionic surfactant comprises a block co-polymer of the poloxamer or poloxamine series or polyethylene glycol.

3. The use of claim 1 or 2 wherein the non-ionic surfactant has a molecular weight of 3000 or greater.

4. The use according to any one of claims 1 to 3 wherein the non-ionic surfactant is administered at a dose of 0.3 mg/kg body weight.

5. The use according to any of the preceding claims wherein the non- ionic surfactant causes the blood clearance of at least 25% of the injected dose of a phagocyte-resistant substrate within 3 h after intravenous administration of the substrate.

6. The use according to claim 5 wherein the phagocyte-resistant substrate is a sterically stabilized colloid of 100 nm in diameter or below, or a pathogenic microorganism.

7. The use according to any of the preceding claims wherein the non- ionic surfactant has minimal effect on the activation of human serum complement activity.

8. The use according to claim 7 wherein the residual total complement hemolytic activity of the non-ionic surfactant treated human serum is 50% or less than that of the residual total complement activity of poloxamer- 188 treated human serum.

>

9. A method of stimulating the phagocytic function of macrophages of the reticuloendothelial system comprising administering a composition comprising a non-ionic surfactant having a molecular weight of 1200 or greater or a conjugate of the non-ionic surfactant.