CA2207652C

CA2207652C - Hemolysis prevention by surfactants and emulsions

Info

Publication number: CA2207652C
Application number: CA002207652A
Authority: CA
Inventors: Timothy Harrison
Original assignee: Merck Sharp and Dohme Ltd
Current assignee: Organon Pharma UK Ltd
Priority date: 1994-12-23
Filing date: 1995-12-15
Publication date: 2006-07-25
Anticipated expiration: 2015-12-15
Also published as: CA2207652A1

Abstract

The present invention relates to the use of a non-ionic surface-active agent or an emulsion for the reduction of the hemolytic effects of an amphiphilic compound. There is also provided a method for reducing the hemolytic effects of an amphiphilic compound which comprises formulating said compound with a non-ionic surface-active agent or in the form of an emulsion.

Description

WO 96!19970 PCTlGB95/02936 HEMOLYSIS PREVENTION BY SURFACTANTS AND EMULSIONS
This invention relates to the novel use of a non-ionic surface-active s agent (otherwise known as a surfactant or wetting agent) or an emulsion for the reduction of the hemolytic effects of an amphiphilic compound.
Hemolysis of red blood cells (erythrocytes) is observed when such cells are suspended in a solution which has a lower osmotic pressure than that of the fluid in the red blood cells - i.e. when the solution is hypotonic.
io This has the effect of causing the red blood cells to swell and burst due to diffusion of water into the cells. Conversely, if red blood cells are suspended in a hypertonic solution - i.e. where the osmotic pressure is greater than that of the fluid in the cells - they may lose water and shrink (crenation).
This classical view of hemolysis has resulted in the development of 1s "isotonic solutions" which exert the same osmotic pressure as blood plasma.
In isotonic solution (e.g. 0.9% sodium chloride) red blood cells maintain their "tone" and the solution is said to be isotonic with human erythrocytes.
Hemolysis following parenteral administration of a pharmaceutical formulation may cause localised tissue damage, irritation, anaemia and in 2o extreme cases may lead to organ damage, particularly damage to the kidneys.
It is now apparent, however, that hemolysis is not only caused by the administration of a hypotonic parenteral formulation. It has been observed that amphiphilic compounds will also exhibit hemolytic effects on red blood 2s cells, even when administered in an isotonic solution.
Surprisingly, it has now been found that a non-ionic surtace-active agent or emulsion can be used to dramatically reduce the hemolytic effects of an amphiphilic compound.
There is therefore provided in a first aspect of the present invention, 3o the use of a non-ionic surface-active agent or an emulsion for the reduction of the hemolytic effects of an amphiphilic compound.
SUBSTITUTE SHEET (RULE 26)

-2-In an alternative aspect of the present invention, there is provided a method for reducing the hemolytic effects of an amphiphilic compound which comprises formulating said compound with a non-ionic surface-active agent or in the form of an emulsion.
Surface-active agents, otherwise known as surfactants or wetting agents are generally molecules which comprise a hydrophobic portion (e.g.
an alkyl chain) and a hydrophilic portion (e.g. a polar or ionic group).
Surface-active agents may be divided into a number of classes including anionic agents, cationic agents, amphoteric agents and non-ionic to agents, of which the non-ionic agents are particularly useful in the present invention.
Non-ionic agents include fatty alcohols; glyceryl esters such as the naturally occurring mono-, di- and triglycerides; fatty acid esters of fatty, and other, alcohols such as propylene glycol, polyethylene glycol, sorbitan, is sucrose and cholesterol; polyoxyethylene sorbitan fatty acid esters; and polyoxyethylene ethers.
Particularly preferred non-ionic surface-active agents are the polyoxyethylene sorbitan fatty acid esters which are commercially available under the trade-name TweenTM, for example, the monooleate ester (Tween 20 8~TM
The precise choice of non-ionic surface-active agent and the concentration used will depend largely upon the physical properties of the chosen agent. It is believed that the masking of the hemolytic nature of an amphiphilic compound in accordance with the present invention is effected by 2s the formation of micelles by the surface-active agent. Micelle formation occurs over a range of concentration of the surface-active agent known as the critical micellization concentration (cmc). It is therefore desirable to use in the present invention a concentration of the non-ionic surface-active agent which falls within the cmc range for the chosen agent. Micelles are formed by 3o surface-active agents due to the lack of affinity of the hydrophobic regions of the surfactant molecules for their aqueous environment. An aggregate is SUBSTITUTE SHEET (RULE 26)

-3-therefore formed by strong hydrophobic chain-chain interactions in the "core"
of the micelle, presenting the hydrophilic (polar) region of the molecules on the surface of the micelle. The lipophilic or non-polar region of the hemolytic amphiphilic compound is believed to be held in the hydrophobic centre of the s micelle thus preventing its damaging interaction with erythrocyte cell membranes immediately upon injection.
Compositions with a non-ionic surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent and preferably between 0.1 and 2.5%.
io Formulation as an emulsion may be effected by mixing the amphiphilic compound with a commercially available fat emulsion, such as IntralipidT"", LiposynT"", Infonutrol T"', Lipofundin T"", and Lipiphysan T"". The compound may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, is sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with an emulsifier such as a phospholipid (e.g. egg phospholipids, soybean phospholipids, or soybean lecithin) and water.
The administration of an amphiphilic compound in an emulsion is believed to mask hemolysis in a similar fashion to that described for surface 2o active agent micelles, above. The hemolytic amphiphilic compound is believed to be dissolved in fat droplets which are suspended in an aqueous medium in the form of an emulsion. The amphiphilic compound is therefore prevented from interacting with erythrocyte cell membranes immediately upon injection.
2s Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion will preferably comprise fat droplets between 0.1 and 1.OE~m, particularly 0.1 and 0.5~~m.
Particularly preferred emulsion compositions are those prepared by mixing the amphiphilic compound with IntralipidT"' or the components thereof 30 (soybean oil, egg phospholipid, glycerol and water).
SUBSTITUTE SHEET (RULE 26)

-4-It will be appreciated that other ingredients may be added to the formulation. Thus, for instance, bulking substances or tonicity modifiers may be added such as glycerin, lactose, mannitol, dextrose, sodium or potassium chloride, sodium sulphate and sorbitol, in general at a concentration up to 5%
s depending upon the chosen substance.
Other additives include antimicrobial preservatives, including benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, chlorocresol, cresol, methyl p-hydroxybenzoate, phenol, phenylethyl alcohol, phenylmercuric nitrate and acetate, propyl p-io hydroxybenzoate, and thimerosal; antioxidants, including acetone sodium bisulphite, ascorbic acid, ascorbic acid esters, butylhydroanisol, butylhydroxytoluene, cysteine, nordihydroguaiaretic acid, sodium bisulphite, sodium formaldehyde sulphoxylate, sodium metabisulphite, and tocopherols;
and buffers, including acetic acid and a salt, citric acid and a salt, glutamic is acid, and phosphoric acid salts.
As used herein, the term "amphiphilic compound" means a molecule which comprises both a hydrophilic (e.g. polar) region and a hydrophobic (e.g. non-polar) region. The hydrophobic region of the molecule will exhibit a greater lipid solubility than the hydrophilic region hence erythrocyte 2o membranes may be disrupted by such amphiphilic compounds.
The hydrophilic region of a molecule will typically be characterised by a polar or ionic group such as an amino moiety, for example, a teriary amino group such as dimethylamino. Such groups may be affected by local pH.
The hydrophobic region may be, for example, an alkyl chain or a part of the 2s molecule bearing one or more aryl moieties such as phenyl groups.
It will be appreciated that there are countless amphiphilic compounds which rely upon their physical properties in order to reach their intended site of action. For instance, drug molecules which act centrally on the central nervous system (CNS) must be able to cross the blood-brain barrier in order 3o to penetrate the brain. This may be facilitated by their lipophilic nature.
These properties only become a problem when the amphiphilic compound is SUBSTITUTE SHEET (RULE 26)

-5-administered by injection and the concentration of the compound in the blood immediately surrounding the needle-tip is high and therefore hemolysis is more likely to occur. Hemolysis can, in such instances, be avoided by slow infusion of the drug substance or by increasing the dose volume if possible, s however, these procedures may be inconvenient or impractical and place an extra burden upon the attendant clinical physicians.
Hemolysis may be measured by the following method in vivo:
Male Sprague-Dawley rats (280 to 450g) are anaesthetised with isoflurane. The tail artery and tail vein are cannulated with polythene tubing io (0.58mm internal diameter, 0.96mm external diameter) and a small amount of heparinized saline (0.2 to 0.5m1) is washed in (heparin concentration 10 units/ml). The rats are placed in restrainers and left to recover for 30 minutes to 1 hour. A baseline blood sample is taken 10 minutes before dosing with the test compound. The rats are then dosed with the test compound at t=0 is minutes. Typically, 1-10m1/kg of a 1-10mg/ml solution is injected through the tail vein catheter over a period of 2 minutes and washed in with heparinized saline.
After dosing, small blood samples (approximately 400y1) are taken at intervals over 1 hour (typically t=5, 10, 15, 30 and 60 minutes) from the tail 2o artery catheter ensuring that the catheter is cleared to get fresh blood which is uncontaminated by the previous sample. Blood withdrawn is replaced with equal volumes of physiological saline. Blood samples are transferred directly into heparinized tubes (typically MicrotainersT"") and put on ice.
The assay for hemolysis is effected by centrifuging the blood samples 2s to obtain a plasma fraction. The plasma is then decanted into plastic tubes and scored by eye for hemolysis (colorimetric cuvettes may be used for measurement of free haemoglobin using a spectrophotometer). Samples of plasma may be frozen at -18°C or below if needed for later analyses.
3o Hemolysis is scored by the following system:
SUBSTITUTE SHEET (RULE 26}

-6-no difference +~_ - borderline: very slight pink colouration + - mild: slight pink colouration ++ - moderate: pink-red colouration +++ - marked: red colouration ++++ = severe: very dark red colouration The following table shows the scores obtained using the compound 2_ (R)-( 1-(R)-bis(trifluoromethyl)phenyl)ethoxy)-4-(5-(N, N-dimethylaminomethyl)-io 1,2,3-triazol_4-yl)methyl-3-(S)-phenylmorpholine (Compound A), and the effect of pertorming the assay using Compound A in varying concentrations of Tween 8OT"" (polyoxyethylene sorbitan monooleate) or Compound A in IntralipidT"" (soybean oil, egg phospholipid, glycerol and water). 5% Mannitol is included in each vehicle to adjust the tonicity.
SUBSTITUTE SHEET (RULE 26) -7_ TABLE
test compositionsolution dose volume dose hemolysis (in 5% mannitol)conc. (mglml)(mllkg) (mg/kg) score Compound A 1 10 10 +++

Compound A in 2 10 20 +/-1.0% Tween 80T"' Compound A in 2.5 10 25 -1.5% Tween 80T""

Compound A in 5 5 25 ++

2.0% Tween 80T""

Compound A in 2 10 20 -10% IntralipidT""

Compound A in 4 5 20 -10% IntralipidT"' Compound A in 10 2 20 ++

10% IntralipidT""

s From the above results it is clear that Compound A, which exhibits marked hemolysis in the above in vivo assay when administered alone in 5%
mannitol, is not hemolytic when formulated with a non-ionicsurface active agent (with Tween 80T"") or in an emulsion (with IntralipidT""). As shown by the above results, moderate hemolysis is only observed when the solution io concentration (i.e. the concentration of Compound A at the needle tip on injection) is elevated (at 5mglml with Tween 8OT"" and at 10mg/ml with Intralipid T"" ) SUBSTITUTE SHEET (RUI:E 26) -g_ The following Examples illustrate pharmaceutical compositions which may be used in accordance with the present invention:
Example 1 - (Surface-Active Aaent) Infection Formulation Active Ingredients) up to 10mglkg Tween 80TM up to 2.5%
(in 5% aqueous mannitol (isotonic)]
to The active ingredients) is (are) dissolved directly in a solution of the commercially available Tween 80TM (polyoxyethylenesorbitan monooleate) and 5% aqueous mannitol (isotonic).
Example 2 - (Emulsion) Infection Formulation Active Ingredients) up to 30mg/ml IntralipidTM (10-20%) The active ingredients) is (are) dissolved directly in the commercially 2o available IntralipidTM (10 or 20%) to form an emulsion.
Example 3 - Alternative (Emulsion) Iniectable Formulation Amount Active Ingredients) 0.1 - 10mg 2s Soybean oil 100mg Egg Phospholipid 6mg Glycerol 22mg Water for injection to 1 ml so All materials are sterilized and pyrogen free. The active ingredients) is (are) dissolved in soybean oil. An emulsion is then formed by mixing this SUBSTITUTE SHEET (RULE 26) solution with the egg phospholipid, glycerol and water. The emulsion is then sealed in sterile vials.
SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS:

1. Use of a polyoxyethylene sorbitan fatty acid ester for the reduction of the hemolytic effects of an amphiphilic compound.

2. Use as claimed in claim 1, wherein the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monooleate.

3. Use as claimed in claim 1 or 2, wherein said amphiphilic compound is formulated with said polyoxyethylene sorbitan fatty acid.

4. Use as claimed in claim 3, wherein said amphiphilic compound is formulated to form a formulation which comprises between 0.05 and 5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.

5. Use as claimed in claim 4, wherein said formulation comprises between 0.1 and 2.5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.

6. Use of a polyoxyethylene sorbitan fatty acid ester for manufacture of a medicament for the reduction of the hemolytic effects of an amphiphilic compound.

7. Use as claimed in claim 6, wherein the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monooleate.

8. Use as claimed in claim 6 or 7, wherein said amphiphilic compound is formulated with said polyoxyethylene sorbitan fatty acid.

9. Use as claimed in claim 8, wherein said amphiphilic compound is formulated to form a formulation which comprises between 0.05 and 5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.

10. Use as claimed in claim 9, wherein said formulation comprises between 0.1 and 2.5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.

11. A pharmaceutical composition comprising an amphiphilic compound and a polyoxyethylene sorbitan fatty acid ester for the reduction of hemolytic effects of said amphiphilic compound, in association with a pharmaceutically acceptable liquid carrier.

12. A pharmaceutical composition as claimed in claim 11, wherein the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monooleate.

13. A pharmaceutical composition as claimed in claim 11 or 12, which comprises between 0.05 and 5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.

14. A pharmaceutical composition as claimed in claim 13, which comprises between 0.1 and 2.5%, by volume, of said polyoxyethylene sorbitan fatty acid ester.