GB2261219A - Bromofluorochemicals useful in synthetic blood - Google Patents

Abstract

Novel dibromodecafluoro-pentamethylene or hexamethylene-tetramine is capable in the presence of emulsifying agents of forming blood substitutes stable at room temperature and possessing enhanced oxygen carrying capacity.

Description

BROMOFLUOROCHEMICALS USEFUL IN SYNTHETIC BLOOD This invention relates to synthetic blood, and more particularly to a blood substitute offering improvements in oxygen carrying capacity and stability, as well as lessened risk of anaphylactoid reaction, and an image enhancement capability.

The facile transport of oxygen through Teflon (polyperfluorothylene membrane has been well known for many years. The realization of the compatibility of perfluorochemicals with oxygen led to their use as oxygen carriers in a new generation of blood substitutes.

Initial work by Leland Clark of Cincinnati Childrens Hospital, Robert Geyer of Harvard and Henry Sloviter of the University of Pennsylvania, continued and extended by Naito and co-workers, led to a preparation (Fluosol DA 20%) produced for clinical testing by Green Cross of Osaka, Japan. Fluosol DA functioned as an oxygen carrier in animal experiments and showed considerable promise for human use.

However, Fluosol DA (perfluorodecalin) had several significant drawbacks. First, the emulsion of fluorochemical droplets in an aqueous phase was inherently unstable, both thermodynamically and kinetically, necessitating storage of the emulsion in the frozen state.

This instability also entailed a laborious and time consuming blending of the emulsion with other accessory solutions immediately before use. A second major problem with Fluosol DA was the necessity of maintaining the patient on 70 to 100% oxygen to ensure sufficient oxygen supply and exchange in the tissues. Finally, limited clinical experience with Fluosol DA showed an incidence of transfusion reactions and, in order to avoid this problem, led to the pretreatment of patients with steroids in the event a small test dose indicated sensitivity; this type of sensitivity appeared in 3% or less of all cases.

US Patent No. 4,900,824 describes the use of certain fluoro or perfluorochemicals in the preparation of an improved blood substitute. In particularly this patent specification claims the use of perfluorohexamethylenetetramine in synethetic blood.

It is one object of the invention to provide an improved blood substitute, employing bromofluorochemicals capable in the presence of suitable emulsifying agents of forming emulsions stable at room temperature and possessing enhanced oxygen carrying capacity. It is another object of the invention to overcome the toxic (anaphylactoid) reaction problem by the use of synthetic phospholipids in the substitute blood in which such bromofluorochemicals are employed.

It has been found, unexpectedly, that, by removing one carbon bond from perfluorohexamethylenetetramine and changing two of the fluorine atoms in this compound to bromine atoms, a compound can be produced which has two advantages over the compounds disclosed in US 4,900,824.

Firstly, the compound has greater compressibility (coefficient of compressibility), a feature which allows more of the oxygen contained in the molecule to be released as the molecule flows through the capillaries, and secondly, the molecule can be imaged by existing diagnostic imaging systems, therefore identifying very small cancer cells early in their development.

According to one aspect of the invention there is provided a compound of formula (1)

wherein X is a fluorine atom or a bond joining the N atom to a CF2 group.

Falling within the definition of formula (1) are the compounds dibromodecafluoropentamethylenetetramine and dibromodecafluorohexamethylenetetramine.

The solubility of oxygen in bromofluorochemicals is correlated with the isothermal compressibility of the liquid bromofluorochemical. The oxygen molecules pack into voids or cavities in the liquid structure in the process of solution, but do not interact significantly with the molecules as evidenced by the quite small enthalpies of solution.

As an illustration, formula (2) shows dibromodecafluorohexamethylenetetramine with 02 molecules in structured voids.

Oxygen molecules can also be transported by voids or interstices formed by close packing of the structures, a simple illustration being oxygen carried in the void formed by three close-packed balls.

Cancer cells have a great affinity for attracting oxygen, therefore oxygen carrying molecules will naturally concentrate in the cancer cell area. The addition of bromine to the fluorochemical compound of US Patent No.

4,900,824 makes it radiopaque in x-ray and CT scans. The molecule also reflects sound waves, so that it is useful in enhancing ultrasound images. The unemulsified molecule contains no hydrogen atoms, thus it can serve as a signalvoid bound contrast agent for MR1 imaging, a technology which creates images of internal structures based on hydrogen content. The molecules of the present invention thus allow all three major imaging technologies to be used with contrasting agents developed.

Such chemicals or mixtures of such chemicals with appropriate surfactants, when emulsified in water along with electrolytes and colloids compatible with natural blood, typically produce droplets which are suspended in solution and which are storable and stable at room temperature, the solution then being directly usable as an oxygen carrying blood substitute. 02 molecules are easily loosely retained for transport in the "basket" areas of the molecules.

The emulsion contains a non-toxic fraction derived from Pluronic F-68 or equivalent, together with one or more synthetic phospholipids as emulsifiers or surfactants to stabilize the emulsion. The fraction from Pluronic F-68 is prepared by fractional precipitation with organic solvents or salts or by absorbtion or partition chromatography, starting in either case with commercially available Pluronic F-68. Pluronic F-68* is not a uniform molecular species but instead consists of a mixture of molecules of differing molecular weight. The effectiveness of these different molecular species as emulsifying agents is a function of molecular weight or chain length. It is for reason that in the present process, highly refined fractions of optimal molecular weight are used in making bromofluorochemical emulsion.

In addition, the fractionation employed to prepare these purified materials tends to remove any residual materials toxic to humans or deleterious to red cells. The synthetic phospholipids differ from one another as to whether the overall structure corresponds to that of a lecithin, cephalin, plasmalogen or sphingovelin and in the nature of the fatty acid side chains in the structure.

The fatty acids differ in the number of carbon atoms, the number and placement of double bonds and in the presence or absence of alicyclic, aromatic or heterocyclic rings.

Synthetic phospholipds, unlike yolk phospholipids contain no trace of egg proteins which in many individuals are highly allergenic.

*Polyxyperopylene-polyoxyethelyne block co-polymer The structure of a typical lecithin is as follows:

where Rl and R2 are fatty acids selected from the group stearic acid, linoleic acid, eicosapentaenoic acid and dogosaheyaenoic acid.

In preparing and storing bromofluorochemical emulsions it is essential to prevent degradative reactions involving any of the components. If such reactions are allowed to occur, emulsion instability and/or toxicity may result.

Several types of such reactions are either known to occur, or may be logically expected to occur, if proper preventative measures are ignored. First, certain bromofluorochemicals, under the energetic influence of homogenization or sonication, especially in the presence of oxygen, can degrade to yield fluoride ion which is quite toxic. Second, any unsaturation in the fatty acid side chains of the phospholipid emulsifiers may result in the formation of peroxides if oxygen is present and if such reactions are not inhibited. For these reasons, in the present process, oxygen is excluded and, in addition, antioxidants such as vitamin E or other tocopherols are added to provide stabilization for oxygen-labile components.

An emulsion embodying the above described compounds prepared for intravenous administration, and also containing a synthetic phospholipid, is as follows: grams/100 ml.

(a) Dibromodecafluoro Hexamethylene tetramine 10-60 (b) Perfluoro (3.3.3) propellane 0-50 (c) Substance selected from the group consisting of: (i) hydroxyethylstarch (ii) polyvinylpyrolidane (iii) modified gelatin (iv) dextran (v) other polymer to supply colloidal osmotic (oncotic) pressure (d) Pluronic F-68 fraction about 2.7 (e) Glycerin USP (glycerol) (optional) about 0.8 (if used) (f) NaCl USP about 0.6 (g) Synthetic phospholipids 0.2-1.0 (h) Sodium bicarbonate about 0.21 (i) Dextrose about 0.18 (j) Magnesium chloride 6H20 about 0.043 (k) Calcium chloride 2H20 about 0.036 (1) Potassium chloride about 0.034 (m) Water for injection gs.

The following are specific examples, with constituents the same as listed above in (a)-(m): Constituents 1 2 3 4 5 6 (a) 20 20 25 25 30 30 (b) 40 40 35 35 30 30 (c) 3.0 3.0 3.0 3.0 3.0 3.0 (d) 2.7 2.7 2.7 2.7 2.7 2.7 (e) 0.8 0 0.8 0 0.8 0 (f) 0.6 0.6 0.6 0.6 0.6 0.6 (g) 0.4 0.4 0.4 0.4 0.4 0.4 (h) 0.21 0.21 0.21 0.21 0.21 0.21 (i) 0.18 0.18 0.18 0.18 0.18 0.18 (j) 0.043 0.043 0.043 0.043 0.043 0.043 (k) 0.036 0.036 0.036 0.036 0.036 0.036 (1) 0.034 0.034 0.034 0.034 0.034 0.034 (m) qs. qs. qs. qs. qs. qs.

In the above, the synthetic phospholipids are of the structure (3), above.

An increase in molecular weight of fluorochemical is commonly observed to result in an increase in emulsion stability. At the same time, if the fluorochemicals are of too high molecular weight, they are retained for excessive periods of time in the body; and if the molecular weight is too low, the fluorochemical can form bubbles of vapor within the circulation and can produce emboli. These conflicting factors have led other workers to restrict the useful molecular weight range of prior art fluorochemicals to 460 to 520. The molecular weight of dibromodecafluoropentamethylenetetramine is 466 and that for dibromodecafluorohexamethylenetratramine is 478, thus both fall within this ideal molecular weight range.

It is important to note that the molecular weight of the artificial blood most ordinarily lies in the range 450525. Below the 450 level, 02 is not efficiently trapped and has unwanted tendency to "Boil Off". It is also difficult to emulsify. Above the higher molecular weight level, the molecule is too large to be removed from the body, primarily via the lungs.

Oxygen carriage or transport occurs in two ways, i.e.

in the molecular "basket" (see position of 2 in molecular form (2)) and 02 entrapment between the molecules.

Consider the following diagram, for example, wherein the bromofluoro molecules are denoted by large circles, moving in a capillary, and the oxygen molecules are denoted by dots in the interstices between the large molecules.

(Also note the oxygen molecules within the circles, i.e.

the first way of 02 transport referred to above).

Advantageous results include greater 02 transport, whereby inbreathing of excessive oxygen by the patient is not required i.e. the patient can breath ordinary air, exclusively.

The introduction of fluorine into the various structures shown may be carried out after the molecular skeleton has been completed or in certain cases before the entire molecule is assembled. As an example of the latter, it may prove preferable in preparing the macrocyclic esters and amides shown to carry out the fluorination before the ester or amide bonds are formed, as for example by an appropriate protective group of alcohol, carboxylic acid or amine to permit fluorination of the methelene groups followed by removal of the protective groups and formation of the ester or amide.

Alternatively, the terminal carbons of the constituent chain to be subsequently coupled together can be chlorinated to prevent fluorination of the terminal carbons, the chlorines then later removed by hydrolysis to permit the desired functional group to be introduced along with bromine.

Fluorination can be accomplished by means of any one of several fluorination reagents or conditions. The exact choice depends upon the degree of fluorination desired, the stability of the carbon skeleton and to a minor degree on convenience and cost.

If it is desired to fluorinate a molecule only partially, then chlorine may be substituted into locations where fluorine is not desired; thereafter, the chlorine is replaced by bromine by means of reduction leaving the fluorination intact.

To fluorinate the structures shown requires powerful fluorinating agents such as fluorine itself at very low temperatures either added directly or produced by the electrolysis of hydrogen fluoride. Somewhat milder reagents such as xenon hexafluoride are useful in the first stages of fluorination followed gradually by bromofluorination or near bromofluorination by a more potent reagent.

As a preliminary step towards adding bromine to the molecules we suggest the addition of iodized compounds before the replacement brominization takes place by replacement.

Recommended methods of procedure would consist of utilization of one or several of the following: 1. Iodic Acid HIO3 2. Metaper Iodic Acid HIO4 3. Orthoparaper Acid HIO4. 2H20 4 Iodine MonoBromide IBr 5. Iodine TriBromide IBr3 6. Iodine HeptaFluoride IF7 7. Iodine PentaFluoride IF5 8. Iodious Acid-Hypo HO1 The proportions are determined precisely in the same manner as those described above for the displacement of fluorine by chlorine.

The invention will be further described by way of reference to the following example which illustrates the preparation of another bromofluorochemical compound.

EXAMPLE Conventional fluorination procedures known in the literature (see references (1) to (7) following this example), use a stream of hydrofluoric and hydrobromic acids in the ratio of five (5) parts to one (1) at a density of approximately 1.4, (HBr p = 2.71 plus HF p = 1.15 x 5 = 5.75 equating to a final density of 1.41). At a temperature of between -400C and 180C preferably about OOC. The above reactants are continuously fed into the reaction vessel along with the hexamethylenetetramine in finely divided solid form. The feed rates are such, that chemically equivalent amounts of the acid and amino are fed, per unit time, to the reaction vessel, and on a continuous basis. The reactants in the vessel are stirred and the amino particles are allowed to dissolve.The stirred solution is electrolized utilizing a nickel anode and a carbon cathode at approximately 6 volts D.C.

Withdraw C2N4FlOBr2 is withdrawn from the bottom of the vessel at a density of 1.4. Any evolution of Br2 or F2 is withdrawn as gases from the top of the vessel and reprocessed accordingly.

The compound, perfluoro (3.3.3) propellane has been disclosed as an oxygen carrier.

Synthetic methods for obtaining propellanes have developed rapidly over the last decade since the first definitive works in this area appeared (Ginsburg, D., Propellanes, Verlag Chemic (1975); Greenberg, A. and Liebman, J.F., Academic Press, New York (1978)).

The synthesis of the present compound proceeds in three stages: 1. Formation of the (3.3.3) diketone.

2. Removal of the two keto groups.

3. Perfluoroination.

The first step is accomplished by condensation of an acetone dicarboxylic ester with 1,2-diketocyclopentane.

The reaction proceeds smoothly at pH 5 in water: The second step is accomplished by the Wolf f Kishner reaction in DMSO (dimethyl sulfoxide) at about 1000C., or by a vapor pulse photochemically activated U.V. reaction.

In this reaction one may use either activation with Hg vapor at 2537 Ad or to activate at the wavelength of maximum abosrbtion of the hydrazone group. The thermodynamic driving force for this reaction may be attributed largely to the large positive free energy of formation of hydrazine.

The chlorobromofluorination is characteristically carried out by the procedure used in the synthesis of bromofluorohexamethylenetetramine, e.g. and can be extrapolated accordingly to the other molecules (3) - (14).

1. Mellor, J.W. Comprehensive Treatise on Inorganic and Theoretical Chemistry, Suppplemental II, part 1, pp.120, 129, 135. Longmans Green and Co., London (1956).

2. Simons, J.H. Chem. Rev. 8 213 (1931).

3. Simons, J.H. U.S. No. 2,519,983 (Aug. 22, 1950).

4. Simons, J.H. U.S. No. 2,490,098 (Dec. 6, 1949).

5. Simmons, T.C., et al., J.Am. Chem. Soc. 79 3429 (1957).

6. Gervasi, J.A., et al. J.Am. Chem. Soc. 78 1679 (1956).

7. Hazeldine, R.N.J., Chem. Soc. p.1966 (1950); p.102 (1951).

Claims (8)

CLAIMS:

1. A compound of formula (1)

wherein X is a fluorine atom or a bond joining the N atom to a CF2 group.

2. The compound dibromodecafluoropentamethylene- tetramine.

3. The compound dibromodecafluorohexamethylenetetramine.

4. A blood substitute comprising a compound of formula (1) as defined in claim 1, together with a physiologically acceptable emulsifying agent.

5. A blood substitute according to claim 4, wherein the compound of formula (1) is dibromodecafluoropentamethylenetetramine.

6. A blood substitute according to claim 4, wherein the compound of formula (1) is dibromodecafluorohexamethylenetetramine.

7. An aqueous emulsion of a compound according to any one of claims 1, 2 and 3, for use in therapy, e.g. as a blood substitute.

8. Use of a compound according to any one of claims 1, 2 and 3 for the manufacture of a medicament for use as a blood substitute.