CA1154684A - Hemocompatible addition solutions and use thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Blood may be stored in vitro for a period of days at a reduced temperature in the container which also includes a sufficient quantity of an emulsion of anti-hemolytic agent to cause a reduction in the hemolysis of the stored blood, when compared with blood under similar storage conditions to the absence of the emulsion. Preferably, the emulsion may include a fatty ester containing at least two ester linkages each comprising a fatty hydrocarbon group, with the emulsion containing sufficient hemo-compatible surfactant to stabil-ize the emulsion.

Description

BACKGROUND Ol~' lrllE INV~Nrl'ION
.. . .
In copending Canadlan ~pplication No. 33~,~67 filed October 25, 1979 by Ulrich D. Geissler, et al. for "Blood Compatible Polymers and Medlcal Devices Made Therefrom"
it is taught that, most surprisingly, common plasticizers for plastic materials such as di-2-ethylhexylphthalate and di-2-ethylhexyladipate are anti-hemolytic agents. In their presence, stored blood exhibits significantly lowered hemolysis over the period of storage than in the absence of such materials.
While the commercially-available blood bags have been made of polyvinyl chloride formulations which include di-2-ethylhexylphthalate, other candidate blood bag materials were free of such ester-type plasticizers, in-tentionally so, because it had been suggested from some experts that the ester-type plasticizers were undesirable,' and should not be present in the plastic containers used for storage o~f blood.
It most surprisingly turned out that the various materials which were free of the ester-type plasticizers exhibited an undesirably high plasma hemoglobin content after, for example, 21 days of storage, indicating that the lysis rate of the red blood cells was high.
After this phenomenon was identified, further studies indicated that it was apparently necessary to incorporate the ester plasticizer into plastic material in contact with the stored blood, to get the anti-hemolytic effect.
~his could be done-either by formulating the material into the blood container itself, or a plastic insert member to dm~ 2 -l~Lc~ 3~
the container in which the insert member contains the ester-l:ype p1asticlYer in accordance with copending Canadian Paterlt Application No. 33~3,~0, filed October 25, 1979 by llenry W.
Collins and enti~led "Blood Bags Havinq an Insert Member", which application is assigned to the assignee of this application.
The initial research, however, also indicated that the addition of quantities per se of the liquid ester material such as di-2-ethylhexylphthalate to the blood container did not exhibit the anti-hemolytic effect obtained when the ester material is incorporated into the plastic material of the blood container or an insert within the container.
Attempts to emulsify the ester material were also unsuccessful, resulting in emulsions that were basically unstable.
By this invention, it has been surprisingly found that stabilized emulsions of anti-hemolytlc agents as described herein provide further significantly improved anti-hemolytic effect, whe~n placed in the presence of stored blood, to make it possible to even reduce the hemolysis of stored biood to less than that which is conventional for the commercially-available, ester-plasticized polyvinyl chloride blood bags. The emulsion may be stabilized in accordance with this invention by a specific technique as described below.
DESC~IPTION OF THE_INVENTION
In accordance with this invention, blood may be stored in vitro for a period of more than two days at a reduced temperature in a hemocompatible container, which also dm ~i~ 3 contalns a suf~icient quantity of arl emulsion of an anti-~
hemolytic agent, ~or examplc, a fatty es~er containlng at leas~ tt70 es~er linkages comprising fa~ty hydrocarbon groups of four to twelve carbon atoms each, with the ester linkages being preferably spaced closer together than the 1,3 relation, to cause a reduction in the hemolysis of the s~ored blood, when compared with blood under similar storage conditions in the absence of the emulsion.
Preferably, the emulsion contains sufficient quantity of a hemocompatible surfactant to stabilize the emulsion for a period of time, at least equal to the duration of storage of the blood. Of course, indefinitely stable emulsions are pre~
ferred. Such emulsions may generally be made by mixing the anti-hemolytic agent, for example the above-described fatty ester, with the hemocompatible surfactant in the substantial absence of water. After intimate mixing, the resulting mater-ial is added to the water ingredient to form the emulsion, which may be indefinitely stable, contrary to the results with other emulsification techniques.
The fatty hydrocarbon groups in the ester linkage ,0.
(e.g., R-OC' -) are preferably alkyl radicals of 4 to 12 car-bon atoms. The ester linkages are preferably spaced closer than the 1,3 relation, by which is meant that the ester link-ages are preferably bonded to the same or adjacent carbon or other atoms such as phosphorous. Most preferably, fatty esters which are on adjacent carbon atoms, or the same phos-phorous atom, appear to be the most active in their anti-hemo-lytic effect, but fatty ester linkages which are separated farther apart on highly mobile hydrocarbon chains can also be very active, fnr exampie linear alkylene-chains, forming compounds such as di-2-ethylhexylad:Lpate, which tend to diminish in activity as the ester groups are spaced ~arther apart tllan ; in that compound. ~referably, such linear hydrocarbon chains contain no more than 8 carbon atoms, excluding the ester linkage carbon atoms. Also, maleate esters and related materials are active.
It is also preferable fo~ the preferred organic radi-cals of the ester linkages to be alkyl of 7 to 10 carbon atoms, e.g., octyl groups, for example N-octyl, heptyl, nonyl, decyl, or 2 ethylhexyl. However, ot~er radicals such as hexyl or do-decyl may also be used. Also, similar alkenyl radicals such as octenyl, nonenyl, or decenyl containing one or more unsaturated linkages may be used.
Examples of these preferred ester materials are the 1~ various dioctyl phthalates and dioctyl adipates, dioctyl mal-eates, and trioctylphosphate, which of course is an ester of phosphoric acid. Other anti-hemolytic agents which may be used include tri-functional esters such as tri-ethylhexyl trimelli-tate, and other esters in which the fatty acid groups are not adjacent on the carbon atoms such as dioctylterephthalate.
Generally, the antihemolytic agent may be rendered effective by bringing it into contact with the stored blood in a highly dispersed manner, so that a diffusion exchange may slowly take place between the antihemolytic agent and the blood.
The emulsions used herein are preferred because, through them, precisely controlled amounts of the antihemolytic agents may be added to the blood, to provide a concentration in the blood of preferably 50 to 100 parts per million. Other gross techniques o~ adding the antihemolytic agents to the blood may cause insuf-ficien~ quantities of the-antihemolytic agent to ~o into the blood phase, especially in the first few days of blood storage.

~ t~

On the other hand, gross excesses of t:he antlhemolytic agent may be placed into the blood by uncontrolled techniques, such as coating the sides of the container with the antihemolytic agent prior to adding the blood to the container.
Any blood-compatible, non-toxic emulsifying agent may ~`
be used herein, for example, polysorbate 80 (as identified in the U.S. Pharmacapoea), which is a complex mixture of poly-oxyethylene ethers of mixed partial oleic esters of sorbitan anhydrides, sold for example as Tween 80 by I.C.I. Americas, Inc. Another suitable material is sold by the same company under the name Tween 40, being polyoxyethylene (20) sorbitan monopalmitate. Other examples of possible emulsifiers includes cholesterol and lecithin, which are advantageous, since they are normally found in the body.
In use, the emulsions of this invention may be inserted into a conventional blood bag in combination with a preservative-nutrient conventionally found in blood bags, such as CPD or ACD.
The examples below are offered for illustrative purposes only, and are not intended to limit the scope of the invention of this application, which is as defined in the claims below.
Example 1. Emulsions were made of 2 ml. of Tween 80 (polysorbate 80), respectively with 2 ml. each of the following materials: di-2-ethylhexylphthalate, dioctylterephthalate, and tri-2-ethylhexyltrimellitate.
~ fter mixing the Tween 80 with the respective ester materials, 0.3 ml~ of each of the mixtures was added to separate portions of 150 ml. of sterile, 0.9 weight percent saline solution, with shaking, to form a stable emulsion.
One half ml. of each of these emulsions was added dm~ 6 -to separate polypropylene test tubes, alon~ with 7.5 ml.
of well-mi~ed, freshly drawn, whole human blood, anticoag-ulated with ~he the known CPD material.
The blood was drawn from a human donor into a vinyl blood bag plasticized wi~h tri-2-ethylhexyltrimelli-tate, which is a non-leachable plasticizer, to prevent the early contact of the blood with any leachable plasticizer.
The polypropylene test tubes were inverted twice after closing with stoppers, and refrigerated at 4C. for 21 days.
Two control tubes included a tube only of blood as control No. 1, and a tube which contained blood and a correspondin~ concentration of the Tween~80 in saline solu~
tion, as control No. 2.
After the 21 days> the plasma hemoglobin content of each of the samples was determined with the results being as indicated in Table I below.

TA~LE I
Plasma Hemoglobin Emulsion Sample TestedContent (mg./Deciliter) Di-2-ethylhexylphthalate 8 ~ 1 Dioctylterephthalate 20 ~ 2 Tri-2-ethylhexyltrimellitate12 + 1 Control No. 1 26 ~ 2 Control No. 2 24 + 1 Example 2. Further blood samples were made for testing in the manner identical to that previously des-cribed in Example 1 above and stored for 28 days. The re-sults were as indicated in the Table II below, TABLE II
Plasma Hemoglobill ~3e/~ L3~3b5~3~ Content ~mg./Deci1iter) Di-2-ethy-lhexylphthalate 10 + 1 Dioctylterephthalate 26 + 1 Tri-2-ethylhexyltrimellitate 16 + 1 Control No. 1 32 + 1 Control No. 2 31 + 1 It can be seen that the emulsions of all three of the ester materials exhibit an antihemolytic effect, with the di-2-ethylhexylphthalate exhibiting the strongest effect.

Example 3. The experiment of Example 1 above was repeated, but substituting the surfactant Tween~ 40 for the Tween~80 of the previous example. Blood was processed and stored in similar manner with, respectively, an equal concen-tration of the emulsion containing, respectively, di-2-ethyl-hexylphthalate and tri-2-ethylhexyltrimellitate.
Table III below shows the plasma hemoglobin in the blood after respectively 21 days and 28 days of storage for each of the emulsions of the two different ester materials.

TABLE III
. _ Plasma Hemoglobin _(mv/Deciliter)_ Emulsion of Emulsion of Di-2-ethylhexylphthalate Tri-2-ethylhexyltri-Storage Time mellitate . _ .
21 Days 9 + 1 16 ~ 1 28 Days 12 + 1 21 ~ 1 The previous control results are believed to be effective for this experiment as well, sin-ce identical condi-tions were used except for the change of surfactant.

c~

Example 4. Emulsions were made of two ml. of Tween~80 (polysorbate 80), respectively with two ml. each of the ester materials as listed below in Table IV.
After mixing the Tween~80 with the respective : 5 ester materials, 0.2 ml. o each of the mixed materials was added to separate portions of 83 ml. of sterile, 0.9 weight percent saline solution, with shaking, to form a stable emulsion.
One half ml. of each of these emulsions was added to separate polypropylene test tubes, along with 7.5 ml. of well-mixed, freshly-drawn, whole human blood, anti-coagulated with the known CPD material.
The blood was drawn from a human donor in the manner pre~iously described in Example 1. The polypro- ;
pylene test tubes were inverted twice after closing with stoppers, and refrigerated at 4C. for 21 days.
One control tube contained blood in a corres-ponding concentration of the Tween~80 in saline solution.
Another control tube contained just blood, but also con-taining a strip of polyvinyl chloride plasticized with ~ di-2-ethylhexylphthalate.
! . . After 21 days, the plasma hemoglobin content of each of the samples was determined, with results being as indicated in lable LV below.

~.

~ . .

TABLI' ~V
Plasma ~lemoglobin Content Emulsioll Sample Tested (m~,/Decili-t r) _ _ Tri-2-ethylhexylphosphate 5 ~ 1 Diisononylphthalate 7 ~ 1 Diisodecylphthalate 8 Di 2-ethylhexylphthalate . 9 ~ 2 Tri-2-ethylhexyltrirnellitate 11 -~ 1 Di-2-ethylhexylmaleate 14 ~ 1 Dihexylphthalate 15 ~ 3 Di-2-ethylhexylisophthaiate 20 ~ 2 Didodecylphthalate 18 ~ 1 ~i-2-ethylhexylazelate 19 -~ 2:
Dibutylphthalate 18 ~ 2 Control tube containing blood plus plasticized polyvinyl chht~o~itde containing di-2-ethylhexyl_ 16 -~ 2 Blank control containing Tween~
80 without ester material 24 ~ 2 This application is a divi.sion of Canadian Patent Appli~ation Serial No. 345,163, filed February 6, 1980.

~10-

Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A hemocompatible additive solution for reducing the hemolysis of stored blood, said solution comprising an effective amount of a phosphate ester containing at least two ester linkages comprising fatty hydrocarbon groups, each of four to twelve carbon atoms, said ester linkages being bonded to the same phosphorous atom.

2. An additive solution according to claim 1 wherein said phosphate ester is tri-2-ethylhexylphosphate.

3. An additive solution according to claim 1 wherein said fatty hydrocarbon groups are selected from the group consisting of alkyl and alkenyl.

4. An additive solution according to claim 1 wherein said fatty hydrocarbon groups are alkyl.