WO1993004731A1

WO1993004731A1 - Iodine-iodide treatment of red blood cells

Info

Publication number: WO1993004731A1
Application number: PCT/US1992/007233
Authority: WO
Inventors: Edward Shanbrom
Original assignee: Edward Shanbrom
Priority date: 1991-09-03
Filing date: 1992-08-26
Publication date: 1993-03-18

Abstract

Red blood cell containing preparations are treated with iodide and iodine, the iodide serving to enhance the antimicrobial effect iodine, for preparing pathogen-free red blood cell containing preparations

Description

IODINE-IODIDE TREATMENT OF RED BLOOD CELLS

Field of the Invention This invention relates to the treatment and preservation of red blood cells. Background of the Invention

Red blood cells are transfused into patients as a component of whole blood and in various red blood cell concentrates. The risk of transmitting pathogenic microorganisms to the recipient remains serious in spite of efforts to provide a safe blood supply. A new method of treating red blood cells to inactivate microorganisms which may be present in red blood cell transfusion preparations is disclosed and claimed herein.

In describing the invention, various terms are used in the sense defined below.

Povidone (USP) is used in the sense that it is used in the U.S. Pharmacopeia to describe grades of polyvinyl pyrrolidone (PVP) suitable for introduction into the human body.

The term "molecular iodine compound" is used in this patent to mean and include molecular iodine, I, diatomic iodine, I,, or a compound or a mixture of compounds which either comprises iodine available in molecular form, typically as diatomic I₂, or which reacts with or in the presence of the sample to produce such iodine. Povidone-iodine is the principal example of such compounds.

Iodide is used to describe a compound which disassociates in aqueous solution to produce iodide ions. Potassium iodide and sodium iodide are considered to be the optimum iodides suitable for use in this invention.

It has been discovered that the antimicrobial effect iodine in red blood cell containing preparations is greatly enhanced by pre-treating the red blood cells with iodide or treating the red blood cells with iodide and iodine contemporaneously. The cytophylactic effect of povidone in protecting whole blood and packed or clotted red blood cells from the cytolitic effect of iodine is enhanced by the generally contemporaneous treatment of the cells with certain salts when the ratio of PVP to I, is sufficiently high, at least about 4:1 (by weight).

This invention relates to the treatment of whole blood to inactivate or destroy infective pathogenic microorganisms found in animal fluids and cells. The term "inactivate" as applied to the inactivation of virus in accordance with the present invention means that the pathogenic microorganisms are rendered non-infectious to the patient.

Whole blood and red blood cell transfusions exemplify generally one application of this invention. This invention is generally applicable to the transfer of red blood cells and other cells which bind iodine from one species to the same species and, to the extent immunological incompatibility problems can be overcome, to inter species transfer.

The Epste n-Barr virus (EBV) and/or cytomegalovirus (CMV) are commonly found pathogenic viruses. Other pathogenic viruses frequently found in red blood cell containing preparations include hepatitis and human immunodeficiency virus (HIV). Many other less life-threatening viruses are also found in red blood cell containing preparations.

Another organism which is frequently present in blood and blood products or fractions and which presents a serious risk in certain procedures is the bacteria Yersinia enterocolitica which is become a serious contaminant, surpassing Salmonella and Campylobacter as a cause of acute bacterial gastroenteritis. A significant increase in transfusion related infections of Y. enterocolitica has been reported, Tipple, et al., Transfusion 30, 3, p.207 (1990).

Y. enterocolitica and other bacteria which propagate at relatively low temperatures, e.g. Staphylococcus epidermidis and Legionella pneumophila, present, potentially, a serious threat in blood products.

Bacterial infections, generally, are a continuing concern to blood bankers and those who produce transfusion products from blood. Indeed, a national surveillance system for transfusion-associated bacterial infections has been called for, Editorial, Transfusion 30, 3, p. 193 (1990).

In addition to the risk of transmitting infectious disease via blood or blood products, the growth of bacteria in blood and blood products at various stages of production and processing introduces pyrogens into the blood component or product which must be removed before the product can be used in therapy. Introduction of molecular iodine, e.g. povidone-I₂, at an early stage in processing of blood products greatly reduces or eliminates the pyrogen-load of the ultimate product or fraction.

Protozoa give rise to many diseases, some of great medical and economic importance. Examples of such protozoa are the genus Plasmodium, e.g. P. falcipamm, P. malariae, P. ovale and P. vivax, which causes malaria, Trypanosoma, which causes Chagas' disease, and Leishmania, which cause a variety of leishmaniasis. The method of this invention is effective in eliminating these causative organisms in blood and blood products.

Generally, this invention is applicable to the treatment of donated blood and other red blood cell containing preparations for inactivating virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms.

The risks of infection from whole blood are well-known. Indeed, one of the great tragedies of modern medicine is the infection of many patients, most frequently hemophiliacs who require frequent blood transfusions, with HIV. The purification of the nation's and the world's whole blood for transfusion would constitute a monumental step forward in the history of medicine. The risks of infection from red blood cells is similar to comparable risks associated with whole blood.

The use of elemental iodine as an antiseptic dates back to 1839. It is used today for various medicinal purposes. The combination of iodine with various solubilizing polymers led to a class of new compositions known as iodophors, which dominate the market once satisfied by simple alcoholic or aqueous iodine solutions. The iodine complexes with either nonionic surfactants, eg, polyethylene glycol mono(nonylphenyl) ether, or poly(vinylpyrrolidone) (PVP). The complexes function by rapidly liberating free iodine in water solutions. They exhibit good activity against bacteria, molds, yeasts, protozoa, and many viruses; indeed, of all antiseptic preparations, only povidone-iodine is capable of killing all classes of pathogens encountered in nosocomial infections: gram-positive and gram- negative bacteria, mycobacteria, fungi, yeasts, viruses and protozoa. Most bacteria are killed within 15 to 30 seconds of contact. These iodophors are generally nontoxic, nonirritating, non-sensitizing, and noncorrosive to most metals (except silver and iron alloys). Medicinal povidone-iodine preparations include aerosol sprays, gauze pads, lubricating gels, creams, solutions, douche preparations, suppositories, gargles, perineal wash solutions, shampoos, and skin cleansers and scrubs. Povidone-iodine preparation are applied topically to the skin and to membranes, e.g. vaginal membranes, and in infected wounds and surgical incisions.

The uses continue to be largely medicinal, though some iodophors are used in industrial sanitation and disinfection in hospitals, building maintenance, and food-processing operations. There has been some interest in the use of iodine for purification of potable water and swimming pools. Two other iodine-containing compounds, p-tolyldiϊodomethyl sulfone and p-chlorophenyldiiodomethyl sulfone have been recommended as preservatives. Iodine and iodine-containing compounds and preparations are employed extensively in medicine, eg, as antiseptics, as drugs administered in different combinations in the prophylaxis and treatment of certain diseases, and as therapeutic agents in various thyroid dyscrasias and other abnormalities. Iodine is a highly reactive substance combining with proteins partly by chemical reaction and partly by adsorption. Therefore its antimicrobial action is subject to substantial impairment in the presence of organic matter such as serum, blood., urine, milk, etc. However, where there is no such interference, non-selective microbicidal action is intense and rapid. A saturated aqueous solution of iodine exhibits anti-bacterial properties. However, owing to the low solubility of iodine in water (33 mg/100 ml at 25°C), reaction with bacteria or with extraneous organic matter rapidly depletes the solution of its active content. Iodide ion is often added to increase solubility of iodine in water. This increase takes place by the formation of triiodide, I₂ + I = 13^". An aqueous solution of iodine and iodide at a Ph of less than eight contains mainly free diatomic iodine I₂ and the triiodide I₃ ^". The ratio of I, and I₃ depends upon the concentration of iodide.

An important solubilizing agent and carrier for iodine is polyvinyl pyrrolidone (PVP), one grade of which is identified as povidone USP. Povidone-iodine (PVP-iodine), is widely used externally on humans as an antiseptic. Such products are marketed as Betadine™ and Isodine™ by The Purdue-Frederick Co.). Povidone-iodine products and the preparation of such products are described in U.S. Patents 2,707,701, 2,826,532, and 2,900,305 to Hosmer and Siggia, assigned to GAF Corporation and in a number of GAF Corporation publications; see, e.g. Tableting with Povidone³A povidone USP (1981) and PVP Polyvinylpyπolidone (1982). Povidone-iodine powder contains approximately 85% PVP, 10 %* I, and 5%Iodide. A 10% solution of this powder contains 1% free, available iodine. (Gershenfeld, Am. J. Surgery 94, 938 (1957)).

Under ordinary conditions, PVP is stable as a solid and in solution. The single most attractive property of PVP is its binding capability. This property has permitted utilization in numerous commercial applications. Small quantities of PVP stabilize aqueous emulsions and suspensions, apparently by its absorption as a thin layer on the surface of individual colloidal particles. The single most widely studied and best characterized PVP complex is that of PVP-iodine. For example, hydrogen triiodide forms a complex with PVP that is so stable that there is no appreciable vapor pressure. It is superior to tincture of iodine as a germicide.

Although iodine is less likely to be consumed by proteinaceous substrates than bromine and chlorine, its efficacy as a disinfectant is still reduced at certain antiseptic applications. This is due to a reducing effect of the material to be disinfected which leads to the conversion of iodine into non-bactericidal iodide. Thus, not only the reservoir of available iodine is diminished but also the equilibrium of triiodide is influenced as well. Both of these effects cause a decrease in the proportion of free molecular iodine, the actual anti-microbial agent. When povidone-iodine preparations are contaminated with liquid substrata (e.g. blood, etc.) there is, in addition, the dilution effect characteristic of povidone-iodine systems which causes an increase in the equilibrium concentration of free molecular iodine. To what extent the latter effect compensates for the other two effects depends on the content of reducing substances. Thus with full blood, a strong decrease of the concentration of free molecular iodine occurs, while, in the presence of plasma, it remains practically unchanged. Durmaz, et al, Mikrobiyol. Bui. 22 (3), 1988 (abstract); Gottardi W, Hyg. Med. 12 (4). 1987. 150-154. Nutrient broth and plasma had little inactivating activity but 1 g hemoglobin inactivated 50 mg of free I; experiments with ¹²⁵I showed that uptake of I by [human] red cells occurred rapidly. Optimal antimicrobial effects in clinical use should be achieved in relatively blood-free situations. Povidone iodine produced a potent and sometimes persistent bactericidal effect towards bacteria on healthy skin. Lacey, R. W. J Appl Bacteriol 46 (3). 1979. 443-450. The bactericidal activity of dilute povidone-iodine solutions is inversely proportional to the concentration of the povidone-iodine solutions and is inhibited to the greatest extent by blood, followed by pus, fat and glove powder. Zamora J L; Surgery (St Louis) 98 (1). 1985. 25-29; Zamora, Am. J. Surgery, 151, p.400 (1986); see also, Waheed Sheikh, Current Therapeutic Research 40, No. 6, 1096 (1986). Van Den Broek, et al, Antimicrobial Agents and Chemotherapy, 1982, 593-597, suggests that povidone-iodine is bound to cell wall proteins leaving little for interaction with microorganisms in the liquid phase (See, also, Abdullah, et al., Arzneim.-Forsch./Drug Res. 31 (I), Nr. 5, 828). Ninneman et al, J. of Immunol. 81, 1265 (1981) reported that povidone-iodine was absorbed in serum albumin and it is know that povidone-iodine is bound to albumin but it has been discovered that the antibiotic activity of povidone-iodine is not destroyed by albumin bounding. Whether the activity remains because the albumin povidone-iodine is active or whether povidone-iodine and/or I₂ are released from the albumin- povidone-iodine complex.

The teachings of the prior art suggest that neither elemental (diatomic) iodine nor complexed iodine, e.g. PVP-I,, would be an effective and reliable biocide in a fluid or in a body, e.g. blood, packed or concentrated cells, organs, etc. in which massive amounts of protein are be available to react with the iodine.

Nor is there a teaching or suggestion that the addition of iodides to iodine increases the antimicrobial effect of iodine in the treatment of red blood cell containing preparations.

Summary of the Invention This invention relates to the treatment of red blood cell containing preparations, i.e. blood which is to be transfused as whole blood or tranfusable red blood cell containing products to inactivate or destroy infective pathogenic microorganisms with molecular iodine in the presence of iodide. The iodine is preferably absorbed by or in complex with an organic stabilizer.

Description of the Preferred Embodiments Contrary to the implications of the prior art, infective pathogenic microorganisms are inactivated when molecular iodine compound is added to whole blood in a concentration of from 0.5 wt % to about 1.5 wt %. The preferred range is from 0.5 to 1.0 wt %.^' Such blood can be used as whole transfusion blood used to produce red blood cell containing preparations which are free of pathogenic microorganisms. It has now been discovered that the antimicrobial effect of iodine in such preparations is greatly enhanced if the red blood cell containing preparation has been pretreated with iodide or, to a lesser extent, the iodine treatment is carried out in the presence of iodide.

In carrying out this invention, the iodide and the iodine compound, e.g. povidone-I₂, may be in the blood collection bag at the time the blood is collected. Preferably, however, the iodide is added first, e.g. is in the collection bag, followed by the addition of iodine, which may be in the

"addsol" bag which, commonly, is a separate bag containing an additive solution of a number of reagents which tend to stabilize the blood or prepare the blood for further processing. The capacity of the blood bag being known, e.g. one pint, the quantity of iodide, e.g. KI or Nal, and iodine, e.g. povidone-I₂ necessary to result in a concentration of I, in the blood of about 1 ± 0.5 wt % is provided in the solution bag. The synergistic enhancement of the antimicrobial effect of iodine is not dependent upon the presence of povidone; however, povidone-iodine compositions protect the red blood cells from the cytolytic effect of iodine and is thus preferred as a source of iodine. In the preferred embodiment of the invention the povidone:!, ratio differs very substantially from the standard ratio of about 2:1 povidone to I, and is preferably at least about 4:1 povidone to I,.

Treatment using povidone-I, does not adversely effect the oxygen carrying capacity of the blood nor does it prevent the clotting of blood. The invention is also embodied in the method of treating packed red blood cells with a molecular iodine compound in the presence of iodide or after treatment with iodide. Red blood cells may, of course, be treated as part of whole blood in the manner described above.

It is advantageous, however, to separate and pack the red blood cells and treat the packed cells with iodide and then with povidone-I₂ in a concentration range of from 0.001 to 0.5 wt %. The iodide may be added substantially contemporaneously with the iodine, though somewhat greater antimicrobial enhancement is achieved by pre-treatment of the red blood cell containing preparation with iodide.

Iodide, preferably in the form of potassium iodide or sodium iodide, is added in approximately the same concentration range as the concentration of iodine, i.e. in the range of from 0.001 to 0.5 wt percent, the preferred range being from 0.01 to 0.25 wt %. A concentration of 0.1 wt % I, added as povidone- 1, to red blood cell containing preparations which are previously or contemporaneously treated with iodide results in a total kill of bacteria and virus. The cells may, for example, be washed with an iodide containing solution and then treated with the molecular iodine compound or washed in a solution containing the molecular iodine compound and iodide.

In addition to the discovery that iodide enhances the antimicrobial effect of iodine in red blood cell containing preparation, it has been discovered that iodine can be used alone, as I₃ or as povidone iodine if suitable substances are included in the red blood cell containing preparation to provide an osmotic balance between the interior and the exterior of the red blood cells; i.e. to prevent undue dehydration or swelling of the red blood cells as a result of a differential in osmotic pressure between the interior of the cell and the liquid surrounding the cell. Povidone, it is believed, performs this function directly, i.e. in effecting the osmotic pressure by concentration, and by "sealing" the red blood cell surface membrane and thus reducing the rate of flow through the membrane. Calcium or magnesium salts, sulfate salts, and/or water soluble poylmers, e.g. hydroxyethyl starch, for example, may be used in red blood cell containing preparation treatment solutions. Upon entry into the cell and reaction with a microbe or a constituent of the cell, iodine becomes iodide ion, thereby changing the osmotic balance between the interior and exterior of the cell. Addition of molecules which are too large to enter into the cell, but increase the effective concentration of the fluid outside the cell may be used to restore the proper osmotic balance.

The iodine treatment of red blood cells may be conducted immediately after separation or immediately before transfusion into the patient, or at any intermediate stage.

It is very advantageous to treat the red blood cells with a solution of povidone-I, in which the povidone:L_> ratio is at least about 4:1. It has been found that this povidone:I, ratio there is negligible lysis.

It has also been found that the oxygen-pickup and carrying ability of the red blood cells is preserved by treatment of the cells, alone or as part of blood, with the povidone-I₂ solution described. Following treatment, the cells may then be washed to remove excess molecular iodine compound from the cell milieux.

Povidone-I, is an effective preservative solution, used as described above, for red blood cell preparations used as laboratory standards or panels in blood banking. The red blood cell containing preparations to be purified can be treated with iodide and then caused to contact or flow through a solid which exposes the preparations to molecular iodine. For example, whole blood to which potassium iodide has been added may be contacted with particles, a membrane or surface of solid povidone-iodine. Where a bed of particles is used, the particles must be large enough to permit intimate contact without acting as a filter, i.e. entrapping or binding the cells.

Polyvinyl pyrrolidone used in the preparation of soluble povidone- iodine preparations is polymerized to a molecular weight of from about 10 Kdaltons to 40 Kdaltons, 30 Kdaltons being a typical molecular weight. However, povidone-iodine preparations can be prepared using very much higher molecular weight polymers which only tend to swell rather than to dissolve in aqueous solutions. It is the use of these higher molecular weight Polyvinyl pyrrolidone polymers reacted with I₂ to form solid, substantially water insoluble povidone-iodine compositions that the present invention is directed. In carrying out this facet of the invention, the red blood cell containing preparation is contacted with the solid povidone-iodine. This may be done most efficiently, in most cases, b passing the liquid through a settled or fluidized or packed bed of povidone-iodine particles or by mixing the particles in a container of the liquid or passing the liquid over a surface of the povidone-iodine material, e.g. over a multiple-plate array of sheets of such material. The povidone-iodine may be washed and the I, content therein regenerated between uses.

The mechanism by which iodide enhances the antimicrobial activity of iodine not known. A possible mechanism is, however, postulated without any intention to be bound by the postulate. It is believed that iodine and iodide are bound to the cell surface membrane of red blood cells. Once bound, the iodine is less possibly less available and/or reacts with components of the cell to become iodide, which has little or no antimicrobial activity. Thus, if this postulate is correct, little iodine enters the cell and much of the iodine is not available for reaction with pathogenic microbes. According to the present postulate, iodide binds to the sites on the cell surface membrane, blocking those sites_* and thereby leaving iodine available as an antimicrobial agent.

Industrial Application This invention finds application in medicine and veterinary science.

Claims

WHAT IS CLAIMED IS:

1. A medicament comprising red blood cells treated to prevent the transmission of disease by adding an iodide compound in a concentration of from 0.001 to 0.5 weight percent to a red blood cell containing liquid, and contemporaneously or thereafter adding molecular iodine compound to the resulting red blood cell containing liquid in a concentration of from 0.001 to 0.5 weight percent and thereafter allowing contact of the red blood cell containing preparation with said molecular iodine compound for at least two minutes sufficient to inactivate or destroy infective pathogenic microorganisms.

2. The medicament of Claim 1 further comprising at least one water soluble compound which produces ions or molecules in solution too large to pass into the red blood cells.