CA2072871A1 - Preservation of blood, tissues and biological fluids - Google Patents

Abstract

The treatment and preservation of blood, blood derivatives and other body tissues, fluids and cells with povidone-iodine to kill pathogenic microbes without destroying the utility of the tissues, fluids and cells is disclosed.

Description

;-`` WO ~2/04031 PCl/US91/062 PRESERVATION OF BLOOD, Z~7~87 T~SSU~S AND BlOLO&lCAL FLU~)S
Background of tke invention This invention relates to the treatment and preservation of blood and 5 blood derivatives, the treatment and preparation of other body tissues and cells, the treatment and preparation of tissue cultures and tissue culture products, and the preparation of laboratory reagents, standards and samples. The treatment of this invention is to kill or inactivate virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisrns. The treatment 1() and preparation of human blood, tiss~les, etc. and of the blood, tissues, etc. of other animals are contemplated. In general, the field of this invention lies in medicine and veterinary practice; most examples being related to the practice of medicine for the benefit of human patients, use in analogous fields of veterinary medicine to the extent applicable being within the scope of the 15 invention.
Definitions The following terms, which are used throughout the specification, will be used and understood to have the meaning stated unless another or different meaning is specified or clear from the context.
Blood and blood derivatives. The term "blood" means whole 20 blood and blood fractions, components, and products of blood. Unless "whole blood" or a specific blood derivative, e.g. a blood fraction, component or product of blood is stated, the term "blood" may apply to whole~ blood at the ~ime of collection or a blood derivative at any stage in processing, as indicated by context. Blood derivatives mean blood components such as blood cell 25 concentrates ~red blood cells, platelets, etc.), plasma, and serum and products and factors prepared from blood such as albumin and the blood factors.
Body tisslles ~nd cells. Body tissues and cells means any tissue(s), organ(s~ or cells or ~uids which contain tissue(s), organ(s) or cellsof animal origin. Thus, in a broad sense, body tissues and cells include ~lood 30 and the cellular componen!s of blood; however, for the most part, simply for clarity in presentation, blood is treated as a separate application of the invention. Examples of body tissues and ceJls include sperm, bone marrow, W O 92/04031 PC~r/U~91/06240 Z~7?8t7~.

kidneys, cornea, heart valves, tendons, ligaments, skin, bone and homograf or xenograft implants and prosthesis generally.
Tissue and cell cultllres. Tissue and cell cultures means cells and tissues grown or enhanced in culture media and the culture media per se, but not including nutrients intended for use in cell cultures. An examples of a cultured tissue is cultured s};in tissue for use in burn victims. Cells and cellular producls prepared by standard biological and/or genetic engineering techniques are other examples of tissue cultures.
Laboratory reagents, standards and samples. Laboratory ~0 reagents and standards~ as used in this specification and the claims, meansreagents and standards produced from or comprising human or animal nuids, cells or tissues. Examples of such products are red blood cell panel utilized for typing blood, control sera and chemistry controls. Samples of tissues and fluids to be tested inclllde samples of blood, urine, sputum, cell smears, etc.
Donor. While the term "donor" is not usually applied to the individual fro~ whom such samples are acquired, that term, "donor" will be used here in a more general sense to include the individual from whom any blood, tissue, cells or fluid is obtained for any pu~ose, and such term will be used to refer even to an unwilling donor.
Povidone (USP) is used in the sense that it is used in the U.S.
Pharmacopeia to identify polyvinyl pyrrolidone suitable for use in physiolog-ically acceptable solutions.
Moleclllar Iodine Compound. The term "molecular iodine compound" is used in this patent to mean and include molecular iodine, I, diatomic iodine, 1~, or a compound or a inixture of compounds which either comprises iodine available in molecular form, iypically 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.
Povidone-lodine. Povidone-iodine is a complex of molecular iodine with polyvinyl pyrrolidone. Povidone-iodine complexes of the type under consideration have-been described in the literature and are marketed by The Purdue-Fredericli Co. When percent concentMtions are r~ferred to in ~ .

WO 92/04031 PCr/US~1/06240 :~ Z~ 37 connection with povidone-iodine, the percentage refers to the percent of povidone-iodine by weight, based upon the weight of the solution or material to which the povidone-iodine is added. Thus, a 1 weight percenl (~/o) solution of povidone-iodine indicates that enough povidone-iodine has been dissolved 5 to result in a concentration of l~/~ povidone-iodine. In most instances, povidone-iodine is added as a solution, e.g. 10% solution in water, pH about 1.5, but it can be added as a powder or otherwise. Povidone-iodine powder contains approximately 85% PVP, 10 % 1~ and 5%10dide. A 10% solution of this powder contains 1% free, available iodine. (Gershenfeld, Am. J. Surgery 947 938 (1957)~. The ratio of polyvinyl pyrrolidone to iodine in the povidone-iodine product used in the experiments referred to hereinafter is 8.5 parts of povidone-iodine per I part of active iodine. The product also contains about 0.5 parts of inactive iodine as iodide. Typical stock solutions are 10% (10,000 ppm 1,), 5% (5,000 ppm 1,) and 1% (100 ppm 1~). In those instances in which 15 a povidone to iodine ratio of higher than about 8.5 to l is referred to, additional povidone (polyvinyl pyrrolidone) is added to increase the PVP to 1~
ratio. The concentration of povidone-iodine in such compositions means the concentration of povidone-iodlne added as 8:5 to 1 PVP to l2 povidone-iodine.
GTPD triterpenoid compounds derived glycyrrhiza glabra or 20 analogous to such compolinds, the most important of which are carbenoxolone and glycyrrhizin.
Those who deal with blood and other invasively obtained body fluid samples risk infection from the samples. Those at risk include the doctor, nurse or clinical technician who takes the sample, the technicians who handle 25 the sample and who use the sample in conducting analyses and tests, those whohand1e the sampling and testing eqllipment and apparatus, and the entire chain of individuals who attend to the disposal of sampling apparatus and the like, from the individuals who pick up the used apparatus through those who ultimately dispose of the apparatus, usually in specially designed high 30 temperature filrnaces.
The risk is substantial, as evidenced by the fact that nearly all health care professionals with long experience carry the Epstein-Barr virus ~EBV) - .
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WO 92/04031 PCl /US91/06240 2~7~8~

and/or cytomegalovirlls (CMV), the latter being probably the most ubiquitous of the pathogenic viruses. Other pathogenic viruses to which health care workers, and those who handle blood and fluid sampling and handling apparatus, are exposed include hepatitis and human immunodeficiency virus S (HIV) as well as a large number of less life-threatening viruses.
Another organism which may contaminate blood and blood products or ~ractions and which presents a serious risk is the bacteria Yersinia enterocolitica. lt surpasses Shi~lla and rivals Salmon~lla and Campylobacter /as a cause of acute bacterial gastroenteritis. A significant increase in 10 transfusion related infections of Y. ~nt~r(7colilic~l has been reported, Tipple, et al., Transfusion 30, 3, p.207 (1990). Y. ~nt(~rocolitica and other bacte~ia which propagale at relatively low temperatures, e.g. Staphylococcus epidermis and Legi~n~lla pn~umnnophili~l, present, potentially, a serious threat in blood products.
Bacterial infections are a continuing concern to blood banking industry.
Indeed, a national surveillance system for transfusion-associated bacterial infections has been called for, Editorial, TransfiJsion 30, 3, p. 193 (1990).
]n addition to the risk of transmitting infectious disease via blood or blood products, the growth of bacteria in blood and blood products at various 2n stages of prodllction 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-iodine, at an early stage in processing of blood products greatly reduces or eliminates the pyrogen-load of the ultimate producI or fraction.
Protozoa give rise to many diseases, some of great medical and economic importance. Examples of s~lch protozoa which may be transmi~ted by blood transfusion are the genus Plasmocli~/m, e.g. P. falciparum, P.
malariae, P. ovale and P. viv~lx, which cause malaria, and ~rypanosoma. The method of this invention is considered to be effective in eliminating these causative organisms in blood and blood products.
Some vimses, e.g. hepatitis virus, are detected in the urine of infected individuals. A risk of infection of technicians begins with the collection of the wo 92/04031 P~r/US91/06240 ; .
2i~7~87l.
sample and continues until the sample is disposed of or treated to kill the virus.
This risk is virtually eliminated by the present invention.
Generally, this invention is applicable to the treatment of donated blood and products produced from blood, ~issues and fluids for inactivating virus, 5 bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms.
Among the important potential pathogens to which this invention is applicable is cytomegalovirus (CMV), probably the most ubiquitous of the pathogenic microorganisms found in animal fluids and tissues. CMV is 10 frequently associated with, and may be a causative or contributing factor in,life threatening disease in individuals with suppressed immune systems, and can be a principal causative factor in pneumonia, neurological disorders, febrile illness, ocular disease and hepatitis. CMV infection is a serious limiting factor in the transplantation of organs, tissues and cells and the transfusion of blood15 and plasma from one individual to another. The kidney transplant patient runsa high risk of contracting serious, and not infrequently fatal, CMV infection from CMV introduced by the transplant organ. Recipients of whole blood, plasma, bone marrow, cornea, cardiac, and semen run a serious risk of CMV
infectious disease, the risk being multiplied where the immune system of the 2() recipient is suppressed to prevent rejection of the foreign organ or cells, or where immunosuppression is present from natural causes.
This invention has application in preventing the transmission of herpesviruses generally. Herpesviruses, of which CMV is a member, represent a very large group of viruses which are responsible for, or involved in, cold 25 sores, shingles, a venereal disease, mononucleosis, eye infections, birth de~ects and probably several cancers. Three subfamilies are of particular importance The alpha subfarnily includes HV I (herpes virus simplex 1) which causes cold sores, fever blisters, eye and brain infections, HV 2 (herpes virus simplex 2) which cause genital ulceration, and HV 3 (HV varicella zoster) which causes 3n chicken pox, shingles and brain infections. The beta subfami~ includes HV
5, the principal member of which is CMV discussed ab-~ve. The gamma WO 92/04031 Pcr/us91/062~0 2~7Æ~ l subfamily includes HV 4 (Epstein-Barr) which cause infectious mononucleosis and is involved in Burkitt's lymphoma and nasopharyngeal carcinoma.
My United States Patent 4,891,221 describes and claims a method for inactivating virus in blood samples using glycyrrhizic triterpenoid compounds.
S While the use of glycyrrhizic triterpenoid compounds in blood product treatment is a major step forward, there remains a need for a method of treatment which would kill or inactivate all or nearly all pathogenic organisms,including those in the cells of the blood or blood products.
If a tissue is explanted into the culture media for the purpose of 10 propagating its cells, the procedure is called tissue culture whereas the explanting of individual cells into culture media would be called cell culture;
however, bo~h procedures are often referred to by the term "tissue culture"
procedures without differentiation, unless the distinction is critical for some ancillary reason. This general usage of the term is employed here.
Tissue cultured cells are extremely fragile in many ways, having exacting requirements not only as to nutrients but also to the amount and type of resident organisms which can be tolerated, and culture media are highly susceptible to bacterial and/Or viral infection.
Many viruses, in both animals and humans, may be transmitted by 20 artificial insemination using sperm from infected individuals. Bovine leucosis (Mateva, V. et al, Monatsh. Veterinaermed. 1987, 42(9) 310) and bovine rhinotracheitis virus are transmitted by sperm of infected bulls. (Kupfer-schmied, H. U., et al Tl7eriogenolo~y 1986, 25(3) 439). Singh, E.L. ((lOth Int. C~ . onAnimalRepr. an~/Arfificiallnseminalion, Cong. Proc. V. I-IY, 25 1984) concluded that some viruses, e.g. bluetongue virus (BTV), infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), foot and mouth vims (FMDV), akabane virus (AV) and bovine parvovirus (BPV), were transmitted via seminal fluid rather than in the sperm cell.
HBV DNA was been found in spermatozoa by Hadchouel et al who 30 concluded thal hepatitis B virus could be transmitted by true vertical transmis-sion of HBV via the germ line ~Hadchouel, M. et al J. Mecl. Virol. 1985, 16(1) 61) and Ayoola. E.A., et al (Int. J. Gynaect)l. Obstet. Ig80, 18(3) : ' ~

wo 92/0~031 Pcr/us91~062 7 2~87~
185) concluded that the hepatitis B virus could be transmitted by sexual intercourse via HBV in semen or sperm of the male. Since cytomegalovirus (CMV) infections rnay alter host defense to a variety of pathogens (Miller, S.A., et al, (Infect. Immun 1985, 47(1) 605), it is doubly important to avoid S introduction of (:MV with a transplanted organ or tissue or with sperm cells as the result of artificial insemination.
The treatment of the preparalion and handling of sperm, both human and animal, is fraught with risk of infection. Sperm is quarantined for several months and the donor's health is followed to assure that the donor is not 10 infected with a pathogenic microbe. In the case HIV and hepatitis, for example, and nnany other diseases, the donor may carry the disease-causing organism for months or years without showing any symptoms of the disease It would be an important step forward to be able to inactivate or destroy pa~hogenic microbes to preveni infectlon of the artificial insemination recipient It is, generally, impossible to defme with precision the exact materials required to propagate a given cell line and, therefore, it is common practice touse media based upon or containing serum and to add nutrient serum as needed during the cell propagation. Bovine serum from adult animals may be suitable in some instances, but fetal bovine semm (FBS) (sometimes referred to as fetal 20 calf serum (FCS)) is required for the safe propagation of many cell lines, and where high purity is critical. Even the use of FBS is not, however, a guarantee of freedom from infective agen~s. Indeed, every lot of commercially produced FBS is contaminated with infectious bovine viral diarrhea (BVD) virus and infections with infectious bovine rhinotracheitis (IBR), parainfluenza25 3 (Pl 3) are extremely common. At best, pools of raw serum probably contain at least 10~ infectious BVD vims particles per milliliter.
Semm filtration is a common step in reducing the load of infectious organisms in serum, but serum quality can be damaged by filtration if significant amounts of serum components are adsorbed to the filters or if 30 macromolecules are sheared. Shearing of macromolecules during filtration occurs generally when tangential flow filtration is used and turbulence :
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wo 92/04031 Pcr/us91/l)6240 2~7,?.~3 ,~.~ X
develops. It is cllrrently very diffic~llt to obtain reliable results on the removal of BVD viruses from serum using filtration.
The presence of adventitious viruses in cell cultures is well recognized, and when the cllitures are of primate origin there are serious hazards for the 5 production of human viral vaccines. This is one reason for the increasing use of bovine cell cultures. These cultures, however, are not free from viral contamination. Calf kidney (CK) and calf testis (CT) cells were often infected by non cytopathic mucosal disease virus (MDV): the cells seemed mo?hologi-cally healthy, but nearly all showed fluorescence with BVD antiserum and ]n rabbit anti-bovine conjugate.
Blood plasma is used in the prodllction of many important blood fractions, components and prodllcts. Transfusion plasma, per se, is frequently prepared as a single blood bag prodllct; however, many plasma fractions and products are prod~lced from large pools of plasma. There is a real and serious 15 risk of infection to the technicians who handle individual blood bags and serum bags, and the risk of infection is multiplied many times in the handling of pooled plasma. There is, of course, a serious risk that the recipient of plasma or a plasma fraction or product may be infected unless suitable steps are taken to kill or inactivate potentially pathogenic organisms. Such steps are usually 20 taken far down the chain of processing steps and frequently as the final step before use, storage or Iyophilization, according to the product.
The production of pyrogens in plasma and plasma products during initial handling or handling down-stream in the process chain by the propaga-tion of organisms which, at a later stage in processing, are inactivated or killed 25 constitutes a serious problem to producers of plasma fractions and products.
Pyrogen production could be eliminated or substantially reduced if pyrogen producing organisms were killed early in the process, e.g. in the initial whole blood or in the pooled plasma.
Virus infections, among the most serious being hepatitis, present a 30 constant and serious risk to both handlers and recipients of blood and blood products. It has been shown that fractionation workers, particularly those engaged in the preparation of plasma pools, are a~ high risk of developing wo 92/04031 Pcr/us91/06240 7,~8 hepatitis B. The hi~h ris}~ products are fibrinogen, AHF, and prothrombin complex. The low risli products are ISG, PPF, and albumin. The lack of infectivity of PPF and albumin is attributable to heating the final products at 60~C. for 10 hours; however, such process steps tend to denature certain products and are unsuitable in the preparation of heat sensitive products.
It is now required in the United States that a]l donors of blood or plasma be tested for the presence of hepatitis B surface antigen by radioimmu-noassay or reversed passive hemagglutinalion. This screening reduces but does not prevent the ~ransmission of hepatitis B virlIs. A major problem is the transmission of non-B hepatitis, for which there is no screening test. Recent evidence indicates that non-A, non-B hepatitis is caused by one or more viral agents. Even if adequately sensitive and reliable tests were available, testing,alone, does not result in a pathogen-free blood supply.
Other diseases which can be transmitted from the donor(s) to the patient(s) include the numerous diseasés in which the causative pathogen appears in viable form, at least dllring one stage of development, in the blood,fluids or tissues of the donor. The risk can be reduced by screening potential donors and refusing to accept blood, tissue or fll~ids for transfer to patients;however, the availability of blood, blood fractions and products, tissues and fluids could be very greatly increased and the cost thereof greatly decreased ifall potential donors could be accepted followed by killing all potential pathogens in the donated blood, fluid or tissue.
The risks of infection from whole blood are wel1-known. (:)ne of the great tragedies of modern medicine is the infection of many patients, most frequently hemophiliacs who reqllire frequent blood transfusions, with HIV.
The purification of the nation's and the wor!d's whole blood for transfusion would constitute a monumental step forward in the history of rnedicine. The risks of infection *om red blood cell concentrates is similar to comparable risks associated with whole blood.
The use of elemental iodine as an antiseptic dates back to abou~ 1839.
-~ It is used today for various medicinal purposes. The corr.bination of iodine with various solubilizing polymers led to a class of new compositions known :

WO 92/OqO31 PC~/I,IS9l/062~0 2a;7~8~
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(vinyl-pyrrolidone) (PVP). The complexes function by rapidly liberating free iodine S in water solutions. They exhibit good activity against bacteria, molds, yeasts, protozoa, and many viruses; indeed, of all antiseptic preparations suitable ~or direct use on humans and animals and upon tissues, only povidone-iodine is capable of }~illing all classes of pa~hogens: gram-positive and gram-negative bacteria, mycobacteria, fungi, yeasts, vinlses and protozoa. Most bacteria are 10 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, ga~lze pads, I~lbricating gels, creams, solutions, douche preparations, sllppositories, gargles, perineal wash solutions, shampoos, and 15 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 20 interes~ in the use of iodine for purification of potable water and swimming pools. Two other iodine-containing compounds, p-tolyl diiodomethyl sulfone and p-chlorophenyldiiodomethyl sulfone have been recommended as preserva-tives.
- lodine and iodine-containing compounds and preparations are employed 25 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. Iodineis a highly reactive substance combining with protPins partly by chemical reaction and partly by adsorption. Therefore i~s antimicrobial action is subject30 to substantial impairment in the presence of organic matter such as serum, blood, urine, mil};, etc. However, where there is no such interference, non-selective microbicidal action is intense and rapid. A saturated aqueous solution Wo 92/04031 Pcr/uS91/~6240 1 ] ~ 87~1.
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.
S This increase takes place by the formation of triiodide, I~ + 1 = ]3. An aqueous solution of iodine and iodide at a Ph of less than 8 contains mainly free diatomic iodine 12 and the triiodide 13. The ratio of 12 and 13- 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 BetadineT" and Isodine~. 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 GA~ Corporation publications; see, e.g. Tableting with Povidone USP (1981) and PVP Polyvinylpyrrolidone (1982~. Povidone-iodine powder contains approximately 85% PVP, 10 % I2 and 5 ~Iodide. A 10% solution of this powder contains I % free, available iodine. (Gershenfeld, Am. 1. Surger~ 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 numerolls commercial applications. Small quantities of PVP stabilize aqueous emulsions (qv) and suspensions, apparently by its absorption as a thin layer on the surface of individual colloidal particles The single most widely studied and bes~ characterized PVP complex is that of PVP-iodine. For example, hydrogen triiodide forms a complex with PVP that is so stable ehat 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 W O 92/04031 PC~r/US91/06240 2a~7,~8~7~

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 contami-nated with liquid substrata (e.g. bloocl, etc.) there is, in addition, the dilution effect characteristic of povidone-iodine systems which causes an increase in theequilibrium 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 I0 free molecular iodine occurs, while, in the presence of plasma, it remains practically unchanged. Durmaz, e~ al, Mikrobiyol. Bul. 22 (3), 1988 (abstract);
Gottardi W, Hyg. Med. 12 (4). 1987. 150-154. Nutrient broth and plasma had little inactivating activity bul I g hemoglobin inactivated 50 mg of free I;experiments with '~51 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. Zarnora J L; Surgery (St ~ouis) 98 ~1). 1985. 25-29; Zamora, Am. J. Surgery, I51, 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 celi wall proteins leaving little for interaction wi~h 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, 12~5 (1981) reported that povidone-iodine was absorbed in serum albumin and it is known that povidone-iodine is bound 3n to albumin but it has been discovered that the antimicrobial activity of povidone-iodine is not destroyed by albumin binding. Whether the activity remains because Ihe albumin povidone-iodine is active or whe~her povidone-..

` W O 92/0403~ PC~r/US91/06240 2~7~8'~
iodine and/or iodine are released from the albumin-povidone-iodine complex has not been determined.
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 5 biocide in a fluid or in a body, e.g. biood, packed or concentrated cells, organs, etc. in which massive amolmts of protein are be available to react with the iodine.
The use of povidone-iodine as a spermicide is known and one would not consider povidone-iodine as a candidate for killing pathogenic microbes in 1() sperm-carrying liquids.
Various medical and blood handling procedures are referred to hereinafter. These are all well-l;nown procedures and steps in these procedures are fully described in the literature. The following references are provided forgeneral background and as sources for detailed reference to the literature as toI5 specific procedures: TECHNICAL MANUAL of the American Association of Blood Bankers, 9th Ed. (1985); HLA TE(:HNIQUES FOR BLOOD
BANKERS, Arnerican Association of Blood Bankers (1984); Developments in Biological St;lnd~rdizsltion, Vols. I - 57, S. Karger, Basel; CL~ICAL
IMMUNOCHEMISTRY, The American Association for Clinical Chemistry;
20 MEDICINE, Vols. 1 - 2, Scientific American, New York; Care of the SURGICAL PATIENT, Vols 1 - 2, Scientific American, New York;
CUlRRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley-lnterscience, John Wiley & Sons, New York.
.
Summary of the Invention ~This invention is embodied in, inter alia, a method of testing ~ody fluids wherein body fluids are collected from a donor human or animal into a container and thereafter subjected to testing to determine physical, chemical orbiological characteristics of such fluids or constituents thereof. This is an improvement in the collection of such fluids to prevent the transmission of disease. The improvement comprises removing a sample of body fluid from the donor, mixing the body fluid sample substantially at the time the sample wo 92/04031 Pcr/ US9 1 /Os240 2~87~ 14 is taken wi~h molecular iodine compound in a concentration of from about O. I
to 5W/o (lO0 to 5,000 ppm 1~) and allowing contact with said molecular iodine compound for at least two minutes sllfficient to inactivate or destroy infectivepathogenic microorganisms and thereafter subjecting the sample to testing. In this and all embodiments of the invention the molecular iodine compound is preferably povidone-iodine.
This invention is embodied in, inter alia, a vacuum tube for collecting body fluids to be tested and preventing the transmission of disease from such body fluids. The vacuum tube comprises a sampling tube, means for directing a sample of body fluid into the sampling tube, and molecular iodine compound in the sampling tube in sufficient amount to inactivate or destroy infective pathogenic microorganisms.
This invention is embodied in, inter alia, a method of treating patients with blood cell concentrates comprising the steps of collecting blood from a donor, concentrating selected blood cells, and thereafter infusing the blood cells into the patient to be treated. The improvement of this invention comprises the additional steps of mixing the blood cells with molecular iodine compound in a concentration of from about 0 IW/o to about 2W/o (100 to 2000 ppm 1.) and allowing contact of said blood with said molecular iodine compound for at least two minlltes sufficient to inactivate or destroy infectivepathogenic rnicrobes. The molecular iodine compound is preferably povidone-iodine and, preferably, the step of mixing of the blood with povidone-iodine may be carried out in sub-steps. The sub-steps are, first, introducing povidone-iodine into the blood in a concentration of from about 0. IW/o to about2W/o (100 to 2000 ppm 1,); second, maintaining the blood in contact with the povidone-iodine for a period of about one to two minutes; and, third, again introducing povldone-iodine into the blood in a concentration of from about O.lW/o to about 2"/o (100 to 2000 ppm 1,).
This invention is embodied in, inter alia, a method of treating patients 3n with blood comprising the steps of collecting blood from a donor, and thereafter infusing the blood into the patient to be treated. The improvement of this invention comprises the additional steps of mixing the blood with W O 92/04031 PC~r/US91/06240 ! : .
1S ~7.28~
molecular iodine compound in a concentration of from about o ]W/O to about 5~/o (100 to 5000 ppm 1,), preferably 0.5`'/O to 2U/o (500 to 2000 ppm 12), and allowing contact of said blood with said molecular iodine compound for at least two minutes sufficient to inactivate or destroy infective pathogenic microbes.
S The mixing of the blood cells with po\~idone-iodine may be carried out in sub-steps, namely, first, introducing povidone-iodine into the blood cells in a concentration of from about O.l"/o to abou~ 5wlo (l00 to 5000 ppm I~) preferably 0.5W/o to 2W/o (500 to 2000 ppm l~); second, maintaining the b]ood cells in contac~ with the povidone-iodine for a period of about one to two 10 minutes; and, third, again introducing povidone-iodine into the blood cells in a concentration of from about o lw/o to abollt ~/o (lO0 to 2000 ppm 1~).
This invention is embodied in, inter alia, in an improvement in the treatment of patients using transplant tissue wherein a tissue is collected froma donor, washed and thereafter implanted into the patient under treatment. The 15 improvement comprises infusing said transplant tissue with a solution comprising molecular iodine compound in a concentration of from about 0. lW/o to about lWlo (lO0 to l000 ppm 1.) and allowing contact of said tissue with said rnolecular iodine compound for at least two minutes sufficient to inactivate or destroy infective pathogenic microorganisms.
This invention is embodied in, inter alia, in a method for the indllction of pregnancy into a female comprising the steps of collecting sperm cells from a donor, washing the sperm cells and thereafter inseminating the sperm cells into the uterus of the female. The improvement comprises the steps of washing the sperm cells in a solution of povidone-iodine in aqueous solution, e.g. buffer, in a concentration of from about 0. l`'/o to about lW/o (100 to 100ppm I~) sufficient to kill bacteria, virllses and other pathogenic micro-organisms but insufficient to inactivate the sperm cells.
This invention is embodied in, inter alia, a method of controlling a cell line comprising adding povidone-iocline to the cell line nutrient in a concentra-tion of from about O.lW/o to abo~lt l"/o (100 to 1000 ppm 1~) based on the nutrient sufficient to arrest or inhibit the propagation of the cell line but -. ;, : .: ., .. : :. , . : ~ .. . . - . .... ,. ~ . . .

WO 92/04031 PCI/US91/1~240 2~ ~,287~.

insufficient to kill the cells of the cell line and harvesting the composition of interest after such composition has been expressed by the cell line.
This invention may also be embodied in a method of preserving blood cells comprising adding povidone-iodine to the cell-containing milieu in a S concentration of from abollt O.lW/o to about l`'/o (100 to 1000 ppm 12) sufficient to arrest or inhibit the principal metabolic functions of the blood cells but insufficient to ~;ill the blood cells.
This invention is embodied in, inter alia, a method of purifying liquid or cell-bearin" liqllid comprising contacthlg the liq~lid to be purified into n contact with solid povidone-iodine having sufficient surface area to expose the liquid to sufficient iodine on sllch surface to liill pathogenic organisms therein, and removing the liq~lid from conta~t with the solid povidone-iodine. The method may further comprise reacting the sur~ace of the solid povidone-iodine with iodine between llses to regenerate the iodine content thereof.
This invention is embodied in, inter alia, a method of treating a patient which comprises the step of collecting a transplant or transfusion biological material from another human or animal, preserving the biological material, thawing the biological material and transfllsing or transplanting the biologicalmaterial into the patient. According to the present invention, the step of 2n preserving the biological material comprises disinfecting the transplant or transfusion biological material in a sollltion of from about 0 IW/o to about 1W/o povidone-iodine ~100 to 1000 ppm 1~) and maintaining the biological material under refrigeration emersed in a sollltion of povidone. The biological material may be frozen for preservation in a sollltion of povidone.
This invention is embodied in, inter alia, a method of disinfecting blood derivatives comprising treating blood (using the term in the broad sense ~o include blood derivatives) before separation of the components ~hereof with povidone-iodine to provide from about 0. IW/o to about 2W/o povidone-iodine (11)0 to 2000 ppm 1,) in the blood, preparing a derivative of the blood from thepreceding step and treating the derivative from the next preceding step to provide from abollt 0.1`'/~- to abollt 2"/o (100 to 2000 ppm l,j povidone-iodinein the derivative.
,~'., :' W O 92/04031 PC~r/US91/06240 17 Z~8~
This invention is embodied in, inter alia, a drug delivery material comprising blood cell concentrate wherein the cell walls of the cells have been opened by treiatment with from abollt 1 to abollt 5W/o povidone-iodine, a drug has been introduced into the cells through passages produced by the povidone-S iodine treatment, and the cell walls have been sealed by heating the cells to from about 42 to about 48 C.
Povidone has been follnd to possess a sufficient cytophylactic effect upon blood cells, e.g., red blood cells, and upon other cells and tissues to protect cells and tissues from the cytolytic effect of iodine when the ratio of PVP tO It iS sufficiently high, e.g. abollt 1~:1, preferably at least about IS~
(by weight) or higher. The preferred range of povidone to iodine ratios is from about 15:1 to 30:1, but ratios as higll as 60:1 are considered sui~able Higher ratios may be used but are of no greater advantage.
Povidone has been found to possess a significant virucidal effect without iodine accomplishing a l;ill of virus of from 2 to 5 logs.
Povidone has been found to cause a more rapid sedimentation of cells from liquid and to result in a better separation and differentiation of the cells It has also been discovered that there is an ultimate synergistic antimicrobial effect when It and glycyrrhizic triterpenoid compounds (GTPD's) such as carbenoxolone or glycyrrhizin are used together.
It has also been discovered that povidone-l compounds can be used to protect and preserve blood and blood-derived products which are used in diagnostic tests without altering the test results, with the single exception of an lncrease In lodlne.
Whole blood which is to be transfused as whole blood or frorn which transfusable blood products are produced are treated with molecular iodine, - e.g. povidone-iodine, in one or plural povidone-iodine treatment steps to inactivate or destroy infective pathogenic microorganisms. For example, this invention relates to the treatment of blood cell concentrates to inactivate or destroy infective extracellular and intracellular pathogenic microorganisms withmolecular iodine, preferably absorbed by or in complex with an organic stabilizer s~lch as povidone. Plasma, in sing!e bags or in pools, is treated to .: , ... .. , ~. . .~ . . . .

WO g2/04031 Pcr/US9l/062 ~7,?~ 7~
kill potential pathogenic organisms and organisms which, if not killed early in the chain of processing, introd~lce pyrogens into plasma and blood product.
In a preferred form, the inven~ion comprises the plural-step addition of molecular iodine, preferably in the form of povidone-iodine, to whole blood S initially or specific blood derivatives. The initial addition of povidone-iodine inactivates pathogens and pyrogen-causing organisms which are readily available in the serum and some cell-attached organisms. A significant portion of the first step povidone-iodine addition becomes bound to albumin and cell membranes and is not fully efficacious in the instant kill of microbes. The first 1() povidone-iodine addition is, however, biostatic, i.e. it prevents the multiplica-tion of microbes. The first povidone-iodine also renders the residual microbes more susceptible to the microbicidal effects of iodine. The integrity of the cellular components of blood is protected by the known cell-stabilizing effect of PVP. The second, and subsequent, povidone-iodine additions is very highly lS effective in killing microbes which survive the first step addition of povidone-iodine and has even less tendency to cell-attack, e.g. inducement of hemolysis, than would be the case if the entire load of povidone-iodine were added at one time. If, however, time is an important factor, povidone-iodine having a higher than the normal 2:1 ratio of povidone to iodine may be added at a 20 sufficiently high level, about one percent, to whole blood to effect a complete kill of microbes in both ~he sera and cellular components of blood with minimal hemolysis and without significant detrimental effect on the oxygen-carrying capacity of the red blood cells. It is also within ~he scope of the inventive concept to add moleclllar iodine, e.g. povidone-iodine, to plasma, 25 serum, cryoprecipitate, factors and cell concentrates and any other blood derivative whether or not povidone-iodine has been added previously or to a source of the derivative. In general, concentrations of above about 0.5 weight percent (WlO) (500 ppm 1, ) povidone-iodine, e.g. generally from about 0.8~/o to about 2W/o~ (800 to 2,000 ppm 1~) are reqllired to effect a complete kill of 3n microbes in whole blood. Somewhat lower additions, e.g. 0. IWlo to about 1Wlo(100 to 1000 ppm 17) are sufficiellt to eliminate viable microbes from plasma, semm and other blood derivatives. Two-step additions of povidone-iodine may .

wo 92/04031 PCr/US91/06240 1~ 2~ f'.~8~.
be preferred, the ratio of the first step addition to the second step addition being from about 1:1 ~o 3:1. For example, a first addition of 0.6W/o povidone-iodine (600 ppm 1, 3 followed by a second addition, a few minutes or more later, of 0.2`'/o (200 ppm 1, ) povidone-iodine is preferred over a single 5 addition of ]W/O povidone-iodine. Cell concentrates may be washed with and/or stored in povidone-iodine solutions of from about o IW/o to about IW/o (100 to 1000 ppm 12).
Body tissues and cells are washed with and/or stored, with or without freezing, wilh povidone-iodine. Renal, cardiac, cornea, and marrow tissue, 10 and any other tissue intended for transplantation are washed to kill infective pathogenic microorganisms. Povidone-iodine concentrations of from about 0.1~V/o to about l~/o (100 to 1000 ppm 1,) generally are suitable for washing and/or for storage. Povidone to iodine ratios of 15:1 or higher may be preferred. Complete organs, e.g. kidneys, etc. may be treated according to 15 this invention. Certain organs, e.g. ground bone, are sometimes comminuted used to promote healing and reconstruction in various surgical procedures.
Individual cells such as sperm cells may be treated to kill pathogenic microbes with tolerable or no impact upon the viability and motility of the cells. Such tissues, whether or not complete organs, are prepared, by treatment with 20 povidone-iodine solutions at one or more stages. The organ may, for example, be placed, upon removal, in a solution of from about 0.]W/o to about IW/o, or higher, povidone-iodine andtor treated at a later stage, e.g. during comrninu-tion or other processing, or immediately before use with povidone-iodine. The tissue bathed or saturated in, or substantially entirely wetted by, an infusion or 25 perfusion solution which comprises molecular iodine, preferabiy absorbed by or in complex with an organic stabilizer, e.g. povidone-iodine. ]n addition to the washing or perfusion of tissues and cells, povidone-iodine solutions serve to prese~ve the tissue, organ or cells during storage above freezing, usually atabout 4 C., or during freezing, frozen stoMge and thawing. The povidone-30 iodine treatment may be one-step or involve plural contact with povidone-iodine, such as immediately upon collection or acquisition, andlor during treatment and preparation, and/.,r just before use.

WO 92/04031 Pcr/~Jssl/o624 ;2~7~7~ 2() Biological cells, ~isslles and flIIids from animal or human donors are used in a virtually infinite variety of laboratory tests as controls or slandards or in rea~,ents. lt is important to protect the technicians who prepare and use these ma~erials from infection from pathogenic microbes contained in these biological maIerials. It is, of course, of great ilnportance that the biologicaland biochemical characteristics of importance in the ultimate product be protected during processing to maintain the integrity of the ultimate test.
Povidone-iodine additions to the biological material at the time of collection, typically in a concentration of from abo~lt 0. IWlo to about lW/o protects worksthrough the entire chain of handling. Subsequent additions, or delaying the initial addition of povidone-iodine, can be ~Ised to effect a complete kill of all microbes in a given product being Inanufac~ured from about the original biological material. For example, povidone-iodine added to blood or red blood cell concentrates in a concentration range of from about 0 lW/o to about lW/o effectively kills all pathogenic organisms and not only protects the specific binding determinants on the red blood cells but actually enhances the antigenic reactivity of the cells.
Brief Description of the Drawing Figure l depicts a vacuum tube blood sampling apparatus modified to ~0 embody the present invention.
Figure 2 depicts an alternative form of the vacuum tube of this invention.
Description of the Preferred Embodiments A number of non-limiting exemplary embodiments of the present invention are given hereinafter, it being clearly stated that these are simply examples and are not limiting as to the scope of the inventive concept.
Samples of blood, tissue and body fluids are, preferably, treated with povidone-iodine at the time the sample is taken or shortly thereafter to inactivate or destroy infective pathogenic microorganisms.
Bndy flllids ~ener~llv, e.g. Urine The risk of contMcting AIDS and other microbial infections from urine samples, serum, peritoneal fluid, etc., isvirtually eliminated by collectino the liquid sarnple into a povidone-iodine-.:

.. . . : , . ,.. ; ,,- . .. . : ., , , :, .. . .

3 1 P~r/uC7sl /062 2~'7 ~1 containing solution or adding povidone-iodine, in a concentration of from about 0 1W/o to about P~/o, (lO0 to lO00 ppm 1,) at the time of collection.
nlood and blood derivDtives - Snmples for Testin~. Blood collection is conventionally carried out using a variety of containers and devices.
S Exemplary are the various types of devices known in the industry as "vacuum tubes" which comprise an invasive needle which is inserted into the vein, or other fluid containing body structllre, a test tube for collecting the sample, and a syringe-like sealing device for causing a vacuum to form inside the test tube when the tes~ tube is moved relative to the sealing device. The interior of the test tube is in fluid communication, through the needle, with the vein or fluid containing body structure, thereby causing the blood or fluid to flow into the test tube for further handling and testing. According to this invention, the test tube contains a predetermined amount of a molecular iodine compound.
In carrying out this method, conventional blood collection containersare preferably used; however, the manner of collection is of no consequence vis-a-vis the effectiveness of this invention. lmportantly, the analyses and tests on the samples are carried out without modification or adaptation. Furthermore, the use of povidone-iodine does not interfere with the action of blood additives, e.g. ethylene diamine tetraacetic acid, acid citrate dextrose and heparin.
Figure l depicts a vacullm tube blood sampling apparatus modified to embody the present invention. The vacullm tube ] 0 is provided with a sealing septum 12 which maintains a vacull1n in the lube, as is conventional and well known. According ~o ~his invention, however, a molecular iodine reagent is also included in the vacuum tube as indicated at 14. In this embodiment, the 2~ molecular iodine reagent is depicted as a crystal or powder which will dissolve in the blood sarnple; however, the compound may be a liquid or solution.
This invention is, thus embodied in a sampling vacuum tube which comprises a chamber for receiving the blood, a septum for receiving and sealing around a needle, the septum sealing the chamber and maintaining the chamber under a reduced pressure, i.e. vacuum, and a molecular iodine compound.

- .

~WO 92/04031 P~r/l~lS9l/062~30 The vacullm tube modified to embody the invention as described is used in conventional manner. For example, the needle assembly 20, which is conventionally received in sleeves protecting ends of the needle 22 maintaining -~sterility and protecting the user from injury, is screwed into the holder cylinder 40, the threaded portion on sleeve 24 being threadably received in the internally threaded passage 26 in the holder 40. The vacuum tube is moved, as shown by the arrow, such that the septllm 12 receives, is punctured by and seals around the needle 22, pushing the resi]ient sleeve 30 off the end of the needle. This operation, which is conventional, is done after the distal end of 10 the needle 2~, shown a~ the left in the figures, is inserted in the patient's blood vessel. The vacullm in the tube draws the sample of blood and the vacuum tube is removed, allowing the slee~!e 30 to re cover the proximal end of needle 22. This may be repeated with as many vacuum tubes as desired for multiple sampling .
In a preferred embodiment, the vacuum tube comprises a gel plug 200 formed of a hydrophilic polymer such as hydroxyethylmethacrylate, methoxy-methylmethacrylate, carboxymethyl celluiose, agar, etc. In use, as the tube is centrifuged, the red blood cells flow by centrifugal force in the annulus between the plug and the interior wall of the tube and are separated from the plasma. These tubes are known in the prior art.
This operation may be carried out using the vacuum tube of this invention in the conventional manner after which the blood sample is thoroughly mixed with the molecular iodine compound in the sampling tube.
Quite surprisingly, it has been discovered that molecular iodine compound, e.g. PVP 1" in concentrations of from about 0. 1 to 5~/o (100 to 5000 ppm 1~) do not alter the results of conventional diagnostic tests nor causesignificant hemolysis or other change in red blood cells. In generally, conventional diagnostic procedures may be followed without alteration to accommodate to the presence of the iodine.
Infective pathogenic microorganisms are inactivated when molecular iodine compound is added to whole blood in a concentration of from about 0.5W/o to about 5V/o (500 to 5000 ppm 1~). The preferred range is from about .. . .. .

WO 92/041)31 PCl'/US91/06240 i 73 Z~7~87~
0.5W/o to 2~/o ~500 tO 2000 ppm 1~). Such blood can be used as whole transfusion blood or fractionated to prodllce an entire family of blood productswhich are free of pathogenic microorganisms.
In carrying out this invention, the povidone-iodine may be in the blood S collection bag at the time the blood is collected bu~, preferably, is the preservative additive solution 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 linown, e.g. one pint, the quantity of povidone-iodine 1() necessary to result in a concentration of iodine in the blood of about 0.5W/o to about 5~/o is provided in the sollltion bag.
In the preferred embodiment of the invention the povidone-iodine ratio differs very substantially from the standard ratio of about 8.5:1 povidone ~o iodine and is preferably at least about I :1 and preferably from about 15:1 to 60:1 povidone to iodine.
Treatment using povidone-iodine does not adversely effect the oxygen carrying capacity of the blood nor does it prevent the clotting of b]ood.
Povidone-iodine is compatible with and its action is not interfered with by the polyrners of which blood collection and handling bags are made.
Blood Cell Concentrates Blood cell concentrates may, likewise, be treated with a molecular iodine compound to kill pathogenic microbes. Blood cells may, of course, be treated as part of whole blood in the manner described above.
It is advantageous, however, to separate and concentrate blood cells and treat the concentrated cells with povidone-iodine in a concentration range of from abou~ 0. lW/o to about 5W/o . The application of from about 0.5W/o to about 2''/o iodine as povidone-iodine results in a total kill of bacteria and virus in packed cells and blood clots. The cells may be washed and then treated with the molecular iodine compound or washed in a solution containing the molecular iodine compound. The iodine treatment of red blood cells may be conducted immediately after separation or immediately before ~ransfusion into wo 92/04031 Pcr/us9l/o62qo ~; the patient, or at any intermediate stage. A povidone to iodine ratio of from about 15:1 to60:1 ispreferred.
Red B!ood Cell Concentrates It is advantageous to treat the red blood cells with a so1ution of povidone-iodine in which the povidone:iodine ratio is S at least about 15:1. lt has been founcl that this povidone:iodine ratio there is negligible lysis. The oxygen-carrying capacity of the red blood cells is preserved by treatment of the cells, alone or as part of blood, with the povidone-iodine solution. Following treatment, the cells may, if desired, be washed to remove excess molecular iodine compound from the cell milieu;
I() however, residual iodine and povidone are well tolerated and, ordinarily, need not be removed.
Povidone-iodine is an effective preservative solution, used as described above, for red blood cell preparations used as laboratory standards or panels in blood banking. It has been discovered that .reatment of red blood cells with 15 povidone-iodine as described does not alter the red blood cell antigen specificity and actually increase the reactivity of these antigens which are on the red blood cell surface to antibody binding.
Tables I and 11 demonstrate that PVP, alone, has viricidal activity and that povidone-iodine is effective in killing virus in whole blood and in cell 20 concentrates.

WO 9~/04031 Pcr/us9l/06240 2~ ~ ,7~

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WO 92/0403l PCI-/US91/06240 . .
` 2t~
TABLE ll - KILL OF VSV IN PLASA1A WITH PVP AND PVP-I
2~7;~, 7~ LOG KILL IN > > > I HOUR 24 HOURS
3 Yo PVP C- 1 5' 1.67 2.0 ~ ~% pVP C-15 3 1 5~o PVP C-15 '~.33 3.0 4 35'o PVP C-lS + 0.2~% PVP-I' 5.33 8+
'~% PVP C-15 + O.'~5C~Q PVP-I 5.0 8+
6 1% PVP C-l~ + 0.'~5C/c PVP-I 4.67 8+
7 3% PVP C-lS + 0.10Yc PVP-I 4.33 5.5 8 :2% PVP C-lS + 0.10% PVP-I 4.33 6.33 9 1% PVPC-IS + 0.10C/C PVP-I 4.33 5.33 3Y~ PVP C-30' 3.33 4.33 70 PVP C-30 3.0 4.
1'~ - I % PVP C-30 3.33 5.0 13 3% PVP C-30 + o.'~5c/L` PVP-I 6.67 8+
14 ~% PVP C-30 + o.'~sci~ PVP-I 7.33 8+
1 % PVP C-30 + 0.'~5~/~ PVP-I 8+ 8+
16 3% PVP C-30 ~ 0.10% PVP-I 4.5 6.67 l7 2% PVP C-30 + 0.105~ PVP-I 4.67 6.5 18 15~o PVP C-30 + 0.10% PVP-I 5.0 6.33 19 3% PVP K-~6-78~ 3.67 5.67 '~% PVP K-~6-'78 3.5 5.33 '71 1 ~ PVP K-'?6-'78 4.0 S.00 2-~ 3% PVP K-76-78 + 0.'~5~ PVP-I 8+ 8~
'~3 '7% PVP K-'76-'~8 + 0.'~5Y~. PVP-I 8+ 8+
'~4 1% PVP K-'~6-~8 + 0.?5C/~ PVP-I 8+ 8+
~5 3C~ PVP K-'~6-'78 + 0.10Y~ PVP-I 5.0 8+
- '76 7% PVP K-'76-'~8 + O.lOCk PVP-I 4.67 7.0 C-15 is polyvinyl pyrrolit~onc havin, a molecular weight of 12,5nO produced by GAF Corporati~?n.
PVP-I is ~ovidone-iodinc ps)w~k:r prc)duce~ hy Purdue-Frederick Company which contains ap~ roximately 85/c PVP, 1() /t 1. and 5~lodi~3e.
3 C-30 is polyvinyl l7yrrolidOnc prodllcl ~I hy CAF Corr~oration having a molecular weight ~f S0.()(1().
K-26-28 is r~olyvinyl r)yrrolidon~ ~)roduced hy GAF Corporation having a molecular wciL~ht of l ctwccn 4(),()()() ~m~l 5().()()(). -WO 92/04031 PtCr/US91/06240 !
7 ;~i~7%~
27 1% PVP K-26-28 + o 10% PVP-I 4.5 6.5 LOG KILL IN > > > I HOUR 24 HOURS
28 0.25% PVP-I 4.67 6.0 ~9 o.lo% PVP-I ~.33 3.67 CONTROL
Virus Presenl Virus Present I HOUR _4 HOURS
POSITIVE CONTROL tvsv/sERuM) 8.0 LOGS 8.0 LOGS

.

.

wo 92/04031 PCr/US~1/062~0 2~ 2 Two very important observations were made based on these and other experiments. First, increasing the povidone to iodine ratio above the ratio found in previous formulations significantly increases the biocidal effect of iodine. Secondly, povidone-iodine in high concentrations, e.g. higher than one 5 percent, typically from about 1 to ~ W/o~ in blood cell concentrates preservesthe basic structure of the cell and generally maintains the integrity of the cell walls. Another observation of less genera1 application was that the povidone-iodine opens pathways through the cell ~,vall which permits certain components of the cell, e.g. potassium salts, to "leali" froln the cell. By the same o mechanism, treatment of red blood cells with from one to about five percent iodine as povidone-iodine opens the cells to "inward lea};ing". Thus, compounds which have a virucidal or other effect in the cell can be introduced into the cell. Povidone-iodine can, for example, be used as described to increase the upta};e of GTPD compollnds which, in turn, prevent the replication s of virus in the cell. The net effect of this procedure is a biological synergism.
A new drug delivery system involves the ~Ise of povidone-iodine to open pathways through the cell wall of red blood cells. Red blood cell concentrates are treated as described to open passageways into the cell. The then permeable cell is emersed in or treated with a dr~lg which is to be delivered to the patient. -20 The cell walls having passages therethro~igh permit the drug to enter into the cell. Thereafter, the iodir,e may be removed and the cell concentrale is heated to 42 - 48 C to seal the cell walls. The concentrated cells are then infused into the patient where they carry o~lt the normal function of such cells. These cells have a finite life. As the cells age, they Iyse, thereby releasing the drug 25 directly into the blood stream where the drug can become effective.
. An interesting discovery was made respecting polyvinyl pyrrolidone, alone, in the course of studying the present invention. It was discovered that polyvinyl pyrrolidone alone was capab3e of killing about 2 to 5 logs of virus in body fluids. Whether or not there is a synergism vis-a-vis this anti-virieidal 30activity and the activity of iodine has not yet been determined. :;
Blood Substitll~es. Povidone-iodine is cross-linked to the hemoglobin to produce a hemoglobin product whlch as a greatly increased life as an oxygen ..

wO 92/04033 PCI /I,IS91 /06240 ( 2'~ 2~7?~
carrier when introduced into the blood circulation system. Povidone-iodine-hemoglobin complex in water or saline solution, thus, constitutes a blood substitute which can be stored with minhnal risk of microbial contamination and used as a blood extender in emergency situa~ions such as may be found on s the battlefield or in remote areas. Ratios of povidone-iodine to hemoglobin offrom about 0.1:1.0 to about 1.0:0.1 are considered satisfactory. povidone-iodiae in which the povidone:iodine ratio is at 1east as high as 4:I may be preferred. The fact that povidone-iodine binds very strongly to hemoglobin allows for the development of a blood substitute. The prior art teaches the o binding of PVP to hemoglobin by a variety of reagents. According to this invention, however, it has been discovered that iodine produces the binding of PVP to hemoglobin and, additionally, sterilizes the blood substitute assuring the absence of viable pathogenic organisms.
Transplan~ Organs Infective pathogenic microorganisms are inactivated when molecular iodine compound is llsed in solution to perfuse tissues and organs after removal from the donor and ~efore transplantation to the recipient The perfusion solution contains molecular iodine compound in a concentration of from about 0. ]`V/~ to about 5`'/~- ( 100 to 5000 ppm 1~), preferably from about 0.25W/o to about 2~/o. After a period of time, most of the unreacted molecular ~o iodine compound is washed away an(i any residual molecular iodine compound is absorbed into the protein or converted to inactive iodides and does not - significantly interfere with acceptance by the recipient.
Sperm Sperm-bearing solutions can be freed of pathogenic microbes by washing and/or storing the sperm in a solution which contains a concentra-2~ tion of povidone-iodine in a concentration is from about 0. IW/o to about l~/o, (I00 to 1000 ppm 1,) and, preferably, wherein polyvinyl pyrrolidone is added to give a polyvinyl pyrrolidone to iodine ratio is at least about 30 to about I,the iodine concentra~ion being sufficient to inactivate bacteria, viruses and other pathogenic organisms, and washing the sperm cells in the solution. It has 30 been discovered that polyvinyl pyrrolidone protects the sperm cells from the spermicidal activity of iodine sufflcient to permit the killing of pathogenic organisms while leaving viable, motile sperm cells suitable for artificial ~`.
-, wo 92/04031 Pcr/~ssl/o624 %~?$~
insemination. The washing is continlled or repeated to assure that substantiallyall of the seminal tluid is replaced with povidone-iodine solution. Other reagents such as are conventionally used in sperm treatment, storage and preparation, or for particular purposes rnay, of course, also be included in the5 infusion solution. If desired, residual iodine may be washed out and any suitable storage fluid, including solu~ions of polyvinyl pyrrolidone, may be used to store and handle the sperm cells.
The invention also contemplates a composition of matter consisting essentially of povidone and iodine wherein the ratio of povidone to iodine is 10 from 15 to 60 parts of povidone to I part of iodine. Such compositions of matter may be powdered povidone-iodine with an increased amount of povidone or solutions of the same.
Solid Povidone-lodine. The above applications in which the material to be purified is a liquid or cells carried in a liquid can be carried out by s flowing the liquid through a bed of solid particles of povidone-iodine of suitable size or by contacting the liquid and/or the cells in the liquid with particles or a membrane or surface of solid povidone-iodine. Where a bed of particles is used with a cell-bearing liquid, the particles must be lar~e enoughto perrnit intimate contact withollt acting as a filter, i.e. entrapping or binding 20 the cells.
Polyvinyl pyrrolidone used in the preparation of soluble povidone-iodine preparations is polymerized to a Inolecular weight of from 10 K daltons to ~0 K daltons, 30 K daltons being a lypical molecular weight. ~owever, povidone-iodine preparations can be prepared using very much higher ~5 molecular weight polymers which only tend to swell rather than to dissolve inaqueous solutions. It is the use of these higher molecular weight Polyvinyl pyrrolidone polymers reacted with iodine to form solid, substantially water insoluble povidone-iodine compositions that the present invention is directed In carrying out this facet of the invention, the liquid or cell-bearing 30 liquid is contacted with the solid povidone-iodine. This may be done most efFciently, in rnost cases, by passing the liquid through a settled or fluidizedor packed bed of povidone-iodine particles; however, such approaches will not, - - .

wO 92t04031 PCl/US91/06240 ~ 1 2~7~
ordinarily, be suitable for trealing cell-bearing liquids. Cell-bearing liquids rnay be treated 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 s and the iodine content therein regenerated between uses.
Industrial Application This invention finds application in medicine and veterinary science.

Claims

WHAT IS CLAIMED IS:
1. The use of povidone-iodine having a povidone:iodine ratio of at least 15:1 and optionally a physiologically acceptable reducing agent for the manufacture of a medicament consisting essentially of a blood cells-containing composition, said blood cells being carried in plasma or another carrier, liquidfor the treatment of disorders wherein the patient requires the transfusion of blood cells, the povidone-iodine being added in an amount in excess of that required to kill or inactivate all microbes therein comprising from 0.1w/o to 5w/o of the medicament.
2. The manufacture of Claim 1 further comprising the addition of a reducing agent to the composition to remove excess iodine.
3. The manufacture of medicament of Claim 1 wherein povidone-iodine is introduced into blood or blood cell concentrate in a concentration of from 0.1w/o to 5w/o, the blood or blood cell concentrate is maintained in contact with the povidone-iodine for a period of at least one to two minutes and povidone-iodine is again introduced into the blood or blood cell concentrate such that the povidone-iodine comprises from 0.1w/o to 5w/o thereof.
4. The manufacture of Claim 3 further comprising the addition of a reducing agent to the composition to remove excess iodine.
5. The use of povidone-iodine and a physiologically acceptable reducing agent in the manufacture of sperm cell-containing compositions for the induction of pregnancy in a female by inseminating the sperm cells into the female, the sperm cells being washed with povidone-iodine in water solution in a concentration of from 0.1w/o to 1w/o, sufficient to kill bacteria,viruses and other pathogenic micro-organisms but insufficient to inactivate the sperm cells.
6. The manufacture of Claim 5 further comprising adding iodine reducing agent in an amount sufficient to reduce substantially all of the iodine.
7. The use of povidone-iodine for the manufacture of liquid or cell-containing liquid biological material from one human or mammal for introduction of such material into another human or mammal by adding povidone-iodine having concentration of from 0.1w/o to 5w/o to said material to kill the microbes therein.
8. The manufacture of Claim 7, further comprising adding an iodine reducing agent to convert any residual iodine to iodide.
9. A method of disinfecting biological material selected from the group consisting of liquids, cell containing liquids, and proteinaceous tissue cells, comprising the steps of:
(a) treating said biological material with povidone-iodine to provide from a concentration of 0.1w/o to 5w/o povidone-iodine in the biological material, and (b) treating the thus treated biological material by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP
to reduce or remove residual iodine.
10. The method of Claim 9 further comprising preparing a derivative of the biological material from step (a), before carrying out step (b) on the thus prepared derivative.
11. The method of Claim 10 wherein the biological material is whole blood.
12. The method of Claim 9 wherein the biological material is whole blood.
13. The method of Claim 9 wherein the biological material is blood plasma.
14. The method of Claim 9 wherein the biological material is body tissue.
15. The method of Claim 9 wherein the biological material is tissue culture nutrient.
16. The method of Claim 9 wherein the biological material is packed red blood cells.
17. The method of Claim 9 wherein the biological material is a cell bearing liquid.
18. The method of Claim 9 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1 19. The method of Claim 18 further comprising preparing a derivative of the biological material from step (a), before carrying out step (b) on the thus prepared derivative.
20. The method of Claim 19 wherein the biological material is whole blood.
21. The method of Claim 18 wherein the biological material is whole blood.
22. The method of Claim 18 wherein the biological material is blood plasma.
23. The method of Claim 18 wherein the biological material is body tissue.
24. The method of Claim 18 wherein the biological material is tissue culture nutrient.
25. The method of Claim 18 wherein the biological material is packed red blood cells.
26. The method of Claim 18 wherein the biological material is a cell bearing liquid.
27. A drug delivery material comprising blood cell concentrate wherein the cell walls of the cells have been opened by treatment with from 1w/o to 5w/o povidone-iodine, a drug has been introduced into the cells through passages produced by the povidone-iodine treatment, the cell walls have between sealed by heating the cells to from 42 to 48°C. and the resulting material having optionally been treated by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.
28. The method of Claim 27 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1.
29. The method of Claim 27 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1 and wherein at least ten percent of the povidone has an average molecular weight of under 15,000.
30. A method of preserving blood cells comprising adding povidone-iodine to the cell-containing milieu in a concentration of from 0.1w/o to 1w/o (100 to 1000 ppm I2) sufficient to arrest or inhibit the principal metabolic functions of the blood cells but insufficient to kill the blood cells and storing the blood cells for future use and thereafter treating the cells by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP
to reduce or remove residual iodine.
31. The method of Claim 30 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1.
32. The method of Claim 30 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1 and wherein at least ten percent of the povidone has an average molecular weight of under 15,000.
33. In the method of treating patients with plasma comprising the steps of collecting plasma from a donor, and thereafter infusing the plasma into the patient lo be treated, the improvement comprising the additional steps of:
mixing the plasma with povidone-iodine sufficient to resulting a povidone-iodine a concentration of from about 0.1w/o to about 5w/o, and allowing contact of said plasma with said povidone-iodine for at least about one-half minute sufficient to inactivate or destroy inactive pathogenic microbes in the plasma and thereafter removing oxidizing iodine from the resulting mixture by passing said mixture into intimate contact with cross-linked povidone or adding a physiologically acceptable reducing agent.
34. The method of Claim 33 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1.
35. The method of Claim 33 wherein the povidone-iodine comprises high povidone:iodine povidone iodine having a povidone to iodine ratio of at least 15:1 and wherein at least ten percent of the povidone has an average molecular weight of under 15,000.
36. In the method of separation of plasma factors by alcohol fraction-ation, the improvement comprising the addition of povidone-iodine to the plasma before fractionation in concentrations to provide from about 0.1w/o to about 10w/o povidone-iodine in the plasma to give higher yields and sharper differentiation, and thereafter removing oxidizing iodine from the fraction by passing said fraction into intimate contact with cross-linked povidone or adding a physiologically acceptable reducing agent.
37. In a method of treating patients with plasma comprising the steps of collecting plasma from a donor, and thereafter infusing the plasma into the patient to be treated, the improvement comprising the additional steps of:
mixing the plasma with povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 15:1, preferably from about 20:1 to 30:1 and optionally up to about 60:1, sufficient to resulting an iodine a concentration of from about 0.1w/o to about 5w/o, and allowing contact of said plasma with said povidone-iodine for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma, and thereafter removing oxidizing iodine from the plasma by passing said plasma into intimate contact with cross-linked povidone or adding a physiologically acceptable reducing agent.
38. The method of Claim 37 wherein at least ten percent of the povidone has an average molecular weight of under 15,000.
39. In a method of separation of plasma fractions by cryoprecipitation, the improvement comprising the addition of povidone-iodine to the plasma before cryoprecipitation, the povidone-iodine with added povidone to give a povidone to iodine ratio in the plasma of at least about 15 to 1, in concentra-tions to provide from about 0.1w/o to about 5w/o povidone-iodine to give hig-her yields and sharper differentiation and optionally thereafter removing oxidizing iodine from the cryoprecipitate or a fraction thereof by passing said cryoprecipitate or fraction thereof into intimate contact with cross-linked povidone or adding a physiologically acceptable reducing agent.
40. An apparatus for treatment of liquid to kill microbes therein comprising a liquid container having, in use an upper reservoir portion for holding said liquid and a lower elutriation portion for recovering liquid and structure defining first and second beds of particulate matter, the first bed comprising substantially insoluble povidone iodine and the second bed consisting essentially of substantially insoluble povidone; the beds being so formed and configured as to permit the passage of the liquid therethrough in intimate contact with the surfaces of the particles forming the respective beds. 41. The apparatus of Claim 40 further comprising an additional layer between the first and second layers, said additional layer comprising substantially insoluble povidone hydrogen peroxide particulate matter.
42. The apparatus of Claim 40 further comprising an additional layer of particulate matter below the second layer comprising an iodine reducing agent.
43. The apparatus of Claim 40 further comprising a layer of soluble povidone iodine on the first layer in the liquid reservoir.
44. The apparatus of Claim 40 wherein the soluble povidone comprises at least about one-fourth polyvinylpyrrolidone having a molecular weight not higher than about 15,000.
45. The apparatus of Claim 40 further comprising soluble povidone-hydrogen peroxide on the top of the first layer for being dissolved into the liquid to be treated.
46. The apparatus of Claim 40 wherein the particulate matter consists essentially of particles having a diameter of from about 10 to about 100 microns.
47. A method of sterilizing an implantable tissue comprising:
(a) placing tissue that is physiologically acceptable for implantation into a human patient into a vacuum chamber;
(b) evacuating said chamber and maintaining a vacuum on said chamber for a period long enough to extract at least about one-half of the unbound water originally present in said tissue; and (c) introducing into said vacuum chamber a solution of povidone iodine for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted;
48. The method of Claim 47 further comprising the step of soaking the thus treated tissue in a solution of an physiologically acceptable iodine reducing agent.