WO1994000011A1

WO1994000011A1 - Starch-iodine-peroxide preservation of blood, tissues and biological fluids

Info

Publication number: WO1994000011A1
Application number: PCT/US1993/006130
Authority: WO
Inventors: Edward Shanbrom
Original assignee: Edward Shanbrom
Priority date: 1992-06-29
Filing date: 1993-06-25
Publication date: 1994-01-06
Also published as: EP0605704A4; EP0605704A1; CN1046691C; CN1139423A; JPH06511040A

Abstract

The treatment and preservation of blood, blood derivatives and other body tissues, fluids and cells with starch-iodine-peroxide or other iodine-binding starch-containing compositions, and equivalents, carrying germicidal iodine and then quenching the oxidizing potential of iodine in the iodine to kill pathogenic microbes without destroying the utility of the tissues, fluids and cells is disclosed.

Description

STARCH-IODINE-PEROXIDE PRESERVATION OF
BLOOD, TISSUES AND BIOLOGICAL FLUIDS
Field of the Invention
This invention relates to the treatment and preservation of blood and 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. According to the invention complexes of iodine with anylose-containing starch per se, or components of starch, e.g. amylose or amylopectin, and analogous iodine-binding polysaccharides and derivatives thereof, all of which are referred to generally and collectively as "starch" hereinafter, unless otherwise indicated by context, are used in the treatment of treating biological materials.

Thereafter a physiologically compatible reducing agent such as an ascorbate salt, as an additive or on a solid support, e.g. in a bed or filter of solid albumin, starch, povidone, etc,, may be used to remove the last traces of oxidizing iodine and also iodides. This invention may, thus, be used to kill or inactivate virus, bacteria, chlanzydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms and to remove all oxidizing iodine.

Tlle treatment and preparation of human blood, tissues, etc. and of tlie 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 invention.

Background of the Invention
Iodine was officially recognized by the Pharmacopeia of the United
States in 1930, also as tincture iodine (tincture of iodine) and linimentum iodi (liniment of iodine). Clinicians and microbiologists described a great number of experimental data and clinical applications, which can be found in numerous surveys. Despite the successes that have been achieved with iodine, it was ascertained early that it also possesses properties unsuitable for practical application, including, for example, the fact that iodine has an unpleasant odor. In addition, it stains the skin with an intensive yellow-brownish color, causes blue stains in the laundry in the presence of starch, and conibines with iron and other metals, its solutions are not stable, it irritates animal tissue, and is a poison.

The adverse side effects of iodine, its painfulness on open wounds and the possibility of allergic reactions in the past 100 years led to the production of a great many iodine compounds (and iodine preparations), with the aim of avoiding these incompatibilities without a significant loss of germicidal efficiency.

ln this connection, the iodophors finally succeeded as near-ideal forms of application. A new iodine complex that has all or most of the advantages of the iodophors, e.g. povidone-iodine is described in this application.

Although exact details about the killing of a living cell by the I2 molecule (or one of the reaction products occurring in aqueous solution) are not known, it can be assumed that iodine reacts:
(1) With basic N-H functions that are parts of some amino acids
(e.g., lysine, llistidiiie, argilliiie) and the bases of nucleotides
(adenine, cytosine, and guanine) forming the corresponding
N-iodo derivatives. By this reaction, important positions for
hydrogen bonding are blocked, and a lethal disorder of the
protein structure may occur.

(2) Oxidizing the S-H group of the amino acid cysteine, through
which the connections of protein chains by disulfide (-S-S-)
bridges, as an important factor in the synthesis of proteins,
are lost.

(3) With the phenolic group of the amino acid tyrosine, forming
mono- or diiodo-derivatives. In this case, the bulk of the
iodine atom(s) in the ortho position may cause a form of
steric hindrance in the hydrogen bonding of the phenolic
OH group.

(4) With the carbon-carbon double bond (C=C) of the
unsaturated fatty acids. This could lead to a change in the
physical properties of the lipids and membrane
immobilization.

Iodine is consumed by proteinaceous substrates and its efficacy as a disinfectant is 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 germicidal iodine, the actual anti-microbial agent.

Iodine is used widely in human medicine is the disinfection of skin, (e.g., tlie preoperative preparations of the skin, the surgical disinfection of hands, the disinfection of the perineum prior to delivery, and the disinfectioii of the skin prior to infections and transfusions). Iodine preparations are also used for therapeutic purposes, e.g., the treatment of infected and burned skin but is a strong irritant. Iodine has also been used for the disinfection of medical equipment, such as catgut, catheters, knife blades, ampules, plastic items, rubber goods, brushes, multiple-dose vials, and thermometers.

The use of "oxidizing iodine" including "compounds incorporating molecules of oxidizing iodine" e.g. absorbed or grafted on a purified vegetable carbon, as blood-contacting reagents having bactericidal and bacteriostatic action are mentioned in passing in connection with an autotransfuser device in U.S. Patent 4,898,572, Surugue nee Lasnier, et al but without any explanation or elucidation.

Iodine is an excellent, prompt, effective microbicide with a broad range of action that includes almost all of the important health-related microorganisms, such as fungi, bacteria, viruses, bacterial viruses, and protozoan cysts, if the sometimes severe limitations inherent in its use are overcome. Mycobacteria and the spores of bacilli and clostridia can be killed by iodine. As to be expected, varying amounts of iodine are necessary to achieve complete disinfection of the different classes or organisms. Within the same class, however, the published data on the disinfecting effect of iodine correspond only to a small extent. In particular, the published killing time of spores and viruses are widely disparate.

Various authors have tried to summarize the disinfecting properties of iodine and the other halogens by reviewing the literature and analyzing the existing data. The most important conclusions are:
(1) A standard destruction (i.e., a 99.999% kill in 10 minutes at
25o C) of enteric bacteria, amoebic cysts, and enteric viruses
requires lX residuals of 0.2, 3.5, and 14.6 ppm, respectively.

(2) On a weight basis, iodine can inactivate viruses more
completely over a wide range of water quality than other
halogens.

(3) In the presence of organic and inorganic nitrogenous
substances, iodine is the cysticide of choice because it does
not produce side reactions that interfere with its disinfecting
properties.

(4) Iodine would require the smallest mg/L dosage compared to
chlorine or bromine to "break any water" to provide a free
residual.

(5) 12 is 2 to 3 times as cysticidal and 6 times as sporicidal as
HO1, while HOl is at least 40 times as virucidal as 12. This
beliavior is explained on the one hand by the higher
diffusibility of germicidal iodine through the cell walls of
cysts and spores and on the other hand by the higher
oxidizing power of HOI.

Gottardi, W. lodiz and Iodine Coin pounds in DISINFECTION,
STERILIZATION, AND PRESERVATION, Third Edition, Block, Seymour
S., Ed., Lea & Febiger, Philadelphia, 1983, and the references cited therein provide more details respecting the background discussed above.

The classic blue starch-iodine complex is well-known and the reaction of iodine with starch is used as an indicator reaction in may diverse types of iodine analysis.

It is also known that starch-iodine complexes have some biocidal activity, although no comprehensive studies of this activity have been identified. The general, and heretofore invariable rule, has been that iodine complexes may provide prolonged contact of iodine with other materials or protect tissues from irritation, etc., the biocidal effect of such complexes is never higher than iodine in tincture or vapor phase.

I have discovered that starch-hydrogen peroxide -iodine complexes have a biocidal activity greater than iodine in any previously known form suitable for treating biological materials. Lower concentrations of iodine are effective in materials and under conditions that heretofore required nlucll liiglicr concelitrations of iodine. This, accordingly, constitutes one facet of the present invention.

Solid beads of cross-linked beads of starch-iodine that absorb moisture from wounds, etc., and also provide a source of iodine are wellknown; see, e.g., Holloway, G. Allen, Jr.; Johansen, Kaj H.; Barnes,
Robert W.; Pierce, George E., Multicenter trial of cadexomer iodine to treat venous stasis ulcer, Western Journal of Medicine, v151 nl, p35(4),
July, 1989. Cadexomer iodine (IODOSORBX PERSTORP CARBOTECO, Perstorp, Sweden]), is a starch polymer bead similar to dextranomer but with iodide (0.9% weight per weight) bonded to the polymer. When fluid contacts the beads, substantial amounts of fluid--as Iiiuch as 6 ml per gram of cadexomer iodine--are absorbed, as well as bacteria within the fluid.

Bonded iodine becomes bactericidal in this milieu.

Other somewhat limited descriptions of the use of starch-iodine have also been published. For example, Glushankof S 1, et al, Russian patent SU 1204575, describes compositions for purification and decontamination of water that contains starch, aluminum sulphate, iodine, iodide and activated charcoal to produce drinking water from open reservoirs in field conditions. A starch-based disinfectant composition prepared by treating starch suspension with potassium permanganate followed by a solution of iodine in potassium permanganate is described by Tatarov P G, et. al., Russian patent SU 979363 821207. One would infer that the product described by Glushankof, et al, is a solid bed of charcoal coated or containing the starch, iodine, and aluminum sulfate.

A protective coating for stored fruit and vegetables containing slurried starch, iodine, potassium iodide and sodium bicarbonate is described by Popova E R, et. al., Russian Patent SU 959733, 820928.

Iodine starch for disinfection is described by Mochnacz, W. et. al.,
Ptitsevodstvo, 1980, 11, p. 37; Zh Vet. 1981, Abstr. No. 38226 (Amyloiodine as bactericides, disin fectants, antiseptics, fungicides, fungistats, virucides and virustats; (CAS REGISTRY NUMBERS: 7553-56-2).

Johansson, J. A. O., U.S. Patent 4,010,259, describes the manufacture of cross-linked, swellable hydrophilic polymer-iodine complexes, including cross-linked starch-iodine complexes. Johansson discloses the following possible uses of tlle insoluble, water-swellable crosslinked compositions he produced:

Cosmetic and pharmaceutical materials in various forums, gels, sprays and powders for example, such as foot powder, baby powder, body deoderants, skin cleansers and creams; disinfectants for skin and wounds, preventing hospital infections and vaginal disinfectants; disinfection of water in, e.g. swimming pools and cooling towers; disinfection of equipment; and in the transportation and storage of commodities such as milk, wine and beer. In all instances, the iodine-containing product contemplated by Johansson is a swellable, insoluble solid material. Removal by filtration, etc., is contemplated in some applications, i.e. in the transportation and storage of commodities.

Alferov, V. V., et al, CA: 82(9)52180,: Tr. Uzb. Nauchno-Issled.

Inst. Vet., 1973, V.21" 238-40, describes an iodinol sperm disinfectant and starch iodinated sperm, and the disinfection of sperm;with starch,iodine complexes. See also, CA: 82(7)39339k, Use of iodinol during artificial inseniination of aniihals, Aliev, N. Ya.; Rakhimov, K IC; Alferov, V. V.;
Pulatov, T., Tr. Uzb. Nauchno-lssled. Inst Vet. 1973 V 21, 241-3.

Iodinated high polymers and their application in medicine and veterinary science, Mokhnach, V. O., Botan., lodonol Med. Vet., 1967 5-20, CA: 68(10)43119s, mentions iodine complexes with polyvinyl alcohols, starch, and polymers.

Bactericidal water filter, Panzer, Hans W. P.; Brown, Jerry Hugh,
France patent FR 1462968, CA: 67(14)67490m, describes water purification by filtration and sterilization by iodine.

The use of soluble organo-iodine compounds in water purification is, of course, well-known. One of the more popular water purifiers is tetraglycine hydroperiodide, which is widely used because of its effectiveness against giardia. iodine has also been used to treat swimming pools, etc., but is objectionable because it irritates the eyes and stains the pool walls an unattractive yellow color.

Mehltretter, C.L., et al, U.S. Patent 3414515 discloses the disiiifection of swimming pools with a non-irritating deeply blue iodine complex of a hydroxyalkyl ether of starch and of starch-containing starches that masks the yellow color of iodine in water.

The production of a tri-iodide of betaamaylose is described by
Minto, W.L., U.S. Patent No. 2540486.

Smith, Frederick R., International Publication No. W085/02422, 6
June 1985, discloses alloy fiber products, including fibers per se and fabrics woven or otherwise formed from fibers, that include iodine. Starch-iodine containing fibers are disclosed. lodization can be conducted after any of fiber formation, fabric formation, article formation or laundering stages.

The following surgical applications are mentioned by White, W085:02422 sponges, towels, dressings, face masks, gowns, drapes, wash cloths, booties,
CSR wraps, scrub suits and air filters. Hospital applications include sleets, pillow cases, towels, bed linens, wash cloths, sponges, incontinent pads, adult diapers, wrapping packs for contagious patients, sterile burn and wound dressings, sterile gloves and draperies.

Consumer item applications include handkerchiefs, tissues, face masks, vaginal tampons, sanitary napkins or pads, bandages, pre-wetted cleaning tissues such as
WET ONES3/4, acne treatment pads, diaper covers, water filters, wiping cloths, Q-TlPS¹, COVETS3/4, absorbent puffs, pill bottle stoppers, socks, shoe linings, pet bed liners, bird cage liners, dog collars, linings and containers for plants and seeds, carpet backing, upholstery, mattress ticking and pads, nursing pads, draperies, diaper backing, diaper absorbents, infant bunting and umbilical stump bands.

Hydrogen peroxide (H2O2), mol wt 34.016, is a weakly acidic, clear colorless liquid, miscible with water in all proportions. The four atoms are covalently bound in a nonpolar H--O--O--H structure. It is now prepared primarily by anthraquinone autoxidation processes. It is used widely to prepare other peroxygen compounds and as a nonpolluting oxidizing agent.

The reactions of hydrogen peroxide are:
Decomposition: 2H2O2 - > 2H2 + O2
Molecular additions: H202 + Y > Y.H2O2
Substitutions: H202 + RX > ROOH + HX
H202 + 2 RX b ROOR + 2 HX
Oxidations: H202 + W b WO + H20 Reductions: H202 + Z - > ZH2 + O2
Hydrogen peroxide may react directly or after it has first ionized or dissociated into free radicals.

In many cases, the reaction mechanism is extremely complex and may involve catalysis or be dependent upon the reaction environment.

Hydrogen peroxide can form free radicals by homolytic cleavage of either an O--H bond or the O--O bond.

HOOH b H. +.OOH (380 Kj/mol or 90 kcal/mol)
HOOH t 2.OH (210 Kj/nzol or 50 kcal/mol)
The last equation predominates in uncatalyzed vapor-phase decompositioll and photocllemically initiated reactions. In catalytic reactions, especially in solution, tlie nature of the reactants determines which reaction is predominant.

Hydrogen peroxide is a strong oxidant and most of its uses and those of its derivatives depend on this property. It oxidizes a wide variety of organic and inorganic compounds, ranging from iodide ions to color bodies of unknown structure in cellulosic fibers. Hydrogen peroxide reduces stronger oxidizing agents such as chlorine.

Aqueous hydrogen peroxide is sold in grades ranging from 3 to 98%, mainly containing 35, 50, 70, or 90% H2O2. The 3-6% H202 solutions for cosmetic and medicinal use are obtained by diluting a more concentrated grade, usually with the addition of extra stabilizer. There is a USP specification for 3% H2O2.

Hydrogen peroxide is irritating to the skin, eyes, and mucous membranes. However, low concentratioiis (3-6%) are used in medicinal and cosmetic applications.

Hydrogen peroxide is used to treat wastewaters and sewage effluents, and to control hydrogeii sulfide generated by the anaerobic reaction of raw sewage in sewer lines or collection points. It has been proposed as a supplemental oxygen source for overloaded activated sludge plants. It reported controls denitrification in secondary clarifiers and improves bulking conditions. It has been used as a flotation assistant. It llas been generated in a wastewater reservoir by the cathodic reduction of oxygen.

Hydrogen peroxide has been used with povidone iodine in biocidal processes. A number of sucli methods are described in tIIe following patents to Witkin, et al.

Simon, Gilbert I; Witkin, Roy T, US Patent 4997625 910305, describe the treatment of dental and medical instruments and appliances to be chemically sterilized by immersion in an mixture of an iodophor sucli as tlie povidone iodine complex or a quaternary ammonium compound such as cetyl pyridinium chloride and a peroxide such as H202, the antimicrobial action of tlie iodine derived from the iodophor being enhanced or potentiated by oxygen released from the peroxide. The invention is applicable also to the chemical sterilization of surgical sites.

Witkin, Roy T, US Patent 4935248 900619, describes applying, spraying, or sponging antimicrobially effective amounts of aqueous iodophor peroxide solutions to the udder surfaces of cows, or other animals to be milked prior to jnilkiiig. The udders are shampooed with a nonstainiiig shampoo containing iodine in a povidone-iodine complex and hydrogen peroxide as a nascent oxygen source.

Simon, Gilbert I; Witkin, Roy T, US Patent 4738840 880419, US
Patent 4592489 860603, and US Patent 4567036 860128, describe pre- and post-operative dental and surgical procedures in and on structures and areas of the oral cavity to maintain sterility by the application of an antimicrobially enhanced aqueous solution of an iodophor constituting a source of iodine and a peroxide as a source of oxygen. Tlie iodophor is preferably a povidone iodine complex soluble in water and the peroxide is preferably hydrogen peroxide, the oxygen frorn the peroxide acting to ellllance tulle antimicrobial activity of the iodine derived from the povidone iodine complex.

Witkin, Roy T, US Patent 5066497 911119, discloses an antimicrobial veterinary composition having enhanced antimicrobial activity against a broad spectrunl of nlicroorganisms afflicting small and large animals. The composition is a solution or mixture of povidone iodine complex and nascent oxygen obtained from a peroxide source.

Hydrogen peroxide has been combined directly with povidone to provide a dry powder source of H2O2.

Garelick Paul; Login, Robert B; Merianos, John J, US Patent 5066488 911119, describe a semi-anhydrous, suspension process for preparing substantially anliydrous complexes of PVP and H202 containing about 18% to about 22 Xs by weight H2O2. The process comprises suspending substantially anhydrous PVP aiid aii aqueous solution of 70 to 85coo H202 in an anhydrous ethyl acetate medium to precipitate a free-flowing, fine white powders of the complex, and filtering and drying under vacuum at about 40-50"C. to form the desired product.

Liebernian, I-lerbert A; Login, Robert B; Merianos, John J, US
Patent 5008106 910416, describe a method of using the product referred to by Garelick, et al, ibid, for reducing tlie microbial content of surfaces which comprises contacting said surface with a microbiocidal amount of a substantially an hydros complex of PVP and H2O2.

Biss Russell B; Cohen Jeffrey; Merianos John J; Taylor Paul D, US
Patent 5077047 911231, also describe a process for the production of pvp-H2O2 products in the fonn of free-flowing powders. A fluidized bed maintained at a reaction temperature of about room temperature to 60"C.

is contacted with finely divided droplets of a 30 to 85% by weight aqueous H202 solution. A 50-70% H202 solution is used, and the feed rate of introduction of the H202 solution is about 5-50 g/minute/kg PVP present.

The pvp-H2O2 product preferably contains about 15-24%, preferably 18-22%, H.O2 (1:1 molar ratio) and less than about 5% water.

The important role of hydrogen peroxide as produced in vivo in the tissues and fluids of living mainnials to kill invading pathogens has been extensively studied. See, e.g.: Sattar S.A.; Springthorpe V. S., Rev. infect.

Dis. (USA) , 1991, 13/3 (430-447) (Abstract); Effects of topical antimicrobial agents on tlie humaii neutrophil respiratory burst, Hansbrougll J F; Zapata-Sirvent RL; Cooper ML, Arch Surg May 1991, 126
(5) p603-8 (abstract); Depression of hydrogen peroxide dependent killing of schistosomula in-vitro by peritoneal exudate cells from schistosoma-inansoni infected mice, Smith J M; Mkoji G M; Prichard R K, Am J Trop Med Hyg 40 (2), 1989, 186-194 (abstract); Oxidative and nonoxidative killing of actinobacillus-actinomycetemcomitans by human neutrophils, Miyasaki K T;

Wilson M E; Brunetti A J; Genco R J, Infect
Immun 53 (1). 1986. 154-160 (abstract); Killing of actino bacil lus-actinomycetemcom itans by the human neutrophil myeloperoxidase-hydrogen peroxide-chloride system, Miyasoki K T; Wilson M E;
Genco R J, infect Immure 53 (1), 1986, 161-165 (abstract); Phagocytes use oxygen to kill bacteria, Baggiolini M, Erpenernia Sep 15 1984, 40 (9) p906-9 (abstract); Regulation of membrane peroxidation in health and disease, Boxer LA; Harris RE;

Baehner RL, Pediatrics Nov 1979, 64 (5 Pt 2 Suppl) p713-8 (abstrad); Oxygell metabolism and the microbicidal activity of macrophages, Johnston RB Jr, Fed Proc Nov 1978, 37 (13) p2759-64 (abstwact); Superoxide radical and the bactericidal action of phagocytes, Fridovich I, N Engl J Med, Mar 14 1974, 290 (11) p624-5, (abstract); Relationsllip between extracellul ar stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes, Leigh P
C J; Nathan C F; Van Den Barselaar M T; Van Furth R, infect Immun 47 (2), 185, 502-507 (abstract); Safety and nmunogenicity of hydrogen peroxide-inactivated pertussis toxoid in 18-month-old children, Siber G.R.;
Thakrar N.;

Yancey B.A.; Herzog L.; Todd C.; Cohen N.; Sekura, R.D.;
Lowe C.U; Vaccine (United Kingdom), 1991, 9/10 (735-740) (abstract); In vitro studies of water activity and bacterial growth inhibition of sucrose-polyethylene glycol 400-hydrogen peroxide and xylose-polyethylene glycol 400-hydrogen peroxide pastes used to treat infected wounds,
Ambrose U.; Middleton K.; Seal D., Antimicrob. Agents Chemother.

(USA) , 1991, 35/9 (1799-1803) (abstract); Bacteriolysis is inhibited by hydrogen peroxide and by proteases, Ginsburg I., Agents Actopms (Switzerland) , 1989, 28/3-4 (238-242) (abstract); Characterization of hydrogen peroxide-potentiating factor, a lymphokine that increases the capacity of human monocytes and monocyte-like cell lines to produce hydrogen peroxide; Gately C.L.; Wahl S.M.; Oppenheim J.J., J. Immunol.

(USA), 1983, 131/6 (2o53-285Ö) (ubslruc); Control of the microbial flora of the vagina by H2O2-generating lactobacilli, Klebanoff, S.J.; Hillier, S.L.;
Eschenbach, D.A.; Waltersdorph, A.M., ivuinal of Infectious Diseases, v164 ill, p94(7), Jltly, 1991 (abstract).

A comprehensive review of oxygen based therapies, including both ozone and hydrogen peroxide therapies, has been published; "Do oxygen therapies work? -- Claims for oxygen as a miracle element tha reports of hydrogen peroxide therapy without scientific controls and many regard the practice as quackery.

Shenep, J.L, et al, in a fairly recent study, reported a lack of antibacterial activity after intravenous hydrogen peroxide infusion in experimental Escherichia coli sepsis. Sheep J.L.; Stokes D.C.; Hughes
W.T., Infect. Iininziii. (USe{), 1985, 48/3 (607-610) (abstract).

Hydrogen peroxide has also been evaluated as a bactericide in poultry chilling water to kill bacteria that reside on the carcasses of poultry prepared for use as a food, Lillard H S; Thomson J E, I Food Sci 48 (1), 1983, 125-126 (abstract). While bacterial kills could be obtained, the poultry carcasses were degraded to the point where they would be unacceptable in tulle marketplace.

The oxidative degradation of tissues both in vivo and in vitro as well as the pain and discomfort resulting from contact of hydrogen peroxide with open wounds and membranes has discouraged workers from using hydrogen peroxide in all but a very limited number of applications. Oral ingestion of hydrogen peroxide has been tried with moderate to severe side reactions, e.g. nausea, vomiting, diarrhoea and physical discomfort and with doubtful benefits, see Thomson, stipra.

1he questionable results front intravenously and orally administered hydrogen peroxide militates against the use of hydrogen peroxide in any circumstance where fragile cells or tissues would be contacted by this strong irritant-oxidant unless the need for a strong, direct contact biocide outweighs all other considerations, as is the case in some infected wounds or infected surgical incisions.

Those who deal with blood and other invasively obtained body fluid samples risk iiifection from tlie samples. Those at risk include the doctor, nurse or cliiiical technician who takes the sample, the technicians who handle the sample and who use tiie sample in conducting analyses and tests, those who handle the sampliiig and testing equipment 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 wlio ulti nately dispose of the apparatus, usually in specially designed high temperature furnaces.

The risk is substantial, as evidenced by the fact that nearly all health care professionals with long experience carry tlie Epstein-Barr virus (EBV) and other pathogenic viruses.

Another organism which may be present in blood and blood products or fractious and which presents a serious risk in certain procedures is tlle bacteria Yersinia ernerocolitica which causes gastroenteritis aiid surpasses Salmottefla 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., Trans.fison 30, 3, p.207 (1990). Y. enlercscolitica and other bacteria which propagate at relatively low temperatures, e.g.

Staphylococcus epidermidis and Lebwionellu pneumcsphilu, present, potentially, a serious threat in blood products.

It is generally recognized that proteinaceous materials destroy the biocidal effectiveness of iodine and iodopliors such as PVP-I. This factor has been considered a niajor impediment to the use of iodine and iodophors in the presence of large amounts of biological materials.

Albumin has been identified as having and extremely high capability of deactivating the biocidal power of iodine and iodophors. For example, Batts
W N; Landolt M L; Wintoii J R, Appl Environ Microbiol 57 (5). 1991, 1379-1385, reported the results of using iodine in fishery waters to kill virus that iodine efficacy decreased when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Batts, et al, also noted that sodium thiosulfate effectively neutralized free iodine.

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 germicidal iodine, e.g. povidone 12, 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. fakiparitni, P. ,nalariae, P. ovale and P. vivar, which causes malaria, Tiypanosoi;ia, which causes Chagas' disease, and Lesshmaniu, which cause a variety of leislinianiasis. 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 products produced from blood, tissues and fluids for illactivatilig virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorgall isms.

As used here, the tenii "blood" means whole 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. Thus, the term "blood" may apply to whole blood at the time of collection or a blood derivative at any stage in processing, as indicated by context. Blood derivatives mean blood components such as blood cell concentrates (red blood cells, platelets, etc.), plasma, and serum and products and factors prepared from blood such as albumin and the blood factors. Body tissues and cells means any tissue(s), organ(s) or cells or fluids which contain tissue(s), organ(s) or cells of animal origin.

Thus, in a broad sense, body tissues and cells include blood and the cellular components 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 cells include bone marrow, kidneys, cornea, heart valves, tendons, ligaments, skin, homograft or xenograft implants and prosthesis generally. Tissue and cell cultures means cells and tissues grown or enhanced in culture media and the culture media per se, but not including nutrieiits intended for use in cell cultures. Examples of a cultured tissue is cultured skin tissue for use in burn victims, cells and cellular products prepared by standard biological and/or genetic engineering techniques are other examples of tissue cultures. Laboratory reagents and standards, as used in this specification and the claims, means reagents and standards produced from or comprising human or animal fluids, 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 include samples of blood, urine, sputum, cell smears, etc. While the term "donor" is not usually applied to the individual from 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 purpose, and such term will be used to refer even to an unwilling donor.

If a tissue is explanted into the culture media for the purpose of propagating its cells, the procedure is called tissue culture whereas the explanting of individual cells into culture media would be called cell culture; however, both 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. One of the great values of tlie present invention is that biocidal effect can be accomplished with very low levels of iodine, thereby reducing cellular deterioration. The presence of excess amylose, 20 to 100 or more percent than can be saturated by iodine, provides a very geiitle milieu for killing or inactivating virus and bacteria and, if present, other microbes.

It is, generally, impossible to define with precision the exact materials required to propagate a given cell line and, therefore, it is common practice to use media based upon or containing serum and to add nutrient serum as needed during the cell propagation. Bovine serum from adult animas may be suitable in some instances, but fetal bovine serum (FBS) (sometimes referred to as fetal 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 agents. Indeed, every lot of commercially produced FBS is contaminated with infectious bovine viral diarrhea (BVD) virus and infections with infectious bovine rhinotracheitis (IBR), parainfluenza 3 (PI 3) are extremely common.

At best, pools of raw serum probably contain at least ]04 infectious BVD virus particles per milliliter. Serum 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 macromolecules are sheared.

Shearing of macromolecules during filtration occurs generally when tangential flow filtration is used and turbulence develops. It is currently very difficult 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 cultures are of primate origin there are serious hazards for the 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 (cut) cells were often infected by non cytopathic mucosal disease virus (MDV): the cells seemed morphologically healthy, but nearly all showed fluoresceiice with BVD antiseruni and rabbit anti-bovine conjugate.

The risks of infection from whole blood are well-known. One of tlie great tragedies of modern medicine is the infection of many patients, most frequently hemophiliacs who require frequent blood transfusions, with serious debilitating or fatal diseases such as hepititus 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 cell concentrates is similar to comparable risks associated with whole blood. The fundamental problem is that, heretofore, it has not been possible to kill pathogenic microbes in blood without doing substantial injury to blood cells - red blood cell hemolysis being an ever present problem. According to this invention, very low amounts of iodine are effective, iodine is regenerated
The teachings of the prior art suggest that neither elemental (diatomic) iodine nor complexed iodine 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.

Starch-iodine-hydrogen peroxide (Starch-I-112O2) is, to the best of applicant's knowledge, a new composition of matter.

It is an object of this invention to provide a method for sterilizing therapeutic biological fluids and tissues, including cell-containing fluids such as blood, an to rninimize the addition of foreign chemicals thereto by the use of starch-iodine-hydrogen peroxide.

It is also an object of this invention to provide a method for killing or inactivating a broad spectrum of virus, bacteria, etc. in therapeutic biological fluids by the use of starch-iodine-hydrogen peroxide.

It is a further object of this invention to provide a method for minimizing the injury to cells and proteins in therapeutic biological fluids and, at the same time, kill or inactivate pathogenic microorganisms therein by the use of starch-iodine-hydrogen peroxide.

Various medical and blood handling procedures are referred to hereinafter. These are all well-known procedures and steps in these procedures are fully described in the literature. The following references are provided for general background and as sources for detailed reference to the literature as to specific procedures: TECHNICAL MANUAL of the
American Association of Blood Bankers, 9th Ed. (1985); HLA TECHNIQUES FOR BLOOD BANKERS, American Association of Blood
Bankers (1984); Developments in Biological Standardization, Vols. 1 - 57,
S.

Karger, Basel; CLINICAL IMMUNOCHEMISTRY, The American
Association for Clinical Chemistry; MEDICINE, Vols. 1 - 2, Scientific
American, New York; Care of the SURGICAL PATIENT, Vols 1 - 2,
Scientific American, New York; CURRENT PROTOCOLS IN Germicidal
BIOLOGY, Greene Publishing Associates and Wiley-Interscience, John
Wiley & Sons, New York.

Summarv of tile Invention
The present invention encompases processes of treating a liquid composition that contains bacteria, virus, or other pathogenic organisms to sterilized the same by reaction with starch-iodine-hydrogen peroxide in the liquid phase or contacting the liquid with starch-iodine-hydrogen peroxide such as passing the liquid through solid starch-iodine-hydrogen peroxide filters or columns.

Starch-iodine-hydrogen peroxide may be iodine and hydrogen peroxide complexed with essentially the entire starch molecule, of any of the great variety of amylose-containing starches available from different sources, or with a polysaccharide component, or derivative thereof, of starch, or all equivalent polysaccharide, that binds to starch, starch an amylopectin being the most widely available of these components. Unless specified or otherwise in dictated from context, the term "starch" is used here to describe compositions of complex or simple starches that bind iodine, components and derivatives thereof, amylose and amylose compounds, polymers and derivatives, and amylopectins. The invention, in various of its facets can be carried out using either soluble or insoluble starch, as described.

Certain facets require soluble starch, other facets require insoluble, water-swelled or water-swellable, starches while other facets can be carried out using either soluble or insoluble starch.

Thereafter, if it is desired to assure total iodine removal and removal of hydrogen peroxide, the solution can be passed into contact with solid starch, e.g. filtered thorough a bed or column of solid starch particles that are less tlian saturated with iodine and hydrogen peroxide, preferably having no more than a trace of iodine, or another absorbing material, such as cross-linked povidone to remove the excess iodine. Cross-linked starch and amylose products such as those described by Holloway, G.

Allen,
Jr.; Johansen, Kaj H.; Barnes, Robert W.; Pierce, George E., Multicenter trial of cadexomer iodine to treat venous stasis ulcer, Western Jouriial of
Medicine, v151 nl, p35(4), July, 1989; Johansson, J. A. O., U.S. Patent 4,010,259,; and White, Frederick R., lnternational Publication No.

W085/02422, without added iodine and hydrogen peroxide, may be used to remove iodine. Likewise a reduciiig agent such as a reducing sugar, ascorbate, sodium sulfite, etc., may be added to eliminate the last traces of iodine and peroxide. Reducing sugars, ascorbic acid (Vitamin C) and its salts, and sodium sulfite are well-known, readily available reducing agents that are physiologically acceptable. However, any physiologically acceptable reducing agents may be used.

The invention is embodied in a method of disinfecting biological materials. The steps of the method include treating biological material before separation of the components thereof with starch-iodine-hydrogen peroxide to provide from a concelltratioll of 0.1W/o to 10w/o (or higher) starch-iodine-hydrogen peroxide in said material before separation of the components thereof. A derivative of the material resulting from the preceding step is prepared aid, optionally, also treated with starch-iodine hydlogell peroxide to provide from 0.1who to 1OW/o (or higher) starcll iodine-hydrogen peroxide in the derivative.

Also optionally the derivative may be treated by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.

These methods are applicable, for example, to whole blood, plasma, tissue, culture nutrient, packed red blood cells and cell-bearing liquids or noncell-bearing biological liquids
Also included in the invention is the improved method of treating patients with plasma comprising the steps of collecting plasma from a donor, and thereafter infusing tlle plasma into the patient to be treated, of mixing the plasma with starch-iodine-hydrogen peroxide sufficient to result in a starcll-iodine-llydrogen peroxide a concentration of from about 0.lW/o to about lowlo, or higher, even up to 50%, though no significant advantage results from the use.of-higher than about 5-10%,

and allowing contact of said plasma with said starch-iodine-hydrogen peroxide for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma and optionally thereafter removing iodine from the resulting mixture by passing said mixture into intimate contact with cross-linked PVP or albumin or adding a physiologically acceptable reducing agent. lodides may be oxidized to iodine using peroxides, e.g. hydrogen peroxide or PVP-hydrogen peroxide, and the resulting iodine removed in the same manner.

An apparatus for treatment of liquid to kill microbes is also provided. The apparatus is in the form of 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 starch-iodine-llydrogen peroxide and the second bed consisting essentially of substantially insoluble PVP, starch or albumin; the beds being so fonned and configured as to permit the passage of the liquid therethrough in intimate contact with tulle surfaces of the particles forming the respective beds. The first bed may be cross-linked PVP.

The apparatus may comprising a third layer between the first and second layers, the third layer comprising substantially insoluble PVP-hydrogen peroxide or starchhydrogen peroxide particulate matter. The apparatus may contain a layer of particulate matter comprising an iodine reducing agent. A layer of soluble starch-iodine-hydrogen peroxide may be provided on the first layer in the liquid reservoir. All or only part of the layers, after the first and second layers, may be provided.

One method of sterilizing an implantable tissue in accordance with this invention comprises placing tissue that is physiologically acceptable for implantation into a human patient into a vacuum chamber, evacuating the chamber and maintaining a vacuum on the chamber for a period long enough to extract at least about one-half of the unbound water originally present in said tissue, and introducing into tlie vacuum chamber a solution of starch-iodine-hydrogen peroxide for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted.

Optionally, iodine may be removed by washing or reconstituting the tissue witli a reducing agent such as ascorbic acid or a salt thereof or sodium sulfite, for example.

Brief Description of the Drawings
Figure 1 depicts an apparatus for contacting a liquid material with starch-iodine-hydrogen peroxide and with either or both of (a) an iodine absorbing material and/or (b) an iodine reducing material, and for providing other materials for processing biological liquids, in particular, according to this invention.

Figure 2 depicts, largely schematically, an apparatus for treating solid tissue samples.

Description of the Preferred Embodiments
Starch-iodine-hydrogen peroxide may be prepared in any of a large number of methods. Substantially pure starch, that may have traces of other biological materials or be essentially free of contaminants is available commercially. Starch, amylose and amylopectin, components of starch and equivalent polysaccharides, encompassed in the term "starch" as used in the general sense indicated above, are also available in high purity. The chemistry of starch and polysaccharides generally is well-developed and a large number of high purity starch and polysaceharide compositions are available commercially.

The term "pure," and its derivatives are used in the sense commonly used in reference to biologically isolates that inherently contain some biologicals other than the principal constituent. Trace amounts of other materials is not per se detrimental to the present invention, and can be tolerated unless they interfere with the iodine-starch reaction or reaction of iodine with microbes.

When iodide, e.g. Nal or Kl, solutions are added to starch or starch solutions of high starch concentration, the iodide tends to become associated with the starch and appears to cause the starch to form larger particles by crystallization or cross-linking. Small amounts of iodide are apparently converted to iodine upon binding to starch. No explanation for this phenomenon has been found: The addition of hydrogen peroxide, in solution or as a gas, results in starch-iodine-hydrogen peroxide by oxidation of the iodide which, along with the hydrogen peroxide binds to the starch . Starch may be reacted first with hydrogen peroxide followed by reaction of iodide salt or iodine, both resulting direct coupling of the peroxide and iodine to the amylose component of the starch.

Reactions may be carried out in solution or by gas phase reaction of hydrogen peroxide and iodine with starch. Ratios of iodine and hydrogen peroxide can be controlled. For example, iodine equivalent to 5 weight percent of the starch may be reacted with the starch either as iodide or iodine.

Hydrogen peroxide, in excess of the reactable amount, is then reacted with the starch-iodine, resulting in 5w/o iodine in starch saturated with hydrogen peroxide.

The binding capacity of starches vary, as would be expected, but it is a simple matter to identify the maximum binding capacity of any given batch of starch. The starch is weighed, saturated with iodine, excess iodine being carefully washed away, and tie end product weighed.

Successive amounts of iodine can be added and the ultimate capacity closely estimated by extrapolating from data derived as one approaches saturation. It is desirable in some instances to ascertain the capacity of a given soluble starch, add iodide in an amount insufficient to result in a full saturation of the starch, i.e. 20% to 200%, or more, starch being present than is needed to absorb the iodine produced upon oxidation of the iodide. Hydrogen peroxide is added to saturation or less than saturation.

This results, when the product is used, in an excess presence of starch for carrying hydrogen peroxide to the reaction and also provides a protective excess of starch.

There is a distinct synergism as to the biocidal power of starchiodine-hydrogen peroxide. The iodine, it is believed, acts most directly on microorganisms, being converted to iodide. The iodide is then converted to iodine by the peroxide, and again kills the microorganisms. The hydrogen peroxide is also biocidal directly, but is believed to be very much less of a factor than tlie iodine. It has been discovered, however, that starch-iodine-hydrogen peroxide is more effective biocidally that either starch iodine or starch hydrogen peroxide alone, having comparable amounts of iodine and hydrogen peroxide, respectively, in the starch. Far lower concentrations of iodine are necessary to accomplish a total kill of virus or bacteria than would be required using povidone iodine or another form of iodine.

The starch-iodine-hyd rogen peroxide may be water soluble or water insoluble. Water soluble starch-iodine-hydrogen peroxide is best formed in solution. Water insoluble starch-iodine-hydrogen peroxide is best formed in very high concentration solutions or suspensions of starch or with starch and added moisture.

The use of starch-iodine-hydrogen peroxide, optionally followed by treatment with a physiologically acceptable reducing agent for the manufacture of a medicament is contemplated by this invention. Such a medicament may, for example, consist essentially of blood cells in plasma or another carrier liquid.

Such medicaments may be used for the treatment of disorders wherein the patient requires the transfusion of blood cells. Starch-l-H2O2 is added in an amount in excess of that required to kill or inactivate all microbes is added. Starch-I-H2O2 may comprise, for example, from about 0.005 t used for tlie treatment of disorders wherein the patient requires the transfusion of blood cells. Either simultaneously therewith, or afterward, starch-I-H2O2 in an amount in excess of that required to kill or inactivate all microbes is added.

Starch-I-H2O2 may comprise, for example, from about 0.005 to 10 weight percent, preferably from O.lW/o to 10W/o of the niedicament. The starch-l-H2O2 is allowed to remain in contact with the blood cells or plasma, or other biological material being prepared to be a niedicament, for a period of at least about a half a minute sufficient to kill tlie microbes, but not long enough to denature or otherwise injure the biological material. Usually, contact of under an hour is preferred.

Accordingly, the contact times will be referred to as from one-half minute to one hour with the caveat that longer contact is not necessary or beneficial and may result in injury to the biological, but would, nevertlieless, be within the scope of the invention. The mixture resulting from the above is then contacted with an iodine absorbing reagent such as cross-linked PVP, or coagulated albumin or solid starch, to remove the iodine. If desired, a reducing agent may thereafter be added in an amount to reduce any iodine that may not have been absorbed. The contact with the iodine absorbing material is preferably accomplished by passing the material undergoing treatment through a layer, i.e. a bed or filter, of solid, substantially insoluble albumin or starch.

A second treatment as described may be performed to assure total sterilization, if desired.

Likewise, a second similar treatment may be performed on a product or fraction of the initial biological material treated as described above.

In a similar manner, the "addition" of a reducing agent to the material undergoing treatment may be accomplished by passing the material through a layer of substantially insoluble material that has active reducing sites thereon or equilibrates with the liquid material undergoing treatment to partially dissolve into such liquid, or make readily available in said liquid (as by swelling, for example) reducing moieties. A bed of beads or fibers, for example, that expose on the surface thereof reducing sugar moieties may be used very conveniently.

Reference is made to Figure 1 of the drawing for a better understanding of the invention in one form. Figure 1 depicts an apparatus for contacting a liquid material with starch-I-H2O2 and with either or both of (a) an iodine absorbing material and/or (b) an iodine reducing material, and for providing other materials for processing biological liquids, in particular, according to this invention. The apparatus, being shown and described in a generally schematic fashion, may be in any of maiiy configurations. The only significant structure, insofar as this invention relates is to tile arrangement of the layers
The apparatus 10 may be viewed as a filter funnel or a column.

As those in the art understand, the difference between a filter and a column is often insignificant in that both "filter" a liquid and both cause the liquid to contact solid material. A filter may, indeed must, remove only part of the material. For example, either a filter or a column may let small cells or particles pass but retain larger cells, or it may permit only liquid and extremely small particles pass. The apparatus comprises cylindrical portion 12 that, in part, defines a reservoir portion. The reservoir may be large or very small as desired. The apparatus, in the configuration depicted comprises a second, smaller cylindrical tube portion 14 and a conical transition zone 16 connecting the two cylindrical portions as is conventional in funnel manufacture.

It is again emphasized, however, treat it is immaterial whether the apparatus defines a reservoir and or funnel portion of any particular size or configuration.

The apparatus defines a first layer 20 and a second layer 22. The first layer is made up of substantially insoluble starch-I-H2O2. This layer is described as being made up.of'particulate materials in that the use or particulates in one way or another is usually involved. Particles of solid, insoluble starch-l-H2O2, e.g. cross-linked starch-1-H2O2, in the form of a layer or bed of particles, either supported directly by a layer below or by way of another support, e.g. being bonded to or entrapped within a layer of fibers or particles, is contemplated.

The first layer may also contain some soluble starch-l-H2O2. A frit made of particles bound together adhesively, by heat or pressure would also be within the disclosure and invention. The starch-l-H2O2 may be formed in situ by iodinating a layer of starch or the layer may be made up of pre-synthesized starch-I-H2O2.

The second layer is downstream of the first layer, i.e. the liquid to be treated flows through tlie first layer and then the second layer. The second layer may comprise an insoluble iodine absorbent, e.g. cross-linked povidone, or insolublized albumin or starch, or an iodine reducing agent, or a mixture of both, or be a multiple sub-layer structure with a sublayer of iodine absorbent first and then a sublayer of iodine reductant. Again, tlie layer may be a self-supporting frit or other structure or may be supported by a support or other layer.

The essential function of the apparatus is to cause a liquid that is to be treated to pass, with or witbout cells or other particles therein, first through a layer of starch-I-H2O2 and, thereafter, to contact such liquid with absorbent to remove the iodine and/or reductant to reduce the iodine. Hence, the layers may be quite deep or quite thin, adjacent each other or spaced from each other, as is necessary or desirable to provide adequate contact of the liquid with each of the layers or beds.

Such an apparatus is conveniently suited for the treatment of liquid to kill microbes in the liquid. The liquid container that is generally defined by the overall apparatus in the simplified, schematic example of
Figure 1, and has an upper or liquid inflow reservoir portion for holding liquid to be treated. This may be a very small reservoir or quite large.

Tlie reservoir may displaced from the beds or layers by a very large distance, though this is not generally beneficial. The apparatus has a lower or elutriation or recovery portion for recovering liquid that has been treated. Between these portions, first and second beds of particulate matter are defined by suitable structure. The first bed or layer comprises substantially insoluble starcll-l-H702. The second bed consists essentially of substantially insoluble albumin or starch, or other iodine absorbent, and/or iodine reducing agent. The beds are 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.

The usual and most common iodine absorbent is cross-linked povidone.

The apparatus may desirably further comprise a third layer 24 between the first and second layers. The third layer comprises substantially insoluble povidone hydrogen peroxide particulate matter.

Tlie presence of the third layer entraps and regenerates iodine and significantly increases the biocidal activity of iodine.

A fourth layer 26, wliich may be in the form of a sublayer within the second layer, comprising particulate iodine reducing agent may be provided downstream from the second layer to provide for the reduction of any residual iodine from 12 to iodide, or, if reduction is earlier provided, to add a safety step to assure that all oxidizing iodine has been reduced.

In many applications, it may be desirable to provide a fifth layer 28 of soluble starch-l-H202 on the first layer in the liquid reservoir to permit tlie actual dissolution into the liquid of substantial amounts of starch-I H202 and thereby provide a greater reservoir of more available iodine to the liquid.

The first and second layers are essential to the full and proper functioning of the apparatus. After those layers or beds, however, any number of additional layers or additives may be provided, so long as they do not interfere with the combined function of the first and second beds or layers.

All of the layers just described may, conveniently but not necessarily, be supported by a layer 30 that may be a frit, a filter paper or a porous layer. The thickness of the beds may be the same or greatly different. lt is a simple matter to calculate contact time in a column and to provide suitable beds of materials therein.

Any of the beds may be made up the active material, e.g. starch-I H2O2, reducing sugar, etc., attached to carrier particles, such as ground glass, charcoal, ion exchange resin, cellulose derivatives, etc. The particulate matter may, in a preferred form, consist essentially of particles having a diameter of from about 10 to about 100 microns, but any size that permits suitable flow rates and assures intimate contact may be used.

The use of starcli-l-H2O2 and a physiologically acceptable reducing agent for the manufacture of transfusion biological material from one human or mammal for transfusion of such material to another human or mammal, or the transplant or transfusion biological material is a part of this invention. The transfusion or transplant is disinfected with a starch-l- H202 solution having concentration of from about 0.005 to 10 weight percent, preferably 0.1W/o to 10W/o, and thereafter treated with the reducing agent to reduce the residual iodine. Liquid materials may be treated in any suitable manner, such as has been described.

Solid tissue samples may be treated simply be soaking, by infusing or by vacuum infusing. Figure 2 depicts, largely schematically, an apparatus for treating solid tissue samples. The apparatus comprises a chamber system 100 capable of withstanding the forces of a vacuum. In the merely exemplary form shown, a cylinder 102 is closed at the respective ends by end covers 104 and 106, tlie end 106 being removable to gain access to the inside of the chamber. For example, a portion 108 of the end 106 may be slipped into the cylinder 102 and sealed using "0" rings, etc., to provide a vacuum tight seal. A vacuum line 110 through valve 112 and line 114 permits evacuation of the chamber. An input line 120, coupled to valve 122 and line 124 permits the introduction of liquid into the chamber. A platform 126, secured to the end 106, supports a tissue sample 130.

The tissue sample is placed in the chamber, the chamber evacuated and then liquid is introduced, thereby substantially replacing water in the sample with the liquid introduced.

ln another embodiment, a filter of starch may be used to remove iodide, either to purify the substance being treated, or to form a oxidizable species for generating iodine in sits, or both. Quite surprisingly, it has been found that certain cotton materials have a very strong affinity for and capacity to hold starch-iodine and iodine. A product known as "cotton wool" has been found to be an excellent filter substrate. A bed or column of cotton wool treated with iodine-hydrogen peroxide acts in a manner nearly comparable to a column of solid starch beads or particles treated with iodine-hydrogen peroxide.

It has also been found that excess amylose is cytophilic. Thus, cellcontaining materials, e.g. blood, blood cell concentrates, and the like, and tissues, are protected from injury by iodine by the presence of excess soluble amylose or amylose polymers or amylose derivatives that retain the iodophilic characteristics of amylose.

lmplantable tissues may be treated to kill microbes, i.e. "sterilized" by placing tissue that is physiologically acceptable for implantation into a human patient into a vacuum chamber, evacuating the chamber and Inaintainiilg a vacuum for a period long enough to extract at least about one-llalf of the unbound water originally present in said tissue and then introduciiig into said vacuum chamber a solution of starch-I-H2O2 for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted. The thus treated tissue may then be soaked iii a solution of an physiologically acceptable iodine reducing agent.

Alternatively, the chamber may again be evacuated to extract the starch-I H202 solution from the tissue and a solution of physiologically acceptable iodine reducing agent introduced into the vacuum chamber for saturating the tissue for reducing any residual iodine.

As a method of disinfecting blood derivatives, the invention may comprise treating blood before separation of the components thereof with starch-l-H202 to provide from a concentration of from about 0.005 to 10 weight percent, preferably 0.1who to 10W/o, starch-I-H2O2 in the blood, preparing a derivative of the blood from step, treating the derivative with starch-I-H2O2 to provide from about 0.005 to 10 weight percent, preferably 0.iW/o to 10W/o, iodine in the derivative thereafter treating the derivative by addition of a pliysiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.

Infective pathogenic micioorganisms are believed to be inactivated when starch-l-H2O2 is used in solution to perfuse tissues and organs after removal from the donor and before transplantation to the recipient. The perfusion solution comprises starch-l-H2O2 in a concentration of from about 0.005 to 10 weight percent, preferably 0.1W/o to about 10W/o (100 to 5000 ppm 12), preferably from about 0.25who to about 2W/o.

After a period of time, most of the unreacted starch-I-H2O2 is washed away and any residual iodine is absorbed into the protein or converted to inactive iodides, e.g. using ascorbate or other reducing agent as described, and does not significantly interfere with acceptance by the recipient.

The above applications in which tulle material to be purified is a liquid or cells carried in a liquid can be carried out by flowing the liquid through a bed (e.g. the conventional filter structure of solid particles on a porous or foraminous support) of solid particles of starch-I-H2O2 of suitable size or by contacting the liquid and/or the cells in the liquid with particles or a membrane or surface of solid starch-I-H202. Where a bed of particles is used with a cell-bearing liquid, the particles must be large enougll to permit intimate contact without entrapping or binding the cells.

The liquid may then be passed thorough a layer or in contact with solid phase starcl'-I-H202 to assure complete biocidal effect. Thereafter, the liquid is passed through or into intimate contact with cross-linked PVP to absorb the germicidal iodine from the liquid. Finally, a reducing agent such as ascorbate may be added if considered necessary as a precaution.

In carrying out this facet of the invention, the liquid or cell-bearing liquid is contacted with the solid starch-l-H202. This may be done most efficiently, in most cases, by passing the liquid through a settled or fluidized or packed bed of starch-I-H2O2 particles; however, such approaches will not, ordinarily, be suitable for treating cell-bearing liquids.

Cell-bearing liquids may be treated by mixing the particles in a container of the liquid or passing tlie liquid over a surface of the starch-I
H202 material, e.g. over a niultiple-plate array of sheets of such material.

Tlie starch-I-H2O2 may be washed aiid the iodine content therein regenerated between uses.

In general a solution of reducing agent , e.g. a reducing sugar (or mixtures of reducing sugars), ascorbic acid or ascorbate, a sulfite, e.g.

sodium sulfite, etc. in which the agent is in a concentration of 0.001 to 1 percent is suitable and such is implicit unless otherwise noted.

Solvent extraction of iodine with a solvent for iodine that is substantially inert to blood and biological materials generally may also be used to remove iodine. N-heptane is a good solvent for iodine and has minimal effect, on short exposure, to blood and blood cells. Close nalkane analogues and vegetable oils may also be used. Cotton seed oil, corn oil, etc. are generally biologically inert as to blood and blood cells and are also suitable solvents for iodine. The solvent extraction may be carried out in any suitable vessel that will permit intimate mixing of the blood and solvent and decantation of the hydrophobic phase from the top or withdrawal of the blood or biological liquid from the bottom.

The process results in transfusion quality whole blood that is safe, being free of pathogenic microbes, and which is also free of any added chemicals except for iodide
Industrial Application
This invention finds application in medicine and veterinary science.

Claims

WHAT IS CLAIMED 1S:

1. The use of starch-I-H2O2 for the manufacture of a medicament consisting essentially of blood cells in plasma or another carrier liquid for the treatment of disorders wherein the patient requires the transfusion of blood cells, the starch-1-H2O2 being added in an amount in excess of that required to kill or inactivate all microbes therein comprising from about 0.005who to about 10W/o of the medicament.

2. The use of starch-i-H2O2 and a physiologically acceptable reducing agent for the manufacture of transplantation or transfusion biological material from one human or mammal for transfusion of such material to another human or mammal, the transplantion or transfusion biological material being disinfected with a starch-l-H202 solution having concentration of from about 0.005who to about low/o and optionally thereafter adding said reducing agent to reduce the residual iodine.

3. A method of disinfecting biological materials derived from humans that are suitable for use in human therapy by transfusion or transplantation comprising the steps of: (a) treating said human transfusion or transplantation biological material before separation of the components thereof with starch-I-H2O, to provide from a concentration of about 0.005W/o to about ioW/o starch-I-H2O2 in said material before separation of the components thereof; (b) preparing a derivative of the material resulting from step (a); (c) treating said derivative with starch-I-H2O2 to provide from about 0.005who to about 10W/o starch-l-H202 in the derivative;

and optionally (d) removing residual iodine by reduction, absorption or solvent extraction.

4. The method of Claim 3 wherein the biological material is whole blood.

5. The method of Claim 3 wherein the biological material is blood plasma.

6. The method of Claim 3 wherein the biological material is body tissue.

7. The method of Claim 3 wherein the biological material is tissue culture nutrient.

8. The method of Claim 3 wherein tile biological material is packed red blood cells.

9. The method of Claim 3 wherein the biological material is a cell bearing liquid.

10. 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 to be treated, the improvement comprising the additional steps of: mixing the plasma with starch-I-H2O2 sufficient to resulting a starch-l-H2Ot a concentration of from about 0.005who to about 10who and allowing contact of said plasma with said starch-I-H2O2 for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma and optionally thereafter removing oxidizing iodine from the resulting mixture by passing said mixture into intimate contact with cross-linked PVP, solid starch or albumin or adding a physiologically acceptable reducing agent.

11. 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 starch-I-H2O2 and the second bed consisting essentially of substantially insoluble iodine absorbent material; tlie 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.

12. The apparatus of Claim 11 wherein the substantially insoluble iodine absorbent material is cross-linked PVP, albumin or starch.

13. The apparatus of Claim 11 further comprising an additional layer between the first and second layers, said additional layer comprising substantially insoluble PVP hydrogen peroxide particulate matter.

14. The apparatus of Claim 11 further comprising an additional layer of particulate matter below tlie second layer, said additional layer comprising an iodine reducing agent.

15. The apparatus of Claim 11 further comprising an additional layer of soluble starch-i-H202 on tlie first layer in the liquid reservoir.

16. The apparatus of Claim 11 wherein the insoluble starch-l H202 particles of the first layer are physically supported by a layer of fibrous Inaterial.

17. A method of sterilizing an irnplantable human tissue comprising: (a) placing said 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 starch-l-HzO2 for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted; 18. The method of Claim 17 further comprising the step of soaking the thus treated tissue in a solution of an physiologically acceptable iodine reducing agent.

19. The method of Claim 17 further comprising the following steps after said step (c): (d) evacuating tulle chamber to extract the starch-I-H2O2 solution; and (e) introducing a solution of physiologically acceptable iodine reducing agent iii to the vacuum chamber for saturating the tissue with said solution for reducing any residual iodine.

20. A method of disinfecting biological materials derived from humans that are suitable for use in human therapy by transfusion or transplantation comprising the steps of: (a) treating said human transfusion or transplantation biological material before separation of the components thereof with starch-I-H2O2 to provide from a concentration of about 0.005who to about 1 ow/o st & ch-I-H2O2 in said material before separation of the components thereof; and optionally (b) removing residual iodine by reduction, absorption or solvent extraction.

21. The method of Claim 20 wherein the biological material is whole blood.

22. The method of Claim 20 wherein the biological material is blood plasma.

23. The method of Claim 20 wherein the biological material is body tissue.

24. The method of Claim 20 wherein the biological material is packed red blood cells.

25. The method of Claim 20 wherein the biological material is a cell bearing liquid.

26. A method of disinfecting biological materials derived from humans that are suitable for use in human therapy by transfusion or transplantation comprising the steps of: (a) treating said human transfusion or transplantation biological material with starch-I-H202 to provide from a concentration of about 0.005who to about 10who starch-I-H2O2 in said material; and (b) removing residual iodine by reduction, absorption or solvent extraction.

27. The method of Claim 26 wherein the biological material is whole blood.

28. The method of Claim 26 wherein the biological material is blood plasma.

29. The method of Claim 26 wherein the biological material is body tissue.

30. The method of Claim 26 wherein the biological material is tissue culture nutrient.

31. The method of Claim 26 wherein the biological material is packed red blood cells.

32. The method of Claim 26 wherein the biological material is a cell bearing liquid.

33. A method of disinfecting biological materials derived from humans that are suitable for use in human therapy by transfusion or transplantation comprising the steps of: (a) treating said human transfusion or transplantation biological material with starch-1-H2O2 to provide from a concentration of about 0.005W/o to about low/o starcli-I-H2O2 in said material, the amount of starch being more than 20% greater than that required to absorb the iodine in the starch-I-H2O2; and (b) removing residual iodine by reduction, absorption or solvent extraction.

34. The method of Claim 33 wherein the biological material is whole blood.

35. The method of Claim 33 wherein the biological material is blood plasma.

36. The method of Claim 33 wherein the biological material is body tissue.

37. The method of Claim 33 wherein the biological material is tissue culture nutrient.

38. The method of Claim 33 wherein the biological material is packed red blood cells.

39. The method of Claim 33 wherein the biological material is a cell bearing liquid.