Note: Descriptions are shown in the official language in which they were submitted.
;-`` 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)
- .
~.
.... ....
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
:
',
.. -- . ~ . , i . . ~ . . . ... . . . . . . . . .
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~
12
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~.
16
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-
.:
.. . . : , . ,.. ; ,,- . .. . : ., , , :, .. . .
WO 92/0403 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
728
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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~ o
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U ~ o ~D O O O O O O O O O O O O ~ O
o
m ~ -
~ o co ~ o o + o ~ o u~ m + I~
.q . P P
A ~1 ~
~a '` O v
~3 Z ~ 'C1 ~
IH O O O O O t) U U U U O O O O tJ` tJ'
.- o o o o o m m ~ m o o o o o o
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'~'o' ~- e t~ e ~
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V~ ~; g g ~ g ~ ~ ~ .
P PP B~
~ 0~ 0~O p, .~0 0~O 0~O p,
u~ o ul In o ul P t~
o~ o~O , p, ~
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d P~ ~ ~ tl~ P P P- Il Q. D. p o o
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a I u u
~ H H 1-1 H 1-1 H ~ i H H H pc 1-1 ~3 F~
'~ I I I . ~ I 1 1 1 1 1 ` t P~ I P P
K ~ P ~ P'
0~o P- ~ ~
0~ o~ o~ o~P 0~ ~o ~o ,1~0 o~O ~0 ~0 ~0 ~ ~0 O O ~
~ O ~ Pl P- . . .
.
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
TABLE 11 - CONTINUED
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
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wO 92/04033 PCI /I,IS91 /06240
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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
~`.
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%~?$~
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,
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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.
