Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 96l08965 ' 2 ~ 9 9 3 7 ~ PCT/~JS95/12069
Photodyanmic Inactivation of Viral and Bacterial Blood Contaminants
with Halogenated Coumarin and Furocoumarin Sensitizers
FT~lTln O F T P ~,IrrVrF,r~TIO N
This invention relates to the general field of chemistry, and more
S specifically, the inactivation of viral and bacterial cont~min~tion of blood
and blood products including compositions comprising peripheral blood
cells (red blood cells, platelets, leukocytes, stem cells, etc.), plasma pl otcin
fractions (albumin, clotting factors, etc.) from collected whole blood, the
blood of virally infected subjects, ex vivo media used in the preparation of
anti-viral vaccines, and cell culture media such as fetal bovine serum,
bovine serum or derivatives from these sources.
l~ACKGROUND OF T~l~ VENTION
A major concern in the kansfusion of don~1e~1 stored whole human
blood or the various blood cell or protein fractions isolated the,~e~oll~ is the15 possibility of viral or b~cteri~l co~ ;"~tion. Of particular concern are the
blood-borne viruses that cause Hepatitis (especially Hepatitis A, Hepatitis
B, and Non-A, Non-B Hepatitis (Hepatitis C)) and Acquired Tmmlme
Deficiency Syndrome (AIDS). While any number of cell washing protocols
may reduce the viral cont~min~tion load for samples of blood cells by
physical elution of the much smaller virus particles, such washing alone is
insufficient to reduce viral cont~min~tion to safe levels. In fact, some
viruses are believed to be cell-associated, and are unlikely to be removed by
extensive washing and centrifugal pelleting of the cells. Current theory
suggests that safe levels require at least a 6 log (6 orders of m~gnit~lde)
demonskated reduction in infectious viral titer for cellular blood
components. This 6 log threshold may be greater for plasma protein
components, especially the clotting factors (Factor VIII, Factor IX) that are
~lmini~tered throughout the life of some hemophilia patients.
= =
WO 9"~5~ 3 7 ~ PCT/US95/12069
A11 blood collected in the United States is currently screened for six
infectious agents: HIV-l, HIV-2, HTLV-l, Hepatitis B Virus, Hepatitis C
Virus and Syphilis. Additionally, donors are screened for risk factors, and
potential donors are elimin~ted that are considered at risk for HIV. Despite
these measures, the risk of becoming infected by a potentially deadly virus
or bacteria via the transfusion of blood or blood products remains serious.
Screens for cont~min~nt~ are by nature not foolproof. There is also the
likely occurrence of new infectious agents that will enter the blood supply
before their significance is known. For example, by the end of June 1992,
the Center for Disease Conkol reports that 4,959 AIDS cases could be
traced directly to the receipt of blood transfusions, blood components or
tissue.
Viral inactivation by stringent sterilization is not acceptable since
this method can also destroy the functional components of the blood,
particularly the erythrocytes (red blood cells), thrombocytes (platelets) and
the labile plasma proteins, such as clotting factor VIII. Viable red blood
cells (RBC) can be characterized by one or more of the following:
capability of synthesizing ATP; cell morphology, Pso values; filterability or
deformability; oxyhemoglobin, methemoglobin and hemochrome values;
MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival.
Thus, if virally inactivated cells are damaged to the extent that the cells are
not capable of metabolizing or synthesizing ATP, or the cell circulation is
com~lolllised, then their utility in transfusion medicine is coll~ onlised.
Wet steam sterilization also destroys function blood components, in
particular, blood cells and plasma proteins. Dry heat sterilization, like wet
steam, is harmful to blood cells and blood proteins at the levels needed to
reduce viral infectivity. The use of stabilizing agents, for example,
carbohydrates, does not provide sufficient protection to the delicate blood
~ wo gG/C-~ c 2 ~ ~ 9 3 7 2 PCT/lJS95112069
cells and proteins from the general effects of exposure to high temperature
and pressure.
~ Methods that are ~ cnLly employed with purified plasma protein
fractions, often followed by lyophilization of the protein preparation,
include tre~tment with organic solvents and heat, or alternatively, extraction
with detcl~,e~ , to disrupt the lipid coat of membrane enveloped viruses.
Lyophilization, freeze-drying, alone has proven insuff1cient to either
inactivate viruses or render blood proteins sufficiently stable to the effects
of heat sterilization. The organic solvent or d~ elll method employed
with purified blood plvtei"s cannot be used with blood cells as these
chemicals destroy the lipid membrane that surrounds the cells.
Another viral inactivation approach for plasma proteins; first
demonstrated in 1958, involves the use ofthe chemical compound beta-
propiolactone with ultraviolet (UV) irradiation. This method has not found
acceptance in the United States due to concern over the toxicity of beta-
propiolactone in the amounts necessary to achieve some demonstrable viral
inactivation and to unacceptable levels of damage to the proteins caused by
the chemical agents. Concern has been raised over the explosive potential
for beta-propiolactone as well.
There is significant interest in an effective viral inactivation
tre~tment for human blood components, that will not damage the valuable
blood cells or proteins. The treatment must be nontoxic and selective for
viruses, while allowing the intermingled blood cells or proteins to survive
~ unharmed. Thus, an immediate need exists to develop protocols for ~e
inactivation of viruses that can be present in the human red blood cell
supply. For example, only recently has a test been developed for Hepatitis
C, but such screening methods, while reducing the incidence of viral
tr~n~mi~sion, do not make the blood supply completely safe or virus free.
7 ~ '
WO 96/08965 PCT/US95/12069 ~
Current statistics indicate that the transfusion risk per unit of transfused
blood is as high as 1:3,000 for Hepatitis C, and depending on geographic
location, ranges from 1:60,000 to 1:225,000 for HIV. Clearly, it is desirable
to develop a method which inactivates or indiscrimin~tely removes virus
from blood.
Co"t~ tion problems also exist for blood plasma protein
fractions, plasma fractions cont~ining immllne globulins and clotting
factors. For example, new cases of Hepatitis A and Hepatitis C have
occurred in hemophilia patients receiving protein fractions cont~ining
Factor VIII which have been treated for viral inactivation according to
approved methods. Hence, there is a need for improved viral inactivation
treatment of blood protein fractions.
In addition to the common viruses that are included in the category
of enveloped viruses, it is also highly desirable to provide a viral
inactivation protocol that is effective for non-enveloped viruses. The non-
enveloped viruses include Hepatitis A and human Parvovirus B 19. Non-
enveloped viruses do not possess lipid coats but compensate by the presence
of highly impenetrable protein capsids.
~llm~n parvovirus B 19 is a heat-stable single-stranded DNA virus of
the genus Parvovirus. B 19 is the only human parvovirus that produces
clinical illness. In children and young adults, B19 causes erythema
infectiosum, or fifth disease, a common childhood exanthema. However, in
pregnant women, patients with disorders involving increased red blood cell
production and those with either acquired or congenital immunodeficiency
B19 can be life-thre~tt ning. The disease manifestations in these individuals
include, respectively, hydrops fetalis, acute aplastic and hypoplastic anemia,
and chronic anemia. See, Luban(1994) Transfusion 34:821.
Current procedures for inactivating viruses from plasma protein
~ WO 9~ 65 PCT/US9S/12069
r ~ ~ 9 9 3 7 2
del;v~Li~es that have been incorporated into m~nllf~ctllring processes are:
1) dry heat-he~ting in freeze-dried state; 2~ heating in solution-
pasteurization, wet heat (60~C, 10 hours); 3) heating in suspension-n-
heptane; 4) vapor heat-freeze-dried state; 5) solvent detergent -- tri(n-butyl)
S phosphate/cholate, Tween 80, Triton X- 100; and 6) low pH-e.g. pH 4.25
(M.M.Mozen, "Viral Inactivation of Plasma Derivatives", in The Role of
Virus-Inactivated Plasma in Clinical Medic;ne, Bethesda, MD, National
Institutes of Health, March 23, 1994). None of these protocols is effective
for the inactivation of B 19. See, Luban, supra at 823 ("[Recent studies
support] the concept that neither solvent/detergent treatment, heat tre~tment
nor the combination is sufficient to prevent tr~n~mi~sion of B 19").
The ability to inactivate bacterial cont~min~nts from blood and blood
products may be as critical as reducing viral cont~min~nt~. Between 1986
and 1991, the Food and Drug Adminictration reported that 15.9% of all
tr~n~filsion related fatalities were associated with the transfusion of
bacterially cont~min~tç~l blood components. Most of these fatalities were
due to the transfusion of bacterially cont~min~te-l platelets.
Psoralens are naturally occurring compounds which have been used
therapeutically for millennia in Asia and Africa. The action of psoralens
and light has been used to treat vitiligo and psoriasis (PUVA therapy;
Psoralen Ultraviolet A) and more recently various forms of lymphoma.
Psoralen binds to nucleic acid double helices by intercalation
between base pairs; adenine, guanine, cytosine and thymine (DNA) or uracil
(RNA). Upon absorption of WA photons the psoralen excited state has
been shown to react with a thymine or uracil double bond and covalently
attach to both strands of a nucleic acid helix.
The crosslinking reaction is specific for a thymine (DNA) or uracil
(E~NA) base and proceeds only if the psoralen is interc~l~te~l in a site
WO 9G/08965 ~ 3 ar PCTJUS95/12069 ~
containing thymine or uracil. The initial photoadduct absorbs a second
WA photon and reacts with a second thymine or uracil on the opposing
strand of the double helix to crosslink the two strands of the double helix.
DN.
W~
~~r~
~f W~
~ O-
-- I O N O Db~ c
~ %
Lethal damage to a cell or virus occurs when a psoralen interc~l~te-l
into a nucleic acid duple~ in sites co~ g two thymines (or uracils) on
opposing strands sequentially absorb 2 WA photons. This is an ineff1cient
process because two lou probability events are required; the loc~li7~tion of
the psoralen into sites with two thymines (or uracils) present and its
sequential absorption of 2 WA photons.
United States Patent 4,748,120, to Wiesehahn ,is an e~ample ofthe
use of certain substituted psoralens by a photochemical decont~in~tion
process for the treatrnent of blood or blood products. The psoralens
described for use in the process do not include halogenated psoralens, or
psoralens with non-hydrogen binding ionic substituents. Using traditional
1~ psoralens, for example. S-metho~ypsoralen (8-MOP), 4'-aminomethyl-
_
wo 96/08965 ~ 3 7 2 PCTIUS95/IZ069
4,5',8-trimethylpsoralen (AMT) and 4'-hydroxymethy-4,5',8-
trimethylpsoralen (E~IT), it is imperative that certain substances be added
~ into the blood product solution in conjunction with W irradiatlon in order
to scavenge singlet oxygen and other highly reactive oxygen species formed
S by irradiation of the psoralen. Without the addition of reactive oxygen
species scavengers, cellular components and protein components in the
blood product are seriously damaged upon irradiation. (See also, United
States Patent 5,176,921.) It is clear, therefore, that irradiation of kaditionalpsoralens in aqueous solution creates a competition between the inefficient
photocrosslinking reaction and the generation of highly reactive oxygen
species. It is also possible that much of the viral deactivation seen using
these photosensitizers actually results from the action of the reactive oxygen
species ~in~t the viral c- nt~min~nt~ rather than the inefficient
photocrosslinkin~ mech~ni~m
Attempts to inactivate viral cont~min~nt~ using photosensitizers and
light have also been developed using some non-psoralen photosensitizers.
The photosen~iti7ers that have been employed are typically dyes. Examples
include (1ihem~topoll~hylill ether (DHE), Merocyanine 540 (MC540) and
methylene blue.
In any event, an effective radiation photosensitizer must bind
specifically to nucleic acids and must not accumulate in significant amounts
in the lipid bilayers that are common to viruses, erythrocytes, and platelets.
Although evidence shows that psoralens bind to nucleic acids by
intercalation, neutral psoralens such as 8-MOP are uncharged and thus also
have a high affinity for the interior of lipid bilayers and cell membranes.
wo~ r~ ~ 9 ~ 3 7 ~ rcT/vs9s/l2069 ~
UP~ O
(~) ~ o ~ L~.~
~ 1
O C-l O
--c
~ C~
The binding of 8-MOP to cell membranes, shown above, is
acceptable only if the psoralen bound to the lipid is photochemically inert.
However, ~vLidden (W.R. Midden, Psoralen DNA photobiology, Vol II (ed.
F.P. Gaspalloco) CRC press, pp. 1. (1988)) has presented evidence that
S psoralens photoreact with lmc~lrated lipids and photoreact with molecular
oxygen to produce active oxygen species such as supero~ide and singlet
oxygen that cause lethal darna~e to membranes. Thus, 8-MOP is an
unacceptable photosensitizer because it sensitizes indiscrimin~te damaoe to
both cells and viruses.
Positively charged psoralerls, for e~arnple, AMT, do not bind to the
interior of phospholipid bilayer membr~nes bec~use of the presence of the
charge. Ho-ve~er, ~IT contains an acidic hydrogen which binds to the
phospholipid head group by hydrogen bonding, shown below.
o
'P~ R--O C~-2.
o I ~.C~
C .. C-.C ~--a-- -- 5
c 3 1
O~O~C-3
C~3 ~ r,O
U
k-2CH~
~ wo ~ . 2 ~ ~ ~ 3 7 2 PCT~US9S~12069
Thus, AMT is an unacceptable photosensitizer because it indiscrimin~t~ly
sen~iti7es and damages viral membranes and the membranes of ery~rocytes
and platelets.
Studies of the affects of cationic sidechains on furoco-lm~rin.s as
photosen~iti7~rs are reviewed in Psoralen DNA Photobiology, Vol. I, ed. F.
Gaspano, CRC Press, Inc., Boca Raton, Fla., Chapter 2. The following
points can be gleaned from this review:
1) the intent of this line of research is to improve the poor water
solubility of the basic psoralen nucleus;
2) none of the psoralens described are halogenated as are the
photosen~iti7~rs of the present invention;
3) later conducted studies show that a cationic group on a large
linker, when added to the 5 or 8 position on the psoralen ring, gives the
psoralen nucleus i~ )roved binding with native DNA relative to
corresponding 5-MOP and 8-MOP analogues;
4) sidechain substitution at the 5 position is less desirable than
substitution at the 8 position; and
5) a study of 5-aminomethyl derivatives of 8-MOP shows that
most of the amino compounds have a much lower ability to both photobind
and form crosslinks to DNA, as compared to 8-MOP. These reports
actually suggest that the primary amino functionality is the preferred ionic
species for both photobinding and crosslinking
~ United States Patent 5,216,176, to Heindel, describes a large number
of psoralens and coumarins that have some effectiveness as photoactivated
inhibitors of epidermal growth factor. Included among the vast
functionalities that could be included in the psoralen or coumarin backbone
were halogens and amines. The inventors do not recognize the significance
W096/0896S ~ j 7 ~ PCT/US95/12069 ~ '
of either the functionality or the benefits of a photosensitizer including both
functionalities.
United States patent application serial nos. 08/165,305 and
08/091,674, commonly assigned with the present application, and parent
applications to this application, disclose the use of a novel class of psoralen
photosen~iti7~rs that are superior for use with irradiation to inactivate viral
and bacterial cu~ nt~ in blood and blood products. Said psoralens are
characterized by the presence of a halogen substituent and a non-hydrogen
binding ionic substituent to the basic psoralen side chain. See also,
Goodrich et al.(1994) Proc. Natl. Acad. Sci. USA 91:5552.
SUl~ ~ OF Tl~ NVF,l~TION
The present invention provides a method for the inactivation of viral
and bacterial cont~min~nt~ present in blood and blood protein fractions.
The present invention involves lltili7~tion of photosensitizers which
bind selectively to a viral nucleic acid, coat protein or membrane envelope.
The photosensitizer is also a moiety which can be activated upon exposure
to radiation, which may be in the form of ultraviolet radiation or ionizing
radiation, such as X-rays, ~at penetrate the cont~min~te-1 sample.
The present invention is also applicable to the inactivation of blood-
borne bacterial cont~min~nt~ and blood-borne parasitic cont~min~nt~
because such infectious org~ni~m~ rely on nucleic acids for their growth and
propagation. Since purified blood plasma protein fractions are subst~nti~lly
free of human nucleic acids, and mature human peripheral blood cells, in
particular, red blood cells and platelets, lack their own genomic DNA/RNA,
nucleic acid-binding photosensitizers are especially useful for treating the
problem of blood cont~min~nt~.
The present invention may also be applied to viral inactivation of
~ wo~ g~ 2 ~ ~ ~ 3 ~ ~ PCT/US95/12069
tissues and organs used for transplantation, to topical creams or ointnlent~
for treatment of skin disorders and for topical decont~min~tion. The present
~ invention may also be used in the m~nllfActllre of virally-based vaccines for
human or veterin~ry use, in particular, to produce live, nonviable or
~ltenll~te-l virus vaccines. The present invention may also be used in the
treatment of certain proliferative cancers, especially solid loc~li7e-1 tumors
accessible via a fiber optic light device and superficial skin cancers.
The present invention utili7es a class of compounds that have a
selective affinity to nucleic acids. The class of compounds also cont~in~ a
halogen substituent and a water soluble moiety, for example, a quaternary
ammonium ion or phosphonium ion. These materials comprise a relatively
low toxicity class of compounds, which can selectively bind to the nucleic
acid (single-stranded DNA, double-stranded DNA, or RNA) that comprises
the genetic material of viruses. The bound compound can be activated by
exposure to radiation, such as ultraviolet radiation of a defined wavelength
or ionizing radiation such as x-rays, after which the activated compound
damages the bound viral nucleic acid or viral membranes rendering the
virus sterile and non-infectious. Activation of the selectively bound
chemical photosen~iti7~r focuses the photochemistry and radiation
chemistry to the viral nucleic acid or viral membranes and limits exposure
to nearby cellular components or plasma proteins.
The preferred class of photosensitizers for use with the present
invention is characterized, generally, as follows: a) intercalators comprised
of either b) at least one halogen substituent or c) at least one non-hydrogen
bonding ionic substituent. In prefelled embodiments, the photosensitizers
comprise at least one halogen substituent and at least one non-hydrogen
bonding ionic substituent. Particularly preferred photosensitizers are
psoralens and coumarins comprising at least one halogen substituent and at
~ ~ 9 ~ 3 7 ~
WO 96/08965 PCT/US95/12069
Ieast one non-hydrogen bonding ionic substituent.
In one embodiment of the present invention, the preferred
photosensitizers are intercalators that fluoresce and that are comprised of
either a) at least one halogen substituent or b) at least one non-hydrogen
bonding ionic substituent. The preferred photosensitizers according to this
embodiment are the substituted coumarins having the structure as shown
below.
Br
~C~2,C~2C~ (CHLC~3)3
Br~C~L,C~l?C~ C~ 3~
~C~J OCI~ Ct~ C~3 N(C~)~C~3 )3
The photosensitizers disclosed herein are suited for the inactivation
of a variety of viral and bacterial cont~min~nt~ associated with blood and
blood products. The present invention specifically includes the
photoinactivation of blood and blood products contz~min~tecl with Human
Immunodeficiency Virus-1 (HIV-I), Sindbis Virus, Cytome_aLovirus.
~ wos6l0ss6s ~ ~ 9 ~ 3 7 2 PCT/US95~12~169
Vesicular Stomatitis Virus (VSV), and Herpes Simplex Virus Type 1 (HSV-
1), using the photosensitizers ofthe present invention.
~ The present invention also demonstrates the flexibility of adding one
or m~ ore halogen atoms to any cyclic ring structure capable of intercalation
5 between the stacked nucleotide bases in a nucleic acid (either DNA or
RNA) in order to confer new photoactive properties to the intercalator. In
the present invention, essentially any interc~l~tin~ molecule (psoralens,
coumarins, or other polycyclic ring structures) can be selectively modified
by halogenation or addition of non-hydrogen bonding ionic substitllçnt~ to
impart advantages in its reaction photochemistry and its competitive binding
affinity for nucleic acids over cell membranes or charged proteins.
In one embodiment, halogenation of psoralen enables the molecule,
once interc~l~te(l within the nucleic acid, to undergo a strand cleavage
reaction upon light activation that non-halogenated psoralens cannot initiate.
The nucleic acid strand cleavage is attributable to a novel electron transfer
pathway (see Figure 1) created by the breaking of the carbon-halogen bond
upon the application of the a~ u~Jl;ate radiation energy. The mech~ni~m
for this ~ltern~tive chemical reaction requires a single W photon and is
more efficient than the crosslinking reaction that normally occurs with non-
halogenated psoralens. In addition, as shown in Figures 1 and 2, the
electron transfer reaction involves transfer from a donor (usually a guanine
base when the intercalator is inserted in nucleic acid) and an acceptûr (the
carbon radical created by the broken carbûn-halogen bond). Since the
donor and acceptor species must be in close physical proximity for the
transfer reaction to proceed, most damage is limited to the nucleic acid, as is
desired in viral inactivation.
In a second embodiment, halogenation of a coumarin imparts totally
new photoactive properties useful for viral inactivation. Coumarins, unlike
WO 96/0896S ~ , PCT/US95/12069
14
psoralens, do not have an inherent ability to crosslink nucleic acid strands
upon exposure to radiation, and hence have not heretofore found application
as photosensitizers. However, as shown in the present invention (Figure 2),
halogenation of this class of interc~l~ting molecules confers the ability to
undergo the electron transfer mech~ni~m, thereby imparting new properties
to the molecule. Without int~.ncling to limit the present invention, the
inventors believe that the example of coumarin halogenation demonstrates
that the principles disclosed herein can be extended to any intercalating
molecule to confer new photoactive properties.
Due to the flexibility in adding halogen substituents or non-hydrogen
bonding ionic substituents to virtually any cyclic or polycyclic ring
structure, the inventors envision that new and useful molecules can be
created by adapting the present invention to many known classes of ring
compounds, whether those compounds comprise interc~l~tin~ agents or not.
For example, known classes of compounds that may be improved by the
present invention include, porphyrins, phthalocyanines, quinones, hypericin,
and organic dye molecules such as coumarins, for example, merocyanine
dyes, methylene blue and eosin dyes.
Without intending to limit the present invention, the inventors
anticipate that new classes of compounds ~ l~aled according to the
principles of this invention will find application in numerous fields, in
addition to the filed of decont~min~tion of blood and blood products. The
new chemical reaction properties imparted by halogenation and the selective
binding properties imparted by the use of non-hydrogen bonding ionic
substitllent~7 may be grafted onto known classes of molecules to impart
advantageous chemical reaction and taLg~lhlg properties to these molecules.
Psoralens for example, such as 8-methyoxypsoralen (8-MOP) have been
used in therapeutic photophoresis to treat cutaneous T Cell Lymphoma,
~ W096~08965 ~ 3 7 2 pcT~usss~2a6s
Scleroderma, and other cancers and skin disorders. The modif1ed psoralen
derivatives of the present invention, or other classes of compounds modified
according to the present invention, may prove more efficacious in
therapeutic photophoresis applications.
As a second example, organic dyes, for example, methylene blue
which is not considered a nucleic acid intercalating compound, have been
used for viral inactivation treatments of blood plasma with questionable
success. It is c~ eln~lated that such organic dyes, modified according to
the present invention, may prove more efficacious than the unmodified dye
in such an application.
Without intending to limit the present invention, the inventors further
anticipate that the fluorescent coumarin photosensitizers described herein
may also be used in combination with known photosensitizing molecules
that absorb in the visible light wavelength region. Figure 11 shows the
fluorescence emission spectrum of one such coumarin molecule,
Photosen~iti7~r A, having an emission peak at 414 nm in the visible light
spectrum. The emission spectrum of Photosen~iti7er A extends beyond 500
nm, which can overlap the absorbance range of certain visible light
activated molecules. It is therefore anticipated that a combination of a
visible fluorescing photosen~iti7~r with one or more photosen~iti7~rs that
absorb in the visible light region may be utilized for enhanced virucidal or
cytotoxic effect. Examples of photosen~iti7Prs that absorb in the visible
light region include hypericin, pthalocyanines, porphyrins, and organic dyes
~ such as methylene blue. See, for example, International Patent Application
WO/94 14956, wherein hypericin is activated via a chemiluminescent
reaction between luciferin and luciferase.
Other fields of application wherein the present invention may find
application include the preparation of non-infectious viral vaccines,
WO 9610896S 2 ~ ~ ~ 3 7 ~ PCT/US9S/12069 ~
16
therapeutic treatment of immune system disorders by photophoresis,
elimin~tion of viable nucleated cells such as leukocytes via the cytotoxicity
of nucleic acid binding photosensitizers and possible treatment for certain
accessible cancers and tumors exploiting the cytotoxic effects of nucleic
S acid binding photosensitizers.
The inventors further anticipate that the problem of singlet oxygen
production by W irradiation of traditional psoralen molecules can also be
reduced by incorporating a quenching sidechain moiety onto the psoralen
nucleus. An example of such a compound is shown below.
B.7
~ OCX~C~ CL~ztt~ 3
In this compound the non-hydrogen bonding ionic substituent of the
present invention further comprises a q~l~tem~ry ammonium pyridyl group.
~ This ql~tern~ry ammonium pyridyl group acts as a quencher of the W
excited triplet state of the psoralen molecule (see Figure 1).
While not int~ncling to be bound by theory, in principle the
quenching pyridyl group, or a comparable functional group, deactivates the
triplet state of any psoralen or intercalator, thereby preventing formation of
undesired singlet oxygen. The pyridyl group quenches the excited triplet
state by promoting electron transfer. In the presence of the pyridium ion the
halo-intercalator serves as the donor, and carbon-centered radicals are not
formed. The electron is transferred from the halo-intercalator to the
pyridium ion and back. This reversible electron transfer shorts out the
triplet state before it can react to make singlet oxygen. Although, in
principle. the pyridium ions quench the excited singlet state of the halo-
~ wo 9610896~ 2 ~ ~ ~ 3 7 2 PCT/USg~/12069
17
intercalator, the lifetime of the singlet state is so short that little quenching
actually occurs.
Reduction of singlet oxygen production minimi7es damage to lipid
membranes or proteins. Alt~chment of a quenching group directly onto the
S psoralen nucleus provides proximity to the excited psoralen, and obviates
the need for addition of exogenous quenching agents, such as oxygen
scavengers, reducing agents, or sugars, into the medium. Without limiting
the scope of the present invention, the inventors anticipate that quenching
sideçh~in.c that comprise both a non-hydrogen bonding ionic feature and a
triplet quenching feature will be useful for selective viral inactivation of
complex biological systems such as blood, blood plasma, or isolated blood
cell fractions.
The present invention includes methods for the viral inactivation of
non-enveloped viruses such as Hepatitis A and Human Parvovirus B 19.
The method generally includes the irradiation of blood and blood
components in the presence of photosensitizers under operating conditions
that "loosen" or increase the permeability of the viral protein capsid. In the
preferred mode of this embo-1iment non-enveloped viruses found as
cont~min~nt~ in plasmid protein compositions are inactivated by irradiation
of said compositions containing one of the photosen~iti7~rs of the present
invention.
The operating conditions for the irradiation are selected so as to
increase the permeability of the capsid. Operating conditions that may be
adjusted in order to increase access to the nucleic acid core of the non-
enveloped virus include reduced ionic strength, solvent detergent
concentration, pH, chaotrophic agents, reducing agents, freeze-thaw cycles
and elevated temperature. According to a preferred embodiment of the
invention, a photosensitizer is added to the blood product solution under
~ ~ 0 ~ 3 ~ ~
WO 9~ S~ PCT/US95/12069
18
operating conditions which increase the permeability of non-enveloped
viruses cont~min~ting said solution. The solution is then inactivated under
- conditions where subst~nti~lly all ofthe non-enveloped viruses in the
solu,tion are inactivated without substantially in~pai~ g the biological
functions of the components of the solution being treated.
Other features and advantages of the present invention will become
apparent form the following detailed description, taken in conjunction with
the accompanying figures, that illustrate by way of example, the principles
of the instant invention.
l~l?Tli ~ DESCE~TPTION OF THE DR~WINGS
FIGURE 1 depicts the proposed energy diagram of photosensitizer A
of the present invention.
FIGURE 2 depicts the proposed reaction mech~ni.~m for the
inactivation of nucleic acid upon irradiation of photosensitizer A.
FIGURE 3 depicts the inactivation of Human Tmm~-nodeficiency
Virus- 1 (E~V- 1) using long wavelength ultraviolet radiation (WA) in the
presence of different concentrations of photosensitizer B. Viral reduction,
log 10, is plotted versus WA fluence, Joules/cm2.
FIGURE 4 depicts the same data as Figure 3 as described above,
where viral reduction is plotted versus concentration of photosensitizer B.
FIGURE 5 depicts the inactivation of Sindbis Virus with
photosensitizer A and photosensitizer B. Virus inactivation is shown versus
~ WC~ 96/08965 ~ 3 7 2 PCT~US951120C9
concentration of photosçn~iti7~r.
FIGURE 6 depicts the inactivation of Cytomegalovirus using
photosensitizer B and WA. Viral inactivation is plotted versus WA
fluence.
S FIGURE 7 depicts the inactivation of Vesicular Stomatitis Virus
(VSV) in platelet concenkate using photosensitizer B and WA. Viral
inactivation is plotted versus WA fluence.
FIGURE 8 depicts the inactivation of Herpes Simplex Virus Type 1
(HSV- 1 ) in the presence of photosensitizer B and WA. Viral inactivation
is plotted versus WA fluence.
FIGURE 9 depicts the synthetic scheme for the synthesis of
photosensitizer A.
FIGURE 10 depicts the absorption spectrum of photosensitizer A.
FIGURE 11 depicts the fluorescence spectrum of photosen~iti7er A.
FIGURE 12 depicts the inactivation of Sindbis Virus with
photosensitizers B, A, AX, CX, D, DX and E.
FIGURE 13 depicts the synthetic scheme for the synthesis of
photosensitizer D.
WO ~f'.~S'', ~ 3' 7 PCT/US9~112069
.TA~T li~T) D~CRTPTION OF T~F INVhl~TION
It is to be understood that both the foregoing general description and
the following detailed description are exemplary and explanatory only and
are not restrictive of the invention as claimed.
The present invention is directed to methods for reducing viral,
bacterial and other parasitic co~-t~ tion in blood, blood components, cell
cultures or cell culture components by irradiation in the presence of a
chemical photosensitizer. Photosen~iti7f~rs are disclosed which are
particularly useful in the decont~min~tion of liquid or frozen-state liquid
compositions, for example, blood, blood components, reconstituted
lyophili7~d cells and the like, using W radiation.
According to the present invention, a radiation sensitizing chemical
compound is added to a suspension of blood, blood components, cell
cultures or cell culture components cont~min~te-l with virus and/or bacteria
and/or parasites, and the mixture is exposed to W or ionizing radiation.
Assays of viral infectivity demonstrate the effectiveness of the compounds
to inactivate the viruses as compared to radiation treatment alone.
The present invention includes a method for reducing viral, bacterial
and other parasitic cc...l~ tion from a biological sample, for example, a
20 solution. Biological solutions include, but are not limited to, solutions
comprising blood, blood components, cell culture or components of a cell
culture. The method comprises mixing the composition in a liquid state
with a chemical photosensitizer capable of binding to the viral, bacterial or
parasitic cont~min~tion. The chemical photosensitizer is capable of being
25 activated by irradiation under conditions of sufficient wavelength, intensityand period of exposure to inactivate the cont~min~nt while at the same time
the conditions for irradiation are insufficient to produce reactive oxygen
species in the composition at levels which substantially impair the
physiological activity of the treated composition. The composition
o WO 96/08965 ~ ~ 9 9 3 7 2 PCTIUS95/12069
21
c-)nt~inin~ the photosensitizer is then irr~ te~l under conditions where the
concentration of biologically active cont~min~nt is reduced and the
physiological activity ofthe composition is subst~nti~lly unimpaired.
The following definitions will be helpful in underst~n-iin~ the
specification and claims. The definitions provided herein should be borne
in mind when these terms are used in the following examples and
throughout the instant application.
One of the most critical elements of the present invention is the use
of a novel class of photosensitizer. A photosensitizer is defined for the
liO purposes ofthis application as a chemical compound that has a light-
absorbing chromophore that absorbs radiation between 780 and 200 nm, and
is capable of inactivating viral, bacterial or parasitic cont~min~nts in blood
or blood products.
The photoserl~iti7ers of the present invention are characterized by
their ability to bind to the nucleic acid components of the viral or bacterial
co. ~ nt~ that are to be inactivated. The blood and blood product
compositions that are to be treated according to the method of this invention
all contain at least some cellular components or complex ~loteills.
In one embodiment of the invention, the photosçnsiti7~rs of this
invention are characterized as comprising a lipophilic moiety, a hydrophilic
moiety and a photoreactive moiety.
The photosensitizers of this invention are preferably nucleic acid
intercalators that are comprised of either 1 ) at least one halogen atom; and
b) at least one non-hydrogen bonding ionic moiety. Intercalators are
defined broadly herein as any chemical compound that has a specific
affinity to double or single stranded nucleic acid. More specifically,
intercalators are chemicals -- not including nucleic acids, proteins or
peptides -- that locate themselves between neighboring base pairs in nucleic
WO 96/08965 r ~ ~ ~ 9 3 7 ~ PCTIUS95112069 ~
acids. Intercalators are generally characterized by the presence of a
relatively planar rigid, multi-cyclic pi-conjugated chemical backbone.
Those skilled in the art are f~mili~r with a relatively large number of
intercalators and are generally able to predict the types of chemical species
that are able to function as intercalators based on the chemical structure of
the backbone of the chemical species. Psoralens and coumarins, the
p~ ed basic structure for the intercalators of the present invention, are
just two examples of chemical backbone structures capable of nucleic acid
intercalation.
Prefellcd photosensitizers of the present invention comprise at least
one halogen subst~ ent The halogens include F, Cl, Br and I. In the
preferred embodiments of the present invention, the photosensitizer contains
at least one bromine or chlorine atom.
Preferred photosensitizers of the present invention comprise at least
one non-hydrogen bonding ionic substituent. Chemical functionalities that
are ionic and non-hydrogen bonding include quaternary ammonium
functionalities and phosphonium functionalities. A variety of additional
functionalities that are both ionic and non-hydrogen bonding are f~mili~r to
those skilled in the art, and equally applicable for use with this invention.
In the ~lefell~d embo~liment~ of the invention, the non-hydrogen
bonding ionic substituent is linked to the backbone of the chemical
intercalator via a spacer unit. The spacer can be selected from any of a
number of chemical subunits known to those skilled in art, but in the
preferred embodiments is composed of a saturated linear alkoxy group. In
the most preferred embodiment the spacer element is -O(CH2)3-.
The most preferred non-hydrogen bonding ionic functionalities are
quaternary ammonium functionalities, more specifically, trialkyl quaternary
ammonium, and even more specifically, -O(CH2)3 N3(CHzCH3)3.
-
~ wog~a~6s 2 ~ 9 9 3 7 2 PCT/USg5~12069
Two preferred photosensitizers o f the present invention are the
following:
~r
~~ A/CCuzC L~ ~C~c~L~C:~,c 3)3
A B
Compound A is a coumarin based photosensitizer, nd compourld B is a
psoralen or furocournarin based photosen~ 7~r.
Addi~ional preferred embodiments of ~e presen~ invention include
~e following coumarin based photosen~iLi;~a:
CCX. C- '-C.~ ~" c ~ )~
G~5J~ ~OC~C' C~ C." C.U.3''
C
~C~ ~C~C.~,~.CL~,.Z~(C~_C~
The synthesis of photosensitizer A is described in EYample 9 beLow.
accordin~J ~o the scheme shown in Fi~ure 7. The svnthesis of
W O 96/08965 ~ 3 7 ~ PCTrUS95/12069
24
photosensitizer D is described in Example 14 below, according to the
scheme shown in Figure 13.
Upon W irradiation, compound A has been shown to be effective at
viral inactivation while compound B has been shown to be effective at viral
and bacterial inactivation. Compounds A, D and E also fluoresce upon W
irradiation. It is theorized by the present inventors that the fluorescence
pathway for the dispersion of energy from the excited state of irr~ terl
compounds A, D and E, as depicted in Figure 1, acts to reduce the
production of highly reactive oxygen species in blood and blood
components. The proposed reaction mech~ni~m for the inactivation of viral
cont~min~nt~ using compound A and W radiation is shown in Figure 2.
According to the proposed mech~ni~m -- which is speculative and not
intended to limit the scope of the invention -- the photoreaction is initi~te~l
by an electron transfer from a guanine residue to the photosçnciti7er in its
executed singlet state. Electron transfer is followed by Br-C bond
homolysis and the generation of a coumarin radical that can attack the
nucleic acid backbone.
Bromopsoralens, specifically photosensitizer B, do not form free
radicals upon irradiation in solution. A donor is re~uired to activate
photosen~iti7~r B. Using fluorescence spectroscopy it has been shown that
amino acids are not suitable donors to activate photosensitizer B. Thus, any
of these photosensitizers bound or associated with proteins should not
generate radicals capable of ~l~n~ginp proteins.
It is, therefore, one preferred embodiment of the method of the
present invention to use a photosensitizer that is capable of fluorescence.
Coumarins and furocoumarins that fluoresce are known to those skilled in
the art and the screening of photosensitizers to determine fluorescent
properties is easily determined.
,~ wo ~Dr ~ 2 ~ ~ 9 3 7 2 PcTrusgs/l2069
properties is easily determined.
Photosensitizers that are capable of fluorescence appear to be
superior to non-fluorescent varieties. For a photosensitizer to be use~l,
there must be a mech~ni~m for viral and bacterial inactivation. Non-
halogenated psoralens may still function as useful photosensitizers if they
are properly situated in the solution to be treated. Such compounds can
inactivate viruses via the traditional photocrosslinkin~ mech~nism Other
photosensitizers, such as those having the coumarin backbone structure,
must be halogenated in order to accomplish significant viral or bacterial
inactivation. Thus, in this embodiment of the invention the preferred
photosensitizers are intercalators capable of fluorescence and either 1) are
halogenated or 2) have the psoralen backbone structure.
According to an additional embodiment of the present invention, the
photos~n~iti7~r of the invention comprises a quenching sidechain moiety
attached to the interc~l~tin~ backbone. Figure 1 provides a diagr~mm~tic
energy diagrarn for certain halogenated photosensitizers that are capable of
fluorescence. According to the theory expressed herein, the ability to
fluoresce provides a rapid means for the excited singlet state species to
revert to ground energy state that competes with intersystem crossing to the
triplet excited state. For photosensitizers that do not fluoresce in particular~the presence of a quenching moiety attached to the intercalator can also
serve the same function.
An e~ample of a photosensitizer of this embodiment of the invention
is as follows:
B~
o~
~ c~ C~ C,~ 3
WO 96/08965 PCT/US95/12069
2 ~
26
The non-hydrogen bonding ionic substituent comprises a quaternary
ammonium pyridy~ group. Such a compound can be easily prepared by one
skilled in the art without undue experimentation. The quaternary
ammonium pyridyl group can serve as a quencher of the W excited triplet
state of the psoralen compound. While not intending to be bound by theory,
it is proposed that the quenching group will deactivate the triplet state of
any intercalator, thereby preventing formation of undesired singlet oxygen.
The reduction of singlet oxygen production as such minimi7es damage to
lipid membranes or proteins. The proximity of the quenching moiety to the
intercalator should make quenching highly preferred to any reaction with
oxygen in solution, and should also obviate the need for the addition of
exogenous quenching agents (such as oxygen scavengers, reducing agents
or sugars) into the medium. The quenching moiety may be attached to the
backbone of the photosensitizer at any position, and can consist of any
chemical functionality known to those skilled in the art to function as an
excited state quenching agent.
The quaternary ammonium or phosphonium substituted halo-
intercalators described herein do not accllm~ te in the interior of lipid
bilayers (membranes) found in blood and blood products because of the
presence of the charge, nor will they bind to the phospholipid head groups
of the membrane because they lack acidic hydrogen for hydrogen bonding.
Prior art psoralens, for example, 8-MOP and AMT, must often be
used in combination with a quencher (e.g. m~nnitol, dithiothreitol, vitamin
E, etc.) to protect, repair or otherwise offset the deleterious effects of the
photosen~iti7er and light on cell membranes, and to quench the production
of free oxygen radicals in solution that cause indiscrimin~te damage. The
photosensitizers described herein do not accumulate in viral membranes and
as a consequence do not require the presence of a quencher additive to the
~ WO 96108965 ' 2 1 9 9 3 7 2 PCTIUS95112069
blood product. In addition, the photosensitizers described herein cont~ining
halogen generate a minim~l amount of free radicals in solution, thereby
avoiding the need for quenchers.
One preferred class of photosen~iti~rs is selected from the group
S consisting of compounds of the formula (I):
wherein ,u is an integer from 1 to 6; X is an anionic counterion; Z is N or P;
Rl, R2, R3, R4, Rs and R6 are indep~n(lçntly halo; H, linear or branched alkyl
of 1-10 carbon atoms, linear or branched alkoxy of 1-10 carbon atoms;
(CH2)-mO (CH2)pZ-R',R",R'n or -O(CH2)nZ-R',R",R"' wherein n, m and p
are indepen(l~ntly integers from 1 to 10 and R~, RN, and R~n are
indepen(l.ontly H or linear or branched alkyl of 1 to 10 carbon atoms with
the proviso that on each Z atom, not more than two of R~, RN, or R~n may be
H; and at least on one of R" R2, R3, R4, R~ or R6 is
(CH2)mO(CH2)pZ-R',R",R'nor-O(CH2)nZ-R',Rn,R'n. Particularlypreferred
are compounds wherein R4 is -O(CH2)nN-R',R",R~n, especially wherein R~,
R~ and R'n are ethyl and n=3. Preferably, R6, Rs~ R2 and R, are hydrogen
and R3 is H or halo, preferably bromo.
3 ~ ~
WO 96/08965 PCT/US95/12069
'~ 8
An additional plere~ d class of photosensitizers is selected from the
group consisting o~the formula (II).
" R~,~ R3 _~
C '~\ V ~ ~ X ~~ (II)
R~
wherein 11 is an integer from 1 to 6; X is an anionic counterion; Z is N or P;
Rl, R~, R3, R~, R" and R6 are independently halo; H, linear or branched
alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms;
(CH~)-mO (CH,)pZ-R',R",R"' or -O(CH,)nZ'R',R",Rn' wherein n, m and p
are independently integers from 1 to 10 and R', R~, and R~n are
indepen~ntly H or linear or branched alkyl of 1 to 10 carbon atoms with
~e proviso that on each Z atom, not more than two of R', R", or R'N may be
H; and at least on one of Rl, R~, R3, R4, Ra or R6 is
(CH2)mO(CH~)pZ-R',R",R"'or-O(CH,)nZ-R',R/',R///. Particularlypreferred
are compounds wherein R4 is -O(CH,)nN-R',R",R"', especiallv wherein R',
RN and R"' are ethyl and n=3. Preferably, R3, Ra~ R~ and R, are hydrogen
1~ and R3 is H or halo, preferably bromo.
In _eneral, the above compounds are made bv halogenating psoralens
and isolating the appropriately substituted isomers. For compounds wherein
the ring substituent is a qll~tPrn~rv ammonium alko~cy or phosphonium
alkoxy group~ that group may be made from the corresponding hydroxv-
substituted psoralens, as exemplified by the following scheme. sr
oJ~o~ oJ~?
¦ B~
~ WO9. '.~' PCT~US95/12069
~2 ~9~37~
29
Br Br
Br ~?
.. r, x
. ~r
, . ~a ~, O O ~~
~. ~ ~ r- I - n~
As described above, the most preferred photosensitizers of the
present invention are comprised of ionic functionalities that are non-
hydrogen bon~lin~. However, included within the scope of this invention
are photosen~iti7~rs comprised of amine functionalities having one and in
some cases two amine hydrogens. These compounds, of course, are capable
of forming hydrogen bonds. It has been shown that there is a direct
correlation between the number of hydrogens available on the amine and the
cellular destruction caused by a class of psoralen compounds. Goodrich, et
al. (1994) Proc. Nat'l. Acad. Sci. USA, 91:5552. Thus, photosensitizers
cont~ining amine functionalities having two hydrogens are less preferred
than those having one hydrogen, which are in turn less preferred than those
having no hydrogen attached to the amine.
Therefore, according to this invention, sensitizing compounds for
viral inactivation, preferably, do not contain substituents which possess free
hydrogen groups capable of exhibiting hydrogen bonding to the cell
membrane.
From the foregoing description, it will be realized that the present
WO 96/0896S ~ 3 7 ~ PCT/US95/12069
Therefore, according to this invention, sensitizing compounds for
viral inactivation, preferably, do not contain substituents which possess free
~ hydrogen groups capable of exhibiting hydrogen bonding to the cell
membrane.
From the foregoing description, it will be realized that the present
invention can be used to selectively bind a chemical photosensitizer to
blood-tr~n.~mittecl viruses, bacteria, or parasites. Also monoclonal or
polyclonal antibodies directed against specific viral antigens, either coat
proteins or envelope proteins, may be covalently coupled with a
photosensitizer compound.
Since cell compositions also comprise a variety of proteins, the
method of decont~min~tion of cells described herein is also applicable to
protein fractions, particularly blood plasma protein fractions, including, but
not limite-l to, fractions cont~ining clotting factors (such as Factor VIII and
Factor I~, serum albumin and immlme globulins. The viral and bacterial
inactivation may be accomplished by treating a protein fraction with a
photosen.~iti7~r as described herein.
Although described in connection with viruses, it will be understood
that the methods of the present invention are generally also useful to
inactivate any biological cont~min~nt found in stored blood or blood
products, including bacteria and blood-tr~n~mitte~l parasites.
The halogenated psoralens and coumarins according to the present
invention are improved and more efficient photosensitizers because they
require only a single WA photon for activation. The ability of the halogen
photosen~iti7er to react with any base pair imposes no limitation for the site
of intercalation. As shown in Figure 2, absorption of a WA photon by a
bromocoumarin in the presence of guanine (or any nucleotide base) leads to
electron transfer and the formation of bound radicals and ultimately nucleic
WO 96108965 PCT/US95/12069
~ 2 ~ 3 7 ~
31
acid cleavage and viral or cell death. This cleavage mech~nism is more
efficient than the conventional crosslinkin~ reaction of non-halogenated
psoralens.
The coumarin radical (Figure 2) can inflict damage on the nucleic
acid double helix to which it is bonded by abstraction of a ribose (RNA) or
deoxyribose (DNA) sugar carbon hydrogen bond. This leads to DNA
cleavage by known mech~ni~m~. The guanine radical cation shown as an
example is also known to react with molecular oxygen, initiating a series of
reactions which cleave DNA. The byproduct of the bound radical
photochemistry is debromin~te~l coumarin 4 that is incapable of forming
crosslinks to DNA, unlike psoralens.
A preferred class of photosen~iti7Prs comprise nucleic acid
intercalators which may be added to plasma or plasma fractions followed by
W radiation to reduce the viral cont~min~tion therein. According to the
present invention, the reduction of viral cont~min~tion can be unexpectedly
reduced by ~ltili7ing halogenated intercalators. For example, it was
observed that the bromopsoralens are about 200,000 times more effective in
reducing viral activity when co~ aLed to use of their non-bromin~te~l
counterparts.
The brominated intercalators are an improvement over the known
psoralens and other substituted psoralens when used as photosensitizers
because only one photon of light is required to activate the brominated
photosensitizer whereas two photons are required to activate a non-
bromin~te(l photosensitizer. Secondly, a brominated intercalator is effective
in virtually every intercalative site, whereas a non-brominated
photosensitizer is effective only in intercalation sites cont~ining a uracil or
thymine on different strands of the DNA or RNA. The brominated
intercalators are also an improvement over the known coumarins, which
W O 96108965 ~ 3 7 2 PCTrUS9S112069 ~
unlike the known psoralens have no crosslinking ability, and therefore, have
generally not been used previously as photosensitizers for viral inactivation
~ or as light activated drugs in therapeutic photophoresis procedures for
certain cancer treatments and immllne disorders.
Brominated or halogenated intercalators are particularly useful for
inactivation in hydrated systems such as plasma, immune sera, tissue culture
media cont~ining animal serum or serum components, for example, fetal
calf serum, or recombinant products isolated from tissue culture media.
The present invention may be applied to treatment of liquid blood in
0 ex vivo irradiation, such as by methods and al~pa,dl~ls described in U.S.
Patents 4,889,129 and 4,878,891 and 4,613,322.
The photosensitizers disclosed herein may also be utilized in vivo,
delivered in liposomes (artificial cells) or drug-loaded natural cells. After
introduction of the liposome or drug-loaded cell, the patient may be treated
lS by radiation to activate the photosen~iti~r.
The present invention is applicable to cont~min~nt~ which comprise
single or double-stranded nucleic acid chains, including RNA and DNA,
viruses, bacteria and paldsilic co~ tion.
According to one embodiment of the present invention, certain
biological solutions that are cont~min~te~l with non-enveloped viruses are
treated in order to inactivate all viral cont~min~nt.~ in the solution, including
non-enveloped viruses. The treatment required for inactivating non-
enveloped viruses includes the manipulation of operating conditions in
order to loosen or increase the permeability of the capsid surrounding the
genetic core of the virus. Although not limited by theory, the inventors
hereto speculate that the adjustment of operating conditions to increase the
permeability of the capsid allows the photosensitizers of the present
invention access to the genetic material of the virus, theFeby allowing viral
~ wo 96l08965 ~ 2 ~1 9 ~ 3 7 ~ PcT/r3sg5~l2a69
- 33
inactivation to occur -- by h~rming the genetic material of the virus -- upon
irradiation.
Past attempts to inactivate non-enveloped viruses by irradiation in
the presence of photosen~iti7~rs have been almost totally unsuccessful. It is
plesuilled that the reason for this failure is the inability of the
photosensitizers to gain access to the genetic material of the virus.
Moreover, unrelated efforts have been made to determine conditions under
which the capsid surrounding non-enveloped viruses are loosened. Despite
such efforts, none of the commercially employed techniques for treating
compositions for viral inactivation are effective ~ in~t non-enveloped
viruses. Some of the conditions that have been manipulated in order to
loosen the capsid of non-enveloped viruses include: ionic strength of the
solution, pH, solvent detergent treatments, chaotrophic agents such as urea,
reducing agents, chelating agents, freeze-thaw cycles, and ten~eldl~lre. In
1~ one l~lefell~d embodiment ofthe present invention, described in detail inExample 15, adjustment of the pH, ionic strength and freeze-thaw cycles on
a plasma solution cont~inin~ Porcine Parvovirus yielded dramatic
improvements upon irradiation in the presence of one of the
photosen~iti7~rs ofthe present invention.
It is, therefore, an embodiment of this invention to reduce non-
enveloped viral cont~min~tion in a biological solution. This method
encompasses the adjustment of the operating conditions of the solution so as
to loosen the capsid of the virus either prior or subsequent to the addition of
a photosensitizer of the present invention into the solution. The solution is
then irr~ te-l under conditions to inactivate the non-enveloped viruses.
Although bromin~te-1 psoralens with quaternary ammonium side
chains have been tried to inactivate non-enveloped viruses, results obtained
to date show that these compounds are relatively ineffective against non-
3 7 ~
WO 96/0896S PCTIUS95112069
34
enveloped viruses. The psoralen compounds tested are quite hydrophilic
due to the presence of the furan ring. However, experiments performed
using DNA intercalators that are relatively less hydrophilic, the level of
inactivation of Porcine Parvovirus -- a non-enveloped single stranded DNA
virus -- increased. For example, with photosen~iti7Pr B, a bromin~te~l
psoralen photosensitizer, 0.26 log reduction was obtained in Porcine
Parvovirus with an initial titer of 6 logs. On the other hand, with a
brominated coumarin such as photosen~iti7er A, which does not have the
hydrophilic furan ring, 0.56 log reduction was obtained (see Table 17).
These data suggest that DNA intercalators with reduced hydrophilicity may
be more effective in penetrating the tight protein capsid of non-enveloped
viruses such as Porcine Parvovirus.
In an alternate version of this embodiment, osmotic shock is used to
loosen the protein capsid. When a cell or virus is suspended in a low ionic
strength hypotonic solution, the cell will be subjected to an osmotic shock
resulting in volume expansion. In some viruses, hypotonicity may lead to
LlllC of the protein capsid with discharge of their nucleic acid contents.
The present invention includes a method for incorporating photosensitizers
into non-enveloped virus in which a short but intense period of osmotic
stress will cause the virus to become transiently permeable and allows
partial incorporation of photosensitizers with low molecular weights.
In the osmotic shock procedure, the virus is first incubated for a short
time with dimethylsulfoxide (DMSO) or another chemical such as polyol
(i.e., glycerol), or organic solvents in addition to DMSO (i.e., ethanol) that
permeate to viral capsid. Next, the virus is rapidly diluted in a solution
cont~inin~ photosensitizers. The abrupt change in extracellular DMSO
concentration induced by rapid dilution creates an osmotic gradient that
spontaneously decays as DMSO reaches a new equilibrium. This transient
W096/0896~ ~ ~ 9 ~ 3 7 2 PCT/US95/12069
osmotic gradient causes the cells to swell and become permeable for a short
period of time to photosensitizers (the smaller the sensitizers the easier the
- movement through the viral capsid). The composition of the diluent has a
profound effect on the viral capsid during osmotic shock. In one preferred
embodiment, the diluent may contain photosensitizer, inositol
hexaphosphate (IHP), EDTA or EGTA, sodium pyrophosphate or any
polyanion in different combinations. Using distilled water alone without
DMSO to induce osmotic shock in Parvovirus, it is possible to increase the
inactivation of the virus from 0.62 to 2.46 logs. Data to this effect were
obtained when the osmolality of plasma was reduced by 50%. However,
further reduction in osmolality of the medium to either 30, 60, 90 or 120
mOsmol (i.e. 1, 20, 30 and 40% dilution of the native plasma with water)
significantly increases the inactivation of Parvovirus from 2.46 logs to as
high at 4 logs.
This embodiment ofthe invention includes the use of methods for the
inactivation of non-enveloped viruses using the osmotic shock process for
selective incorporation of photosensitizer into the virus. In addition, this
invention also includes the use of small molecular weight nucleic acid
intercalators that are more effective at penetrating the ~roteill capsid of
viruses that have been subjected to osmotic shock, either alone or in
combination with a freeze-thaw cycle, metal chelators, and polyanions or
other operating conditions that help loosen the capsid. Furthermore, this
invention covers the following photosensitizer compounds (or derivatives
thereof) that offer potentially desirable intercalation properties and that are
less hydrophilic than psoralen-based photosen~iti7ers:
1. Cinnamic acid
2. Caffeic acid
3. Ferulic acid
WO 96/08965 r 2 ~ 3 ~ 2 PCT/US95112069
36
4. Coumarins
5. Gallic acid
6. Polyacetylenes
7. Thiophenes
8. Alpha terthienyl
The present invention includes the inactivation of specific viral
species that are found as cont~min~nt~ in blood and blood products.
Fx~mple 1 below describes in great detail the experimental protocol for the
inactivation of HIV- 1 virus in platelet concentrate. The results obtained
from this series of experiments validate the ability of the photosensitizers of
the present invention to inactivate HIV- 1 virus in a blood product. The
results of this study are ~llmm~rized in Table 1. Reductions in viral titer
were obtained by subtracting the viral titer of treated samples from control
samples. Figures 3 and 4 show a graphic representation of the results of the
study. Figure 3 shows the viral reduction versus light intensity for a number
of different concentrations of photosensitizer B, and Figure 4 shows viral
reduction versus concentration of photosensitizer B.
The procedure described in detail in Example 1 for the inactivation
of the HIV-l virus in platelets is typical of the type of experimental protocol
lltili7e~1 to examine the inactivation of a variety of viral species. Example 2
below describes the general protocol used to demonstrate the inactivation of
Sindbis Virus in human plasma. The results of the inactivation using
photosen~iti7~r A and photosensitizer B are depicted in Figure 5. Example
3 below describes the general protocol used to demonstrate the inactivation
of Cytomegalovirus in human platelet concentrates. The results of the
inactivation using photosensitizer B are depicted in Figure 6. Example 4
below describes the general protocol used to demonstrate the inactivation of
Vesicular Stomatitis Virus in human platelet concentrates. The results of
~ WO 96/08965 2 ~ ~ ~ 3 ~ 2 PCT/US95/12069
37
the inactivation using photosensitizer B are depicted in Figure 7. Example 5
below describes the general protocol used to demonstrate the inactivation of
Herpes Simplex Virus Type I. The results of the inactivation using
pkotosensitizer B are depicted in Figure 8.
Because the photosensitizers of the present invention are to be used
to inactivate blood and blood products for use in the transfusion into human
patients, it is imperative that they be safe for transfusion following
irradiation. Example 6 below describes the mutagenicity protocol used to
verify the safeness of the photosen~iti7ers of the present invention.
Example 6 is specific for photosensitizer B, before and after irradiation,
under conditions suitable for the inactivation of viral and bacterial
components in blood and blood products. The results of the mutagenicity
tests for photosen.citi7er B demonstrate that a mixture of photosen~iti7er B
photolysis products and a maximum residual photosensitizer B
concentration of 4.36 ~lg/mL per test plate do not cause any mutagenic
effects in Salmonella strains TA98, TA100, TA1535, TA1537 and TA1538.
The m~x;...l.... residual concentrations of photosensitizer B under use
conditions (25 J/cm2 of WA) correspond to about 3 .4 times the expected
conc~ dlion of photosen~iti7~r B per therapeutic dose of platelet
concentrates of 1.28 ~lg/mL. The results, thus, demonstrate that
photos~n~iti7er B is non-mutagenic when photolyzed in platelet
concentrates such that the initial concentration is reduced by at least 60%
under use conditions (>25 J/cm2 WA and 12.8 ~lg/mL photosensitizer B
plate).
The mutagenicity results for photosensitizer A show that for both
irr~ te~1 and non-irr~ te~l solutions there is no significant increase in
reversion rate with any of the five test strains in either the absence or
presence of S-9 activation.
WO 96/0896~i 2 ~ ~ ~ 3 7 ~ PCT/US95/12069 ~
38
Example 7 describes the mouse fibroblast protocol used to determine
the cytotoxicity of the photosensitizers of the present invention. The results
~ of these tests for photosensitizer B at 72 hr are depicted in Table 2.
Example 8 describes the Chinese hamster ovary hybridoma cell and AE-L
cell protocol used to cletermine the cytotoxicity of the photosensitizers of
the present invention. The results of these tests for photosensitizer B are
depicted in Tables 3 and 4.
Compound A, 3-bromo-7-(r-triethylammonium propyloxy)
coumarin bromide, is one of the most preferred photosensitizers of the
present invention. The synthesis of Compound A is given in Example 9.
The reaction scheme for the synthesis is shown in Figure 9.
One of the best measures of the effectiveness of potential
photosensitizers is the extent to which the photosen~iti7er tends to associate
with nucleic acids rather than to cellular membrane components or proteins
in blood or blood products. Example 10 describes the protocol employed
for analyzing the specificities that a variety of photosensitizers have for
nucleic acids.
Independent of the mech~ni~m of photosensitized inactivation of
viral and bacterial co~t~ nt~ in blood or blood products, it is generally
clear that the greater the preference the photosensitizer has to the nucleic
acid components of the cont~min~nt~ -- as opposed to cellular membranes
or proteins in solution -- the better the performance of the photosensitizer.
The quality of a photosensitizer being determined by the rate of efficiency
of cont~min~nt inactivation, absolute cont~min~nt inactivation and
impairment of the physiological activity of the treated composition. Of
course, these factors are all interrelated. The results of specificity
experiments comparing the photosensitizers of the present invention with
prior art photosensitizers reveal the superior properties of the novel
~ W096108965 ~ 3 7 ~ PCT/US95rl2069
39
photosensitizers disclosed herein. These results are shown in Table 5.
The photosensitizers of the present invention have been examined
with regard to their effects on the constituents of platelet concentrates under
conditions that are sufficient for obtaining complete cont~min~nt
inactivation. The general procedure for condllcting these experiments is
disclosed in Example 11 below.
Table 6 presents a summ~ry of the in vitro platelet properties after
photoactivation in the presence of 300 ,ug/mL of photosensitizer B, with and
without bicarbonate. Bicarbonate is added to offset the effects on the pH of
1~ the solution resulting from irradiation. Table 7 presents a summary of the
phoresed platelet in vitro properties following photoinactivation in the
presence 300 ~lg/mL of photosenciti7er B. Table 8 summarizes the platelet
in vitro properties following photoinactivation in the presence of
photosensitizer A. The pH does not subst~nti~lly change with the use of
photosçnsiti7er A.
Additional experiments were conducted in order to compare the
photosçnsiti~rs of the present invention with two prior art photosensitizers,
8-MOP and AMT. The protocol for this evaluation of photosensitizers
irr~ te~1 in human platelet concentrate is described in Example 12. The
results ofthis comparison can be summarized as follows:
1. complete inactivation of bacteriophage ~6(26 logs of viral
reduction) is obtained with photosensitizer B without
alteration in platelet in vitro properties (HSR, morphology,
aggregation response to collagen) under normal oxygen
content at WA fluence of 7.6 J/cm2,
2. equimolar concentrations of AMT and 8-MOP required 45
W O 96/08965 ~ 3 7 2 PCTrUS9S112069
and 68 J/cm2 of WA energy, respectively, to obtain greater
than 4 logs of viral inactivation and are associated with major
alterations in platelet in vitro properties;
3. photoinactivated platelet concentrates using photosensitizer B
(60 ~lM photosensitizer concentration and 4.5 J/cm2) m~int~in
normal properties following post-tre~tn ent storage for 5 days
in a standard platelet incubator at 22 ~t 2~C; and
4. virucidal efficacy of bromin~te~l psoralen is substantially higher than that of 8-MOP or AMT with respect to
inactivation of non-enveloped bacteriophages such as lambda
and R-17.
Example 13 describes the results of a comparison study of the ability
of a variety of photosensitizers of the present invention to inactivate Sindbis
Virus in human plasma. The compounds tested in this series of experiments
were photosen~iti7Prs A, B, D and E and non-halogenated forms of A, C
and D. These results of these experiments are depicted graphically in
Figure 12. The results show that under the same conditions: 1) the
coumarin-based photosensitizers A, C and D are superior to the psoralen-
based photosensitizer B; 2) the non-halogenated coumarin-based
photosensitizers are not suitable for photoactivated inactivation of virus; and
3) the methylated coumarins, photosensitizers D and E, appear to be the
most efficient photosensitizers for viral inactivation.
Example 14 describes the synthesis of photosensitizer D. The
procedure follows the synthetic scheme depicted in Figure 13. Following
this general procedure, believed to be novel, one skilled in the art may also
~ WO 9~'D8'' J~ 2 ~ 0 9 3 7 2 PCT/USg5/12069
=41
synthesize photosensitizer E and other photosen.~iti7ers of the present
invention. (See,~e.g, Sethna (1945) Chem. Rev. 36:10; Sethna et al. (1953)
~ Organic Reactions 7:1 ).
Example 15 describes the results of a series of experiments showing
the effectiveness of the present invention in inactivating the non-enveloped
Porcine Parvovirus in plasma. Manipulation of the operating conditions --
particularly ionic strength, pH and freeze/thaw cycles -- make it possible to
significantly inactivate Porcine Parvovirus with photosensitizer A and
irradiation.
1~ The following examples serve to explain and illustrate the present
invention. Said examples are not to be construed as limiting of the
invention in anyway. Various modification are possible within the scope of
the invention as described and claimed herein.
F~mple 1: Tn~l~tivation of ~IV-1 Virus ;n Platelet Concentrate
The experimental design for the viral validation studies involves the
addition of photosen~iti7Pr B to platelet concentrates in standard platelet
collection bags and subsequent activation of the photosen~iti7er by
ultraviolet irradiation at 320-400 nm. The following studies were
performed in order to verify the elimin~tion of HIV-l from platelet
conce~ a~es.
PHASE I
Photosensitizer Toxicity Test: This study establishes the degree of
toxicity of the photosensitizer to the indicator cell lines used in the assay
and rules out any interference, by the photosensitizer, with the ability of the
chosen viruses to infect the indicator cell lines.
WO 9C/08965 ~ 9 3 ~ 2 PCT/US9!itl2069
42
Photosensitizer Toxicity to Viral Indicator Cells
Sample Set 1
1. Platelet + Saline + Orbital Shaking + 30 min ambient
2. Platelet + Saline + Orbital Shaking + 30 min WA
3. Platelet + 100 ,ug/mL Photosen~iti7er B + Orbital shaking + 30 min
ambient
4. Platelet + 100 ~Lg/mL Photos~n~iti~er B + Orbital Shaking + 30 min
WA
5. Platelet + 300 ,ug/mL Photosen~iti7~r B + Orbital Shaking + 30 min
1 0 ambient
6. Platelet + 300 ,ug/mL Photosensitizer B + Orbital Shaking + 30 min
WA
Sample Set 2
7. Platelet + Saline + Orbital Shaking + 60 min ambient
8. Platelet + Saline + Orbital Shaking + 60 min UVA
9. Platelet + 100 ~lg/mL Photosensitizer B + Orbital Shaking + 60 min
ambient
10. Platelet + 100 ~lg/mL Photosensitizer B + Orbital Shaking + 60 min
WA
11. Platelet + 300 ~lg/mL Photosensitizer B + Orbital Shaking + 60 min
ambient
12. Platelet + 300 ,ug/mL Photosensitizer B + Orbital Shaking + 60 min
WA
PHASE II
Photosensitizer Dose Response: This study determines the optimum
concentration of photosensitizer for complete inactivation of HIV- 1.
WO 96)08965 2 ~ 9 9 3 7 2 PCT~US9~2069
43
Kinetics of Inactivation: This study establishes the optimal exposure time
for effective inactivation of HIV- l .
Variables Under Investi~tion
1. Dose of Photosensitizer (Dose Response)
2. WA Exposure Time (Kinetics of Inactivation)
Fixed Parameters
1. Light Source: WA
2. Photosensitizer: B
3. Virus: HIV-l
4. Suspending medium: Plasma
5. Light intf~:n~ y (including distance of sample from the light source)
6. Rotational speed for sample platform
7. Viral Titer: 2 X 107
8. Post-photosensitizer incubation time: 10 min~tes
9. WA Reactor: Orbital shaker
PHASE III
li,limin~tion of HIV in Platelet Concentrate - Experimental Conditions
~ Dose Response
~ Kinetics of Inactivation
20 Sample Set 1
13. Platelet + Virus + Saline + Orbital Shaking + 5 min ambient
14. Platelet + Virus + Saline + Orbital Shaking + 5 min UVA
15. Platelet + Virus + 50 ,ug/mL Photosensitizer B + Orbital Shaking + 5
WO 9C~ 9~' - PCTIUS9S/12069 ~
3 7 ~
44
min WA
16. Platelet + Virus + 100 ,ug/mL Photosensitizer B + Orbital Shaking +
~ 5 min WA
17. Platelet + Virus + 200 ~lg/mL Photosen~iti7er B + Orbital Shaking +
5 min WA
18. Platelet + Virus + 300 Il~/mL Photosensitizer B + Orbital Shaking +
5 min WA
1 8A. Platelet + Virus + 400 ,ug/rnL Photosensitizer B + Orbital Shaking +
5 min WA
10 Sample Set 2
19. Platelet + Virus + Saline + Orbital Shaking + 15 min ambient
20. Platelet + Virus + Saline + Orbital Shaking + 15 min WA
21. Platelet + Virus + 50 ~lglmL Photosensitizer B + Orbital Shaking +
15 min WA
22. Platelet + Virus + 100 ,uglmL Photosensitizer B + Orbital Shaking +
15 min WA
23. Platelet + Virus + 200 ~glmL Photose.r~iti7~.r B + Orbital Shaking +
15 min WA
24. Platelet + Virus + 300 ,ug/mL Photosen~iti7.~.r B + Orbital Shaking +
15 min WA
24A. Platelet + Virus + 400 ,ug/mL Photosensitizer B + Orbital Shaking +
15 min WA
Sample Set 3
25. Platelet + Virus + Saline + Orbital Shaking + 30 min ambient
26. Platelet + Virus + Saline + Orbital Shaking + 30 min WA
27. Platelet + Virus + 50 ~lg/mL Photosensitizer B + Orbital Shaking +
~ W096J08965 2~ 3 7 ~ PCT/US95~12069
30 min ambient
28. Platelet +'Virus + 50 ~lg/mL Photosen.~iti7:~r B + Orbital Shaking +
30 min WA
29. Platelet + Virus + 100 ~lg/mL Photosensitizer B + Orbital Shaking +
30 min ambient
30. Platelet + Virus + 100 ~lg/mL Photosen~iti7er B + Orbital Shaking +
30 min WA
31. Platelet + Virus + 200 ~lg/mL Photosensitizer B + Orbital Shaking +
30 min ambient
32. Platelet + Virus + 200 ,ug/mL Photosen.~iti~er B + Orbital Shaking +
30 min WA
33. Platelet + Virus + 300 ~g/mL Photosensitizer B + Orbital Shaking +
30 min ambient
34. Platelet + Virus + 300 ~Lg/mL Photosensitizer B + Orbital Shaking +
30 min WA
34A. Platelet + Virus + 300 ~Lg/mL Photosensitizer B + Orbital Shaking +
30 min UVA
Sample Set 4
35. Platelet + Virus + Saline + Orbital Shaking + 60 min ambient
36. Platelet + Virus + Saline + Orbital Shaking + 60 min WA
37. Platelet + Virus + 50 ,ug/mL Photosensitizer B + Orbital Shaking +
60 min ambient
- 38. Platelet + Virus + 50 ~lg/mL Photosensitizer B + Orbital Shaking +
60 min WA
39. Platelet + Virus + 100 ~lg/mL Photosensitizer B + Orbital Shaking +
60 min ambient
40. Platelet + Virus + 100 ,ug/mL Photosensitizer B + Orbital Shaking +
-
WO 96/08965 ~ ~ 3 7 ~ PCT/US95/12069
46
60 min WA
41. Platelet + Virus + 200 ~g/mL Photosensitizer B + Orbital Shaking +
60 min ambient
42. . Platelet + Virus + 200 ~lg/mL Photosensitizer B + Orbital Shaking +
60 min WA
43. Platelet + Virus + 300 ~g/mL Photosen~iti7~r B + Orbital Shaking +
60 min ambient
44. . Platelet + Virus + 300 ,ug/mL Photosensitizer B + Orbital Shaking +
60 min WA
Note: Ambient means arnbient laboratory light (Non-WA light Source).
METHODS - PHASE I
Selection of uniform UVA exposure area:
Step 1: Place a transparent sample platform equidistance
between the top and bottom WA lamps.
Step 2: Outline a square on the sample platform ofthe reactor.
Step 3: Switch on the top-bank of WA light and turn on the
fan for m~inten~nce of ambient temperature during
photolysis.
Step 4: Place a light intensity meter at both the four corners of
the square and the center. Record the light intensity
meter re~tling.~ at these locations for the top bank of
lights.
Step 5: Repeat step 4 for the bottom bank of lights.
~WO 96/08965 ~ ~ 3 7 2 ' /USg5/12069
47
Step 6: If the light int~n~ity iS different for the various
' locations, redefine the "square" such that light intensity
is the same at all the different sections of the square.
Step 7: P~ alion of Stock Solution Photosensitizer B:
S P~ aLc solution A by dissolving photosensitizer B in
10 mM phosphate buffered saline (PBS) such that the
final concentration is 40 mglmL. Next, prepare 4
working solutions, having final concentrations as
indicated, from solution A:
Solution B: S mg/mL
Solution C: 10 m~/mL
Solution D: 20 mg/mL
Solution E: 30 mg/mL
Step 8: Platelet Concentrate Preparation for WA Irradiation:
Pool four units of ABO compatible platelet
concentrates together in a standard platelet collection
bag to obtain a final volume of about 182 mL of
platelet rich plasma (platelet suspension F). Place 50
mL of platelet concentrates into a standard platelet
collection bag to be used in Phase lA (platelet
suspension G). Set aside the rem~ining 132 mL of
platelet concentrate for Phase II.
Step 9: For samples 1-12, place 7.0 mL aliquots of suspension
G into 15 mL centrifuge tubes labeled for both control
and test samples (100 and 300 ~lg/mL).
WO 96/08965 ~ j 7 ~ PCT/US95/12069
48
Step 10: Pipette 71 ~11 of working solutions C and E and add to
platelet concentrates from step 8 and allow said
~ samples to incubate with photosensitizer at 24DC for 10
minutes in ambient light. Add 71 ,ul of phosphate
burr~,ed saline (PBS) to the control samples and
incubate as described above.
Step 11: Place 3.0 mL aliquots of treated and untreated samples
from Step 10 in covered 35 mm petri dishes and
irradiate samples according to the experimental
conditions as outlined in Phase IA.
Step 12: After irradiation, pour platelet samples into 5 mL test
tubes and test control and treated samples for (1)
cellular toxicity for viral assay system; and (2) viral
i~lelrelellce for assay system.
METHODS-PHASE II:
Selection of uniform WA exposure area: Using the same area of
uniform radiation distribution as in Phase I (i.e., Steps 1-7), prepare stock
solution B.
Step 8: Preparation of Platelet Concentrates with HIV-l for
WA Irradiation
Add 8 mL of HIV-l, at 2 x 107 PFU of HIV-l/mL, to
the remaining 132 mL of platelet concentrate from step
8 of Phase I such that the final HIV- 1 titer is about 1.1
x 106 (platelet-HIV suspension H). Divide platelet
~ W096~08965 2 ~ ~ ~ 3 7 2 PcTlus95ll2o69
49
suspension H into the following aliquots ~or use in the
' different sample sets of the Phase II viral elimin~tion
- studies:
27 mL of Suspension H for Sample Set 1
27 mL of Suspension H for Sample Set 2
39 mL of Suspension H for Sample Set 3
39 mL of Suspension H for Sample Set 4
Step 9: Prepare samples for viral elimin~tion studies.
Step 10: Place 3.0 mL aliquots oftreated and untreated samples
from Step 4 into covered 35 mm petri dishes and
irradiate samples according to the experimental
conditions as outlined above.
Step 11: After irradiation, pour platelet samples into 5 mL test
tubes and detçrmine HIV-1 infectivity in control and
treated samples.
HIV Infectivity Assay:
HIV is generally titrated in vitro by an MT-4 syncytium assay. MT-4 is a
cell line developed specifically to facilitate the recognition of HIV infection.These cells adhere to and abllncl~ntly express the CD4 receptor used by HIV
during the infection of a cell. When infected with HIV, cells develop
easily-detectable multinucleated cells or syncytium forming units.
~uffer Toxicity/Viral Interference
Twenty-four well cluster plates are seeded with MT-4 cells in a total
WO 96/08965 ~ 3 i 2 PCT/US95112069
volume of 1.0 ml/well. Each test dilution is inoculated into 3 wells at 0.1
ml/well and the cultures are incubated at 36OC ~ 1~C. Observations for
cytotoxicity and, if necessary, an estimation of the percentage of cells
affected in each culture are performed on day 5 and day 7 post-inoculation.
Viral ~n~rtivation Assay
The samples are spiked with Human Immunodeficiency Virus- 1.
The spiked samples are then carried through the inactivation process. All
samples are tested undiluted or diluted in RPMI medium (negative control)
at various dilutions. Retained samples are stored frozen at -60~C or below.
Titratio~ of Samples for the Presence of HIV-1
Twenty-four well cluster plates are seeded with MT-4 cells in a total
volume of 1.0 ml/well. Ten fold serial dilutions are made in culture
medium from the spiked sample or positive control,. At each dilution step,
in quadruplicate, a 0.1 ml volume of each of the samples is tested. Cultures
are fed twice a week by removal of 1.0 ml of medium and addition of 1.0 ml
of fresh medium. On days 7, 14 and 28 the cultures are evaluated for
cytopathic effects to ~et~rmine the TCIDso On days 7, 14 and 28, 1.0 ml of
each culture is removed for analysis by HIV-l p24 antigen capture ELISA.
The formula for the final titer calculation of TCID50 is based on the
Karber method:
negative logarithm of the endpoint titer = A - (Sl/100-0.5) x B
wherein, A = negative logarithm of the highest concentration inoculated, S
= sum of the percentage positive at each dilution, and B = logl0 (of the
dilution factor). The values are then converted to TCID50/ml using a sample
inoculum volume of 0.1 ml.
The p24 assay is the Coulter HIV p24 Ag Assay which is an enzyme
W 0 96108965 ~ ~ 3 7 ~ PC~rnUS9SI12069
;... ~n~ for the ~etecti~n of p24 ~ntige~ of EIIV in pl~m~ se~lmn or
- tissue culture media. This assay uses a .. ;.. e monoclonal ~ntibo~ly (anti-
HIV core ~ntjg~n) coated onto ~ u~.~ll strips and binds the ple~l
~ntihorly to the antibody-coated ~ o~.~lls. The bound ~ntig~n iS
recog~ized by biotinylated antibodies to HIV which react with conjugated
,~vidin hul:~c~dish pero~ se, and develop color ~om the re~etior of
the pero~ e with l~y~ peroxide in the presence of
te~m~-thylh~n7i~ine~ to. The;..~ yofthecolordevelopedis
directly ~lopo.Lional to ~e amount of HTV ~nti ~ n ~lesenl in the s~mplP.
The p24 assay negative control is RPMI 1640 and the positive control is
~ntiS~en re~g~nt
Culture fluid from each well is analyzed by the HIV p24 assay and
the al,s.,~l,~ce value is cQ.nl~ ed to the cut offvalue for a positive result.
The cut off value for a positive result is ~ e~l by adding ~e mean
al)so~l.~lce value ofthe ELISA negative control to a pre~let~, ..,j"r~l factor
of 0.0~ The c.~.e.ile~ range of ~e cut offvalue is 0.0~5 to 0.155. If the
absoll,a.lce value for ~e well ~rcee~lc the cut offvalue. then the well is
con~ .ed l.o~ilivc for HIV p24 ~nti~n The level of HIV p24 in each well
is not ~ The TCIDso ofthe sample is t~ ...;..ed from the sum of
the y~,c~ e of wells positive for HIV p24 ~ntiE~on at each dilution using
the st~n~rd form~ , as stated above.
Materi~lc
Positive Control and Spiking Virus: Human imm~lnodeficiency virus
type 1
Strain: IIIB
LotNo.: VP012 H.1/8/93
Titer: 1075 TCIDsOfml
Source: Advanced Biotechnologies, Inc.,
Columbia~ Maryland
. 7 ~ 37 ~ '
WO 96/08965 PCTIUS95/12069
Negative Control Article: RPMI 1640 Medium
Source: Microbiological Associates, Inc.,
Rockville, Maryland
Test System: MT-4 cells (L013-T)
Source: National Institute of Health,
Bethesda, Maryland (Human T
cells isolated from a patient with
adult T cell leukemia; HTLV-I
transformed)
Results
Cytotoxicity is observed wi~ all undiluted samples, however, the
cultures appear to recover from the effects by day 7. Cytotoxicity is
observed with all the sarnples diluted 1:10 on day 3, however, the cultures
recover by day 7. These effects are most likely due to the excessive amount
of cellular material in the samples.
Results for samples taken at various points during the inactivation of
HIV-l study show no evidence of replication competent HIV- 1: 34A, 42
and 44. One well of four inoculated with undiluted sample 34 and sample
32 is positive for CPE on day 28. Two wells of four inoc~ ted with
undiluted sample 40 are positive for CPE on day 28. The rem~inin~
samples have significant levels of replicating HIV-l.
F~ ple 2~ activation of Sindbis Virus in Plasma Solution
Human plasma is spiked with Sindbis Virus to a final concentration
of > 7 log,0 plaque forming units (PFU)/mL. Photosensitizer is then added
to the virus spiked plasma at either 100 or 300 ~lg/mL final concentration.
After a 15 minute room temperature incubation, samples of photosensitizer
treated virus spiked plasma is placed in a ultraviolet (W) irradiator and
exposed to 24 J/cm2 of WA energy. Treated samples are then assayed for
residual infectious virus by plaque assay. Virus reduction (VR) is calculated
~ Wo 96/0896~ ~ 11 9 9 3 7 2 PCT~USg~120G9
by the equation
- Vs~VfVR
wherein, Vs is the starting virus titer, and Vf is the virus titer after treatment.
mple 3: Inactivation of Cytomegalovirus in Human Platelet
Concentrate
Inactivation of Cytomegalovirus (CMV) in human platelet
concenkate is conducted under ambient oxygen tension using a
photosen~iti7er and long wavelength ultraviolet radiation (UVA) at 22
2OC. Dose response and kinetics studies are conducted in order to
~lete.rmine the optimal conditions for inactivation of CMV in human platelet
concenkation. Four to six logs of CMV virus are added to standard units of
human platelet concenkate. The co~t~min~te~l platelet concentrate is
incubated at ambient non-WA laboratory light for 60 ~ ~ minutes with
different concentrations of suspension F (100 - 300 ~LglmL). ~ollowing
incubation, the platelet concentrates are exposed to WA at various fluences
(14 - 43 J/cm2). Inactivation of CMV virus is then evaluated by an
inreclivily assay using MRC-5 cells. Complete inactivation of CMV is
obtained at 100 ,ug/mL using Photose.~iti~er B and a WA fluence of 21.6
J/cm2.
li ~mple 4: Inactivation of Vesicular Stomatitis Virus in Platelet
Concentrates
Inactivation of Vesicular Stomatitis (VSV) in human platelet
concenkate is conducted under ambient oxygen tension using a
photosen~iti7er and long wavelength ulkaviolet light (UVA) at 22 ~t 2~C.
Dose response and kinetics studies are conducted in order to determine the
~ optimal conditions for inactivation of VSV in human platelet concentrate.
Six logs of VSV are added to standard units of human platelet concenkate.
WO 96/08965 F~ 2 ~ ~ ~ 3 7 ~ PCT/US95/12069
54
The cnntSlmin~te~l platelet concentrate is incubated at ambient non-WA
laboratory light for 10 ~t 5 minutes with different concentrations of
photosensitizer B (30 and 150 ,ug/mL). Following incubation, the platelet
concentrate is exposed to WA at various fluences (4.20-8.40 J/cm2).
Inactivation of VSV is then evaluated by an infectivity assay (plaque assay)
using Vero cells. Inactivation of 6 logs of VSV using photosensitizer B is
obtained at a minimllm WA fluence of 4.20 J/cm2.
mple 5: Herpes Sinlplex Virus Type 1 Inactivation in Calf Serum
Inactivation of Herpes Simplex Virus type 1 (HSV- 1 ) in calf serum is
conducted under ambient oxygen tension using a photosensitizer and long
wavelength ultraviolet light (WA) at 22 ~ 2~C. Using a fixed
concentration of photosen~iti7.or B (30 llg/rnL) kinetics studies are
conducted in order to determine the optimal conditions for inactivation of
HSV-1 in calf serum. Three logs of HSV-l virus are added to 100 mL of
calf serum. The cont~min~te~l sera are incubated at ambient non-WA
laboratory light for 10 ~ 5 mimltes. Following incubation, the sera are
exposed to WA at various fluences (4.20-8.40 J/cm2). Inactivation of HSV
virus is evaluated by an infectivity assay. Inactivation of 3 logs of HSV- 1
using photosensitizer B is obtained at a WA fluence of 12.6 J/cm2.
~ mple 6: Measurement of Photosensitizer Mu~enicity by Ames
Mut~enicity Test
The Ames Mutagenicity test is based upon the use of five specially
constructed strains of Salmonella typhimurium cont~inin~ a specific
mutation in the histidine operon. These genetically altered strains, TA98,
TA100, TA1535, TA1537 and TA1538, cannot grow in the absence of
histidine. When they are placed in a histidine-free medium, only those cells
which mutate spontaneously back to their wild type state -- non-histidine-
W0~610896S 2 ~ ~ ~ 3 ~ 2 PCT/US9sll2069
rl~p~n~lPnt by ,~z~ r~r~tl~rin~ their own hi~ti-line -- are able to form colonies.
The spu~ e~ -ls mllt~tion rate, or reversion rate, for any one strain is
relatively c~ , but if a mllt~en is added to the test system, the mutation
rate is significantly il-cleased. Each test strain cont~in~, in additlon to a
mllt~tinn in the hi~ti-1ine operon, two additional mutations that çnh~nce
s~ili~iily to some mut~n~. The rfa mutation results in a cell wall
deficiency that increases the pPrme~bility of the cell to certain classes of
chemicals, for example, those chemicals c~ large ring systems that
are (~LIle, vvise excluded. The second mutation is a deletion in the uvrB gene
r~l-hin~ in a deficient DNA excision-repair system. Test strains TA98 and
TA100 also cû~ the pKM101 plasmid that carries the R-factor. It has
been s~geste~l that the plasmid illc.~ases sensitivity to mllt~çnc by
modifying an e~ tin~ bacterial DNA repair polymerase complex involved
with the mi~m~tch-repair pl'OCe,SS. TA98, TAl ~3 7 and TA153 8 revert from
hicti~line dep~n~nce (auxotro~hy) to histidine independence (prototrophy)
by f~m~shi~ mutations. TA100 reverts by both fr~m~chif~ and base
s~ ;on mutations and TA1535 reverts only by substitution mutations.
;KIMENTAL DESIGN FOR AMES MUTAGENICITY TEST
The ~ llclll is desi~ned such that the concentrations of photosensitizer
B on the agar plate is e~uivalent to the expected final dose in a recipient
given S units of platelet concentrates. Note that 5 units of platelet
concel~ es is e~uivalent to a standard sin~le therapeutic dose ( I TD).
Calculation ofthe theoretical concentration of photost?n~iti7er B is based on
the following deductions ~csllmin~ homogenous distribution of the drug in a
70 kg normal individual:
Normal Blood Volurne = 5600 mL
1 Unit of Platelet Cnnc~ rate = 50 mL
.-- ~
WO 96t08965 ~ ~ ~1 la 2 7 ~D PCTtUS9Stl2069
56
1 Therapeutic Dose (lTD) = 5 Units of Platelet
Concentrates
~ Volume of lTD = 250 mL
., Starting Concentration of Photo-
sensitizer B = 300 ,ug/mL
Irradiation time = sufficient to break
down 90% of
photosen~iti7er B
Based on the above assumptions, if a patient receives 5 Units of platelet
concentrates the final concentration of photosensitizer B in the body is
derived as follows:
Final Body Concen- = 300 ~/mT x Total
tration of Photose~iti7er Vol. of Platelets
Total Blood Vol.
= 300 ~/mL x 250
5580
12.8 ~g/mL of
Photosensitizer
10% Residual photosensitizer = 1.28 ,ug/mL of
B after WA irradiation Photosensitizer
WO 9610896~i PCT/US95112069
2 ~ 3 7 ~
57
A Salmonellalm~nm~ n microsome mutagenicity test is conducted to
determine whether a plasma test solution of photosensitizer B in platelet
~ concentrate causes mutagenic changes in histidine-dependent Ill~ L strains
of Salmonella typhimurium. The Ames mutagenicity test system has been
widely used as a rapid screening procedure for the determination of
mutagenic and potential carcinogenic hazards of pure compounds, complex
compounds and commercial products.
F~ ple 7: Measurement of Pholo~c ~;tizer Cytotoxicity Using Mouse
Fibroblasts
Historically, in vitro m~mm~ n cell culture studies have been used
to evaluate the cytotoxicity of biomaterials and complex chemical
compounds. Mouse fibroblasts (L-929) are grown to confluency in 25 cm2
culture flasks using sterile mi.,i-..l--.. essential medium (MEM)
supplement~l with 5% fetal calf serum and nontoxic concentrations of
l S penicillin, streptomycin and amphotericin B. Confluent monolayers of L-
929 cells are exposed to extract dilutions of photosensitizer B. A standard
solution of photoseIl~iti7~r B is ~l~aled by dissolving 12 mg in 20 mL of
MEM supplemented with 5% bovine serum and then incubated at 37~C for
24 hours. Following incubation, different dilutions (1:2 to 1:16) of sts~/n(1~rdstock of photosensiti7er B are pl~aled with fresh MEM. A S mL aliquot of
the different dilutions of photosen~iti7er B is added to confluent monolayers
of L-929 cells and then incubated at 37OC for 72 hours. A 5 mL MEM
aliquot is added as a negative control. After exposure to photosensitizer B,
the cells are microscopically examined at approximately 100 x, and scored
for cytotoxic effects (CTE) at the end of the 24, 28 and 72 hours of
incubation. Presence (+) or absence (-) of a confluent monolayer,
vacuolization, cellular swelling and the percentage of cellular lysis are also
recorded. CTE is scored as either Nontoxic (N), Intermediate (I) or Toxic
WO 96/0896S - ~ ~ G, PCTIUS95/12069
58
(T). These data are shown in Table 2 and the evaluation criteria are shown
below:
CTE SCORE ~ICROSCOPIC APPEARANCE OF CELLS
Nontoxic (N) A uniform confluent monolayer cont~ining
primarily elongated cells with discrete
intracytoplasmic granules present at 24 hours. At
48 and 72 hours, there should be an increasing
number of rounded cells as cell population
increases and crowding begins. Little or no
vacuolization, crenation or swelling should be
present.
Intermediate (I) Cells may show marked vacuolization, crenation or
swelling. Cytolysis (0-50%) of cells that results in
"floating" cells and debris in the medium may be
present. The rem~inin~ cells are still attached to the
ilask.
Toxic (T) Greater than 50% of all cells have been lysed.
Fxte~ive vacuolization, swelling, or crenation are
usually present in the cells remaining on the flask
surface.
.Y~mple 8: l\Ieasurement of Photosensitizer Cytotoxicity Usin.~
Chinese Hamster Ovary (CHO) Hybridoma Cells and AE-
~. Cells
Chinese hamster ovary and AE-L cells are grown to confluency in 25
cm2 culture flasks using sterile Eagles Minimum Essential Medium
(EMEM) supplemented with 2 mM L-glutamine, 1% proline and 5% calf
serum treated with various concentrations of photosensitizer B (30-150
,ug/rnL) in the presence of WA. Nontoxic concentrations of penicillin,
, . . , , . , ,, _
~ W09'~~9r' 9 9 3 7 2 PCT~US9~/12069
59
streptomycin and amphoteric B are also added to the culture medium to
prevent bacterial growth. Control samples contain non-treated calf serum.
All samples are incubated at 37 ~C for 2 to 7 days. The number of viable
cells are measured at the end of each incubation period. Results show that
the growth and viability of the two cell types are not affected by
e~ en~ of the sera with irr~ te~l and non-irradiated photosen~iti7er
B. The viability of CHO cells as well as the expression of rhCg proteins on
recombinant CHO cell lines, is not affected. Note that upon WA exposure
of 30 mim~tes in the presence of 30 ~lg/mL of photosensitizer B, there are no
io adverse effects to the growth supporting functions of the treated sera or the
expression of rhCg antigens.
.
FY~n~ple 9: Synthesis of 3-Bromo-7-(r-Triethylammonium Propyloxy)
Co~lm~rin Bromide (Photosensitizer A)
Place 15 g of 7-hydroxycoumarin, 15 g of potassium carbonate, 500
mL oftetrahydrofuran (THF) and 70 mL of 1,3-dibromopropane into a 1000
mL round bottom flask c~ il)g a 2.5 cm stirring bar. After stirring at
reflux for 72-96 hours, the solution is filtered, the solids washed 4 times
with 50 mL of dichloromethane and the combined filtrate concentrated by
rotary evaporator. 50 mL of ethyl acetate is added to the concentrate
followed by concentration under reduced pressure (25 in Hg.). Another 50
mL of ethyl acetate is added to the concentrate followed by filtration. The
solids are washed 3 times with 10 mL of a 1:1 mixture of ethyl acetate and
hexane. After drying, 15-20 g ofthe crude product is dissolved in 150-200
mL of dichloromethane and purified by flash chromatography ( 130-150 g
SiO2 (70 - 230 mesh), 35 mm O.D. column, approximately 60 cm in length),
using dichloromethane as the eluting solvent. The fractions are collected in
50-250 mL beakers and monitored by TLC (developing solvent: 4:6 mixture
of ethyl acetate:hexane). The fractions containing product are combined,
W096/08965 ~ 3 7 ~ PCT/US9~112069
concentrated by rotary evaporator and dried.
Synthesi~ of 3-bromo-7-(y-bromopropyloxy)coumarin.
Place 13 g of 7-(~-bromopropyloxy)coumarin and 120-150 mL of
THF into a 500 mL round bottom flask cont~ining a 2.5 cm stirring bar .
S When the 7-( y-bromopropyloxy)coumarin is completely dissolved, 3 mL of
bromine is added by syringe. After stirring for 2-5 hours at room
temperature, the solution is concentrated by rotary evaporator. 50 mL of a
1:1 mixture of ethyl acetate and hexane is added to the concentrate and the
mixture is stirred for 30 minutes at room temperature. The solution is then
filtered and the solids washed 3 times with 1:1 ethyl acetate and hexane and
then dried for 2 hours. To obtain additional product, the filtrate is
concentrated by rotary evaporator and 30 mL of a 1:1 mixture of ethyl
acetate and hexane is added to the concentrate. The resulting mixture is
then filtered, the solids washed three times with 1:1 ethyl acetate and
hexane and then dried for 2-5 hours. The product is checked by TLC (ethyl
acetate:hexane (4:6)).
The crude product (13-16 g) is dissolved in 100-170 mL of
dichloromethane and purified by flash chromatography (100-150 g SiO2 (70
- 230 mesh), 35 mm O.D. column, approximately 60 cm in length), using
dichloromethane as the eluting solvent. Fractions are collected in a 50-250
mL beakers and monitored by TLC (developing solvent: ethyl
acetate:hexane (4:6)). The fractions cont~ining product are combined,
concentrated by rotary evaporator and dried.
Synthesis of 3-bromo-7-(~-triethylammoniull.plol,yloxy)coumarin bromide.
Place 11 g of 3-bromo-7-( y-bromopropyloxy)coumarin, 150-200 mL
of THF and 60-70 mL of triethylamine into a 500 mL round bottom flask
-
~ W~>96108965 2 ~ ~ ~ 3 7 2 PCTIUS95/12069
F-
- 61
cont~ining a 2.5 cm stirring bar . After stirring at reflux for 72-96 hours, thesolution is filtered and the solids washed 3 times with 10 mL of acetone, 3
times with 10 mL of hexane and then dried for 1 hour. The product is
transferred to a 600 mL beaker and 80 mL of acetone is added. The mixture
is stirred for 30 minl1tes, filtered, washed 3 times with 10 mL of acetone and
dried for 3-5 hours. The product is checked by TLC (ethyl acetate:hexane
(4:6)).
It has been found that photose~iti7er A fluoresces when treated with
W radiation. The absorption spectrum of photosensitizer A in water is
shown in Figure 10. The fluorescence spectrum of photosensitizer A is
shown in Figure 11.
F~ ple 10: Measurement of Photosensitizer Mi~ration ;n
~olution
Dialysis experiments are carried out using a custom-made
polystyrene dialysis chamber. The unit consists of three chambers capable
of holding a volume of 10 mL of solution. Each chamber is separated from
the adjoining chamber by a dialysis membrane (MW cut off, 5000, Fisher).
The center chamber is loaded with 100 ,uM photosen.citi7er solution either in
phosphate burreled saline (PBS) or plasma. The other two adjoining
chambers are loaded with solutions containing the agents for which the
binding is to be tested. Liposomes are prepared by vortexing dioleyl
phosphatidylserine (4.0 mglmL, Avanti polar lipids) solution in PBS.
Polyadynelic acid (Poly A; Siga), Calf thymus DNA (DNA, Sigma) and
bovine serum albumin (BSA) solutions are prepared in PBS (4.0 mg/mL).
~ 25 The dialysis cells con~ining solutions are allowed to equilibrate withconstant agitation for a period of 24 hours at room temperature. Next, the
solutions are removed from individual chambers and absorbance is
determined at 350 nm using a spectrophotometer. For experiments
, , , . _ _ _
WO 96/08965 ' ~ 7 ~ PCT/US9Stl2069
62
involving liposomes, 5% Titron X-100 (Sigrna) is used to clarify the
solutions prior to absorbance re~tling QualltiLalive determination of
~ photosen~itizer in plasma and platelets is carried out by high perforrnance
liquid chromatography (HPLC) equipped with a C 18 reverse phase column.
S l~ ple 11: Irradiation of Platelet Concentrates and
Photosensiti7~r
Twenty-four hour old random donor platelet concentrates are
obtained from American Associate of Blood Banks accredited blood banks.
Platelet units are aseptically pooled and subsequently split into controls and
treatments. Ten milliliters of photosensitizer solution in 0.9% saline is
added to 50 mL platelet concentrates in CLX (Miles) containers to obtain
the photosensitizer final preset concenkation. After addition of the
photosensitizer, the platelet units are incubated at room temperature while
mixin~ on a shaker for 10 minlltes. Platelet concentrate samples cont~ining
photosensitizers are WA irr~ te~l from top and bottom in a prototype
WA reactor to deliver 25 J/cm2 fluence. During the irradiation, samples
are placed on a linear shaker. After WA exposure, the samples are stored
in a platelet incubator with ~h~king for an additional 4 days. During storage
3 mL aliquots from each unit are collected and subsequently analyzed for
platelet in vitro properties.
~nlple 12: Comparison Study of Photosensitizer B~ 8-MQP, and
Full units of one day old human platelet concentrate are collected in
Cyrocyte bags (PL 269, Fenwal, Deerfield, IL) according to standard blood
banking procedures (AABB Technical Manual (1989) 13th Ed., p. 136).
Platelet units are spiked with 6 logs of bacteriophage ~6. Equimolar
concentrations (60 ,~LM) of 8-MOP, AMT and brominated psoraien are
WO 96108965 PCT/US95/12069
2 ~ ~ ~ 3 7 ~
63
added to the platelet concentrations and then incubated at 22 ~ 2OC for 10
minutes. Treated samples are irr~ te~l from top and bottom with a
constant total WA source intensity of 7 mW/cm2. During WA exposure
samples are continuously ~git~te~l to ensure adequate mixin~ Virucidal
properties are evaluated using a standard double agar plaque assay
consisting of host bacteria Pseudomonas Syringae. In vitro platelet
properties are evaluated using (1) aggregation response to collagen, (2)
hypotonic shock response; and (3) morphology according to method
described by Kunicki et al. ((1975) Transfusion 15:414). Data represent
the mean ~ standard deviation of n=3.
li,Y~nlple 13: Comparison Studv of Photoinactivation of Sindbis
Virus in H~ n Plasma
A comparison study is performed to evaluate the viral inactivation
properties of photosen~iti7er~ A, B, D, and E of the present invention. Also
ex~mined are non-halogenated forms of photosensitizer A (hereina~er
referred to as photosensitizer AX), photosensitizer C (hereinafter referred to
as photos~n~iti7er CX), and photos~ iLi~el D and E (hereinafter referred to
as photosen~iti~r DX). The TClDso assay is used to measure the affects of
virus inactivation.
The photosensitizer is added to virus spiked plasma. The virus
employed is Sindbis and the plasma is spiked to a working titer of > 1 x 107.
Each test unit is exposed to ultraviolet radiation at 320-400 nm (peak
absorbance 365 nm) for 30 min. to achieve an irradiation of about 24 J/cm2.
Virus inactivation is quanlilated by plaque assay. A monolayer of indicator
cells are grown on a solid support and exposed to sample materials to allow
for virus attachment. A foci of infection develops as a virus replicates and
lyses and released virus diffuses to and infects neighboring cells, or virus
infects neighboring cells via cell-cell fusion. CPE develops after several
W 0 96/08965 ~ 7 ~ PCTrUS95/12069
64
days of incubation. The virus titer in the sample is calculated from number
of units exhibiting CPE. The results of this experiment are shown in Figure
12.
mple 14: Synthesis of 3-Bromo-7-(r-Triethylamino 8-Methyl)
S Coumarin (Photosensitizer D)
Preparation of 7-hydroxy-8-methyl coumarin
2-methyl resorcinol (.161 mmol, 20.024 g) and malic acid (.165
mmol, 22.129 g) are dissolved in concentrated sulfuric acid. The reaction
mixture is stirred at 80~C for 24 h. The resulting solution is then poured
over crushed ice, and the precipitate is collected by vacuum filtration. The
precipitate is then washed with 5% NaHCO3 and again collected by vacuum
filtration yielding an orange-yellow solid in a 20.64% yield.
Preparation of 7-3'-bromopropyloxy-8-methyl coumarin
7-hydroxy-8-methyl coumarin (0.286 mol, 5.045 g) and K2CO3
(.0317 mol, 4.379 g) are added to 50 ml of dibromopropane. The reaction is
stirred at reflux for 24 h. The excess dibromopropane is removed by
~li.still~tion. The reln~inin~ slurry is taken up in CHCl3 and gravity filtered
to remove the K2CO3. The CHCl3 is dried and removed in vacuo. The final
solid is washed with hexanes giving a pale-yellow product in a 76.0% yield.
Preparation of 3 bromo-7-3'-bromopropyloxy-8-methyl coumarin
7-3'-bromopropyloxy-8-methyl coumarin (6.75 mmol, 2.01 g) is dissolved
in THF and cooled to -76OC. Approximately 3 ml of Br2 is slowly added to
the solution. The mixture is stirred for 3h and then allowed to warm to
room temperature. The resulting solution is dissolved in CHCl3 and washed
with a 10% Na2S2O4 solution, a 10% NaHCO3 solution and finally water.
The CHCl3 is dried and removed in vacuo. The resulting pale-yellow solid
is washed with hexanes and collected by vacuum filtration to give a
quantitative yield.
~ wog~ ns~6~ 3 7 ~ rcT/usgsrl2069
Preparation of 3-bromo-7-(y-triethylamino-propyloxy)-8-methyl
coumarin
7-3'-bromopropyloxy-8-methyl coumarin (0.17 mmol, 0.064 g) is
dissolved in 30 ml of CHCI3. Approximately 10 ml oftriethylamine is
S added to the solution. By following TLC, the reaction requires refluxing for
48 h to ensure completion. The CHCl3 and Et3N are removed in vacuo. The
precipitate is washed with hexane, ethyl acetate and acetone several times.
The resulting white solid is dried under high vacuum and obtained in a
65.4% yield.
li~mple 15: Inactivation of Parvovirus in Plasma
This set of experiments identifies the precise combination of factors
and levels that optimize the conditions for the inactivation of Porcine
Parvovirus (PPV, a small non-lipid enveloped single stranded DNA virus)
in human plasma using dirr~le.ll photosensitizers (photosen~iti7~rs B, A, D
and E) in the presence of long wavelength ultraviolet radiation (UVA).
Human parvovirus B 19 is a major concern with respect to the safety
of plasma derived products. This class of virus exhibits high resistance to
chemical reagents such as alcohol, detergents and solvents that are ~ elllly
employed for inactivating viruses in plasma.
Virus PPV
Family Parvoviridae
Genome ss DNA
Lipid Envelope None
Size (nm) 18-2 6
Shape Icosahedral
Resistance to Physioco-Chemical Reagents High
This study involves the increase of PPV susceptibility to inactivation
wo 9~e3~5 ~ ~ ff ~ ~ ~ 7 ~ PCT/US9S/12069
66
using various photosensitizer and WA combinations. Three different
factors are manipulated to achieve such increased susceptibility, including
pH (5.5), freeze-thaw cycle (-30~C), freeze-thaw duration (2 to 8~C) and
low and high ionic strength. In order to obtain low and high ionic strength
plasma, fresh frozen plasma is thawed at 4~C and then centrifuged to
remove any cryo-precipitate. Following centrifugation, the resulting 40 mL
of supem~t~nt plasma is diluted with either distilled water or 2 M sodium
chloride solution to obtain a low- and high ionic strength plasma
respectively. The following experimental conditions are employed:
Sample Set 1: Normal Plasma Samples at pH 7.0 - 7.5
1. Plasma + PPV + pH 7 + Saline + arnbient (50-60 minutes)
2. Plasma + PPV + pH 7 + 300 ~lglmL + 30 J/cm2
Sample Set 2: Normal Plasma: Freeze - at -30OC and Thaw at
+37~c
3. Plasma + PPV + pH 5.5 + Saline + ambient (50-60 minutes)
4. Plasma + PPV + pH 5.5 + 300 llglmL + FT + 30 J/cm2
Sample Set 3: Normal Plasma Diluted 1:1 with Saline at pH 5.5-
6.0
5. Plasma + PPV + pH 5.5 + Saline + ambient (50-60 minutes)
6. Plasma + PPV + pH 5.5 + 600 llg/mL Photosensitizer B + FT 4~C at
30 J/cm2
7. Plasma + PPV + pH 5.5 + 600 ,ug/mL Photosensitizer A + FT 4~C at
30 J/cm2
8. Plasma + PPV + pH 5.5 + 600 ~lglmL Photosensitizer D + FT 4~C at
30 J/cm2
~ WO 96108965 2 1 9 9 3 ~ 2 PCT/USg5/12069
67
9. Plasma + PPV + pH 5.5 + 600 llg/mL Photosen~iti7:~r E + FT 4~C at
30 J/cm2
Sample Set 4: Normal Plasma Diluted 1:1 with Distilled Water
10. Plasma + PPV + pH 5.5 + 600 ,ug/mL Photosen~iti7~r B + FT 4~C at
30 J/cm2
11. Plasma + PPV + pH 5.5 + 600 ~lg/mL Photosensitizer A + FT 4~C at
30 J/cm2
Sample Set 5: Normal Plasma Diluted 1:1 with 2 M Sodium
Chloride
12. Plasma + PPV + pH 5.~ + 600 ~lglmL Photosensitizer B + FT 4~C at
30 J/cm~
13. Plasma + PPV + pH 5.5 + 600 ,ug/mL Photosen~iti7:er A + FT 4~C at
30 J/cm2
Vari~hles lln~l~r Investi~tion
1. Concentration of Sen~iti7er (0, 300 and 600 ~LglmL)
2. T~ eldlule of processing (freeze-thaw: -30~C + 37~C vs. -30~C
to+4~C)
3. Ionic strength (Normal vs. Low vs. High ionic strength)
4. Types of Sensitizers (Photosensitizers B, A, D and E)
20 Fixed Parameters
1. Volume of plasma for irradiation (5 mL)
2. Light source (WA, 320-400 nm with peak intensity at 365 nm)
3. Post-Sensitizer incubation time (60 minutes)
a. Shaker speed: 70 ~t 5 rpm
WO 96/08965 ~ 3 7 2 PCT/US95/12069
68
b. Sample location: six samples
c. Temperature during irradiation (22~C-29~C)
4. WA Fluence (30 J/cm2)
The selections of the variables under investigation are based on a
prelimin~ry review of the m~nllf~cturing processes for the various plasma
fractions as well as current literature on the inactivation of non-enveloped
viruses.
WA kan~alell~ petri dishes are labeled along the sides with the
sample numbers 1-13 (see section 3.2 and attachment 1 for the sample
identif1cation). Two 50 mL sterile centrifuge tubes are labeled (Tube 1 - pH
7, Tube 2 - pH 5.5). 1 unit of fresh frozen plasma is obtained and prepared
according to the recommendation of the American Association of Blood
Banks (AABB). This unit of fresh frozen plasma is placed in a 37~C
waterbath and allowed to thaw.
The thawed plasma is centrifuged at 3000 g for 20 minutes. Using a
50 mL pipette, all the supern~t~nt is transferred into a sterile 500 mL plastic
bottle. With a 50 mL syringe, 40 mL aliquots of plasma are transferred into
tubes 1 and 2. The pH of the plasma in Tube 1 is adjusted to a value
between 7 and 7.5 by dropwise addition of 2 M hydrochloric acid.
Similarly, the pH of plasma in Tube 2 was adjusted to a value between 5 .5
and 6.
Sample Set 1 9 mL of pH 7 plasma into Tube 1
Sample Set 2 9 mL of pH 5.5 plasma into Tube 2
Sample Set 3 12 mL of pH 5.5 plasma into Tube 3
Sample Set 4 4.5 mL of pH 5.5 plasma into Tube 4
Sample Set 5 4.5 mL of pH 5.5 plasma into Tube 5
12 mL of 0.9% sodium chloride solution is added to tube 3 sample set 3; 4.5
mL of sterile distilled water to tube 4 sample set 4; and 4.5 mL of 2 M
~ W096J~965 2 ~ 9 ~ 3 7 ~ PCT/rJS9S/lZ069
69
sodium chloride solution to tube S sample set 5. Each tube is ~git~te~l to
ensure adequate mixing of the contents of the tubes. 1 mL of parvovirus
(PPV) is added to tubes 1, 2, 4 and 5 for sample sets 1, 2, 4 and 5,
respectively; 2.67 mL of PPV to tube 3, sample set 3. The contents of each
S tube are ~l~it~te~l to ensure homogenous dispersion of the virus. Thirteen 50
mL sterile plastic tubes are labeled 1-12 corresponding to the samples
outlined above. 5 mL of PPV-cont~min~tecl plasma is transferred into the
labeled tubes accordingly:
-~ mL of PPV-Plasma is transferred from sample set 1 into tubes 1
and2
-5 mL of PPV-Plasma is transferred from sample set 2 into tubes 3
and 4
-5 mL of PPV-Plasma is transferred from sample set 3 into tubes 5
and 9
-5 mL of PPV-Plasma is transferred from sample set 4 into tubes 10
and 1 1
-5 mL of PPV-Plasma is transferred from sample set 5 into tubes 12
and 13
Saline or photosensitizer solution is added accordingly:
-1 mL of 0.9% sodium chloride solution to sample tube 1
-1 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube 2
-1 mL of 0.9% sodium chloride solution to sample tube 3
-1 mL of 1.8/mglmL of Photosen~iti7er B solution to sample tube 4
-1 mL of 0.9% sodium chloride solution to sample tube 5
-2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube 6
-2.5 mL of 1.8 mg/mL of Photosensitizer A solution to sample tube 7
WO 96/08965 2 ~ ~ ~ 3 ~ 2 PCTIUS95/12069
-2.5 mL of 1.8 mg/mL of Photosen~iti7~r D solution to sample tube 8
-2.5 mL of 1.8 mg/mL of Photosensitizer E solution to sample tube 9
-2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube
5-2.5 mL of 1.8 mg/mL of Photosensitizer A solution to sample tube
11
-2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube
12
-2.5 mL of 1.8 mg/mL of Photosen~iti7~r A solution to sample tube
13
All samples are placed on a shaker and incubated at room
temperature (22-24~C) for 60 minutes. At the end of the incubation period,
samples 4, 6-13 are frozen at temperatures between -20~C and -40~C.
Sample 4 is thawed at 37~C (about 10-15 minlltes at 37~C) and samples 6-
13 at temperatures between 2 and 8~C. Freeze-thaw cycles are repeated 10
times over 24 hour period. At completion of the freeze-thaw cycles, 6 mL
of each sample were exposed to WA at 30 J/cm2. At the end of the 60
minute incubation, 6 mL of the contents from tubes 1, 2, 3 and 5 are
transferred to applopliately labeled petri dishes for photoinactivation
treatment. The results of this exQeriment are shown in Table 17.
While the above description contains many specificities, these
specificities should not be construed as limitations on the scope of the
invention, but rather an exemplification of the preferred embodiment
thereof. That is to say, the foregoing description of the invention is
exemplary for purposes of illustration and explanation. Without departing
i~om the spirit and scope of this invention, one skilled in the art can make
various changes and modifications to the invention to adapt it to various
usages and conditions. As such, these changes and modifications are
-
~ WO 96~' ~9fi~ 2 11 ~ ~ 3 7 2 PCT/USg5/12069
71
properly, equitably, and intended to be within the full range of equivalence
of the following claims. Thus, the scope of the invention should be
~ determined by the appended claims and their legal equivalents, rather than
by the examples given.
W096108965~ 2 ~ 7 ~ '
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