[go: up one dir, main page]

WO1994020090A1 - Decontamination de prelevements cliniques - Google Patents

Decontamination de prelevements cliniques Download PDF

Info

Publication number
WO1994020090A1
WO1994020090A1 PCT/US1994/002607 US9402607W WO9420090A1 WO 1994020090 A1 WO1994020090 A1 WO 1994020090A1 US 9402607 W US9402607 W US 9402607W WO 9420090 A1 WO9420090 A1 WO 9420090A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
dimethyl
methoxypsoralen
amt
aminomethyl
Prior art date
Application number
PCT/US1994/002607
Other languages
English (en)
Inventor
Peter G. Carroll
Stephen T. Isaacs
George D. Cimino
Original Assignee
Steritech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Steritech, Inc. filed Critical Steritech, Inc.
Priority to AU64024/94A priority Critical patent/AU6402494A/en
Publication of WO1994020090A1 publication Critical patent/WO1994020090A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the invention generally relates to the inactivation of contaminants in material intended for in vitro use, and in particular the inactivation of pathogens in human fluids prior to clinical testing.
  • the status of hospital patients is routinely monitored by obtaining and testing human fluids (blood, urine, spinal fluid, etc.). Testing is typically performed at a centralized location, such as a clinical laboratory.
  • each admitted patient has at least a tube of blood collected every day by a phlebotomist.
  • these blood samples are processed by first centrifuging the unopened tube to separate the cells from the serum or plasma. Thereafter, the tube is opened by removing the rubber stopper by hand. To maintain the separation, plastic inserts can be manually pushed down into the serum to a level just above the packed cells.
  • the tubes are placed in a standard test tube rack and delivered to a technologist for automated analysis. At this point, the technologist running the machine pipettes serum from the top of tube into a small sample cup. The sample cup is then placed on the instrument and processed.
  • This intensive handling of potentially infectious human fluids is not without health risk.
  • the Occupational Safety and Healtli Administration (OSHA) estimates that over five million health workers, including hospital laboratory workers, are exposed to bloodborne-pathogen infections in the work place annually. The pathogen responsible for the overwhelming majority of infections is the hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • the present invention relates to methods of inactivating contaminants in material intended for in vitro use.
  • the present invention relates to the inactivation of pathogens in human fluids prior to clinical testing.
  • a nucleic acid binding compound is selectively employed to treat contamination by nucleic acid-containing microorganisms, including pathogenic viruses.
  • the present invention contemplates a method of decontamination, comprising: a) providing, in any order, i) an aminopsoralen; ii) means for activating said aminopsoralen, iii) a biological fluid intended for in vitro testing suspected of being contaminated with one or more pathogens; b) adding said aminopsoralen to said fluid; and c) activating said aminopsoralen, so that said nucleic acid binding compound binds covalently to the nucleic acid of said pathogens.
  • the method of the present invention is particularly useful where the pathogens are bacteria, fungi, mycoplasma, protozoa and viruses.
  • the present invention is employed with success with human serum and human plasma.
  • the intensity of light received by the human fluid is less than 20 mW/cm 2 and the human fluid is exposed to this intensity for less than thirty minutes.
  • the present invention contemplates a method of treating biological material intended for in vitro clinical testing, comprising: a) providing, in any order, i) a container containing one or more aminopsoralens; ii) a photoactivation device; and iii) material intended for in vitro clinical testing suspected of being contaminated with pathogens; b) adding the material to the container; c) photoactivating the aminopsoralens, so that the aminopsoralens bind covalently to the nucleic acid of the pathogens; d) testing the material for the presence of serum analytes.
  • the photoactivation device comprises filters providing wavelength cutoffs at 320 nm, below which no irradiation is transmitted, and at 360 nm, above which no irradiation is transmitted.
  • the material comprises blood.
  • the present invention contemplates the use of 4'-aminomethyl-4,5'-trimethylpsoralen, 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen, radiolabelled 4'-aminomethyl-4,5'-dimethyl-8-methoxypsoralen or biotinylated aminopsoralen.
  • the activating means comprises a photoactivation device. It is also not intended that the invention be limited by the type of container (tube, specimen cup, etc.) or the condition of the container (opened, closed, labelled, unlabelled etc.). It is preferred however that the container be such that it is an ultraviolet light transparent container.
  • the container is a glass vacuum tube having a UV-transparent top and a UV- transparent label.
  • the container is a blood collection tube, such as a standard red top tube, and the rubber stopper is removed before irradiation.
  • the compound can be added dry and then resuspended when the blood is drawn into the tube. In this manner, the existence of the compound would not be apparent to the phlebotomist.
  • the compound is 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen (AMMP).
  • AMMP 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen
  • the present invention also contemplates the salt of AMMP.
  • the present invention further contemplates a stable intermediate, in the synthesis of compounds of the present invention having the following formula:
  • the present invention contemplates binding aminopsoralen compounds to nucleic acid.
  • the present invention contemplates a complex, comprising this compound bound to nucleic acid.
  • the binding is covalent.
  • the binding is non-covalent.
  • the nucleic acid is selected from the group consisting of viral, bacterial, fungal, mycoplasmal and protozoan nucleic acid.
  • RNA and DNA include Human Immunodeficiency Virus nucleic acid. It is not intended that the present invention be limited by the method by which the compounds of the present invention are synthesized.
  • 4'-aminomethyl-4,5'-dimethyl-8-methoxypsoralen is synthesized by the following method: a) providing 4'-chloromethyl-8-methoxy- 4,5'-dimethylpsoralen; b) treating 4'-chloromethyl-8-methoxy-4,5'- dimethylpsoralen with an alkali salt of phthalimide to give 8-methoxy-4,5'- dimethyl-4'-(phthalimidomethyl)psoralen; and c) treating 8-methoxy-4,5'- dimethyl-4'-(phthalimidomethyl)psoralen with hydrazine or methylamine to yield 4'-aminomethyl-4,5'-dimethyl-8-methoxypsoralen.
  • 4'-chloromethyl-8-methoxy-4,5'-dimethylpsoralen may be synthesized by the following method: a) providing 4,5'-dimethyl-8- methoxypsoralen, b) treating 4,5'-dimethyl-8-methoxypsoralen with chloromethyl methyl ether to yield 4'-chloromethyl-8-methoxy-4,5'-dimethylpsoralen.
  • FIG. 1 is a side view of one embodiment of a photoactivation device suitable for the practice of the present invention.
  • FIG. 2 is a side view of the device shown in FIG. 1, in the raised position.
  • FIG. 3 is a side view of the device shown in FIG. 1, in the lowered position.
  • FIG. 4 is a cross sectional view of the device shown in FIG. 2.
  • FIG. 5 is a cross-sectional view of the device shown in FIG. 2 in the closed position.
  • FIG. 6 is a cross-sectional view of the device shown in FIG. 2, in the open position.
  • FIG. 7 is a cross sectional view of the device shown in FIG. 1.
  • FIG. 8 is a cross sectional view of the bottom of the cover of the device shown in FIG. 1.
  • FIG. 9 is a perspective view of a tube holder device of the present invention.
  • FIG. 10 is a cross sectional view of the device shown in FIG. 9.
  • FIG. 11 is a perspective view of a tube holder device of the present invention.
  • FIG. 12 is a perspective view of a tube holder device of the present invention.
  • FIG. 13 is a cross sectional view of the device shown in FIG. 12.
  • FIG. 14 is a perspective view of a tube holder device of the present invention.
  • FIG. 15 is a perspective view of a tube holder device of the present invention.
  • FIG. 16 is a cross sectional view of the device shown in FIG. 15.
  • FIG. 17 is a perspective view of the device shown in FIG. 15.
  • FIG. 18 is a cross sectional view of one embodiment of a photoactivation device suitable for the practice of the present invention.
  • FIG. 19 is a cross sectional view of the top of the device shown in HG. 18.
  • FIG. 20 is a perspective view of one embodiment of the device of the present invention.
  • FIG. 21 is a cross-sectional view of the device shown in Figure 1 along the lines of 2--2.
  • FIG. 22 is a cross-sectional view of the device shown in Figure 1 along the lines of 3—3.
  • FIG. 23 is a cross-sectional view of the device shown in Figure 1 along the lines of 4—4.
  • FIG. 24 details one compound synthesis scheme of the present invention where the starting material is 4,5'-dimethyl-8-methoxypsoralen.
  • FIG. 25 details one compound synthesis scheme of the present invention where the starting material is 4'-chloromethyl-4,5'-dimethyl-8-methoxypsoralen.
  • FIG. 26 is a graph showing the photoaddition of 4'-aminomethyl-4,5',8- trimethylpsoralen (AMT), 4'-aminomethyl-4,5'-dimethyl-8-methoxypsoralen (AMMP) and 8-methoxypsoralen (8-MOP) to nucleic acid after exposure to ultraviolet light.
  • AMT 4'-aminomethyl-4,5',8- trimethylpsoralen
  • AMMP 4'-aminomethyl-4,5'-dimethyl-8-methoxypsoralen
  • 8-MOP 8-methoxypsoralen
  • FIG. 27 is a graph showing the photoaddition of 4'-aminomethyl-4,5',8- trimethylpsoralen (AMT) to calf thymus DNA after irradiation with two different photoactivation devices.
  • FIG. 28 is a graph showing the viral inactivation of cell-associated HIV by
  • AMT 4'-aminomethyl-4,5',8-trimethylpsoralen
  • AMMP 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen
  • 8-MOP 8-methoxypsoralen
  • FIG. 29 is a bar graph showing the clinical testing results for Total Bilirubin following the decontamination of human serum according to the present invention.
  • FIG. 30 is a bar graph showing the clinical testing results for Uric Acid following the decontamination of human serum according to the present invention.
  • FIG. 31 is a graph showing the viral inactivation of cell-associated HIV in medium by 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) when irradiated with and without a liquid filter providing wavelength cutoffs at 320 nm and at 360 nm.
  • AMT 4'-aminomethyl-4,5',8-trimethylpsoralen
  • FIG. 32 is a graph showing the viral inactivation of cell-associated HIV in serum by 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) and 4'-aminomethyl- 4,5'-dimethyl-8-methoxypsoralen (AMMP) when irradiated with and without a liquid filter providing wavelength cutoffs at 320 nm and at 360 nm.
  • AMT 4'-aminomethyl-4,5',8-trimethylpsoralen
  • AMMP 4'-aminomethyl- 4,5'-dimethyl-8-methoxypsoralen
  • FIG. 33 is a graph showing the photoaddition of 4'-aminomethyl-4,5',8- trimethylpsoralen (AMT) to nucleic acid in serum at varied concentrations of AMT.
  • FIG. 34 is a graph showing the photoaddition of 4'-aminomethyl-4,5',8- trimethylpsoralen (AMT) to nucleic acid in medium at varied concentrations of AMT.
  • FIG. 35 is a graph showing the production of active oxygen species by 4'- aminomethyl-4,5',8-trimethylpsoralen (AMT), 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen (AMMP) and 8-methoxypsoralen (8-MOP) over time.
  • AMT 4'- aminomethyl-4,5',8-trimethylpsoralen
  • AMMP 4'-aminomethyl-4,5'-dimethyl-8- methoxypsoralen
  • 8-MOP 8-methoxypsoralen
  • the invention generally relates to the inactivation of contaminants in material intended for in vitro use, and in particular the inactivation of pathogens in human fluids prior to clinical testing.
  • whole blood is received and these samples are processed to separate the cells from the serum or plasma.
  • These steps involves handling of potentially infectious agents.
  • a process that inactivated pathogens prior to handling and testing would be expected to prevent the transmission of disease.
  • the present invention contemplates inactivating human serum after collection but before testing.
  • a nucleic acid binding compound is selectively employed, such as a furocoumarin.
  • the furocoumarin is a psoralen that is activated by a photoactivation device.
  • Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA).
  • UVA long wave ultraviolet light
  • Aminopsoralens are defined as psoralens that have amino-substitutions on the 3-, 4-, 5-, 8-, 4'-, or 5'- carbons.
  • the psoralens may or may not have further substitutions, including substitutions on the nitrogen.
  • Serum analytes are defined here as components sometimes found in blood which are measured in clinical chemistry tests.
  • the present invention contemplates new photoreactive nucleic acid binding compounds and methods of synthesis of new photoreactive nucleic acid binding compounds. Activation of the new compounds does not result in significant damage to serum analytes.
  • the inactivation method of the present invention provides a method of inactivating pathogens in human serum, and in particular, viruses prior to clinical testing. In contrast to previous approaches, the method requires only short irradiation times and there is no need to limit the concentration of molecular oxygen. The method serves as protection against a wide range of pathogens without unduly interfering with laboratory operations.
  • the present invention also contemplates devices for inactivation which activate nucleic acid binding compounds.
  • the device is an inexpensive source of ultraviolet radiation.
  • the device provides rapid photoactivation.
  • the device allows for large sample processing.
  • the present invention contemplates devices which can control the temperature of the irradiated samples. These devices are also configured to ensure the inherent safety of the user.
  • the present invention contemplates devices and methods for photoactivation and specifically, for activation of photoreactive nucleic acid binding compounds.
  • the present invention contemplates devices having an inexpensive source of electromagnetic radiation that is integrated into a unit.
  • the present invention contemplates a photoactivation device for treating photoreactive compounds, comprising: a) means for providing appropriate wavelengths of electromagnetic radiation to cause activation of at least one photoreactive compound; b) means for supporting a plurality of samples in a fixed relationship with the radiation providing means during activation; and c) means for maintaining the temperature of the samples within a desired temperature range during activation.
  • the present invention also contemplates methods, comprising: a) supporting a plurality of sample containers, containing one or more photoreactive compounds, in a fixed relationship with a fluorescent source of electromagnetic radiation; b) irradiating the plurality of sample containers simultaneously with electromagnetic radiation to cause activation of at least one photoreactive compound; and c) maintaining the temperature of the sample within a desired temperature range during activation.
  • the major features of one embodiment of the device of the present invention involve: A) an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample containers, B) rapid photoactivation, C) large sample processing, D) temperature control of the irradiated samples, and E) inherent safety.
  • a preferred photoactivation device of the present invention has an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample vessels.
  • Ultraviolet radiation is a form of energy that occupies a portion of the electromagnetic radiation spectrum (the electromagnetic radiation spectrum ranges from cosmic rays to radio waves).
  • Ultraviolet radiation can come from many natural and artificial sources. Depending on the source of ultraviolet radiation, it may be accompanied by other (non-ultraviolet) types of electromagnetic radiation (e.g. visible light).
  • ultraviolet radiation extends from approximately 180 nm to 400 nm.
  • a radiation source by virtue of filters or other means, does not allow radiation below a particular wavelength (e.g. 320 nm), it is said to have a low end “cutoff” at that wavelength (e.g. "a wavelength cutoff at 300 nanometers”).
  • a radiation source allows only radiation below a particular wavelength (e.g. 360 nm), it is said to have a high end “cutoff” at that wavelength (e.g. "a wavelength cutoff at 360 nanometers”).
  • any photochemical reaction it is desired to eliminate or least minimize any deleterious side reactions.
  • Some of these side reactions can be caused by the excitation of endogenous chromophores that may be present during the photochemical activation procedure.
  • the endogenous chromophores are the nucleic acid bases themselves. Restricting the activation process to wavelengths greater than 320 nm minimizes direct nucleic acid damage since there is very little absorption by nucleic acids above 313 nm.
  • the nucleic acid is typically present together with additional biological constituents. If the biological fluid is just protein, the 320 nm cutoff will be adequate for minimizing side reactions (aromatic amino acids do not absorb above 320 nm). If the biological fluid includes other analytes, there may be constituents that are sensitive to particular wavelengths of light. In view of the presence of these endogenous constituents, it is intended that the device of the present invention be designed to allow for irradiation within a small range of specific and desirable wavelengths, and thus avoid damage to analytes that are to be measured by clinical testing. The preferred range of desirable wavelengths is between 320 and 350 nm.
  • BLB type fluorescent lamps are designed to remove wavelengths above 400 nm. This, however, only provides an upper end cutoff.
  • the device of the present invention comprises an additional filtering means.
  • the filtering means comprises a glass cut-off filter, such as a piece of Cobalt glass.
  • the filtering means comprises a liquid filter solution that transmits only a specific region of the electromagnetic spectrum, such as an aqueous solution of Co(No 3 ) 2 . This salt solution yields a transmission window of 320-400 nm.
  • the aqueous solution of Co(No 3 ) 2 is used in combination with NiS0 4 to remove the 365 nm component of the emission spectrum of the fluorescent or arc source employed.
  • the Co-Ni solution preserves its initial transmission remarkably well even after tens of hours of exposure to the direct light of high energy sources. It is not intended that the present invention be limited by the particular filter employed.
  • Several inorganic salts and glasses satisfy the necessary requirements.
  • cupric sulfate is a most useful general filter for removing the infra-red, when only the ultraviolet is to be isolated. Its stability in intense sources is quite good.
  • Other salts are known to one skilled in the art.
  • Aperture or reflector lamps may also be used to achieve specific wavelengths and intensities.
  • UV radiation When ultraviolet radiation is herein described in terms of irradiation, it is expressed in terms of intensity flux (milliwatts per square centimeter or “mW cm “ 2 ). "Output" is herein defined to encompass both the emission of radiation (yes or no; on or off) as well as the level of irradiation. In a preferred embodiment, intensity is monitored at 4 locations: 2 for each side of the plane of irradiation.
  • a preferred source of ultraviolet radiation is a fluorescent source.
  • Fluorescence is a special case of luminescence. Luminescence involves the absorption of electromagnetic radiation by a substance and the conversion of the energy into radiation of a different wavelength. With fluorescence, the substance that is excited by the electromagnetic radiation returns to its ground state by emitting a quantum of electromagnetic radiation. While fluorescent sources have heretofore been thought to be of too low intensity to be useful for photoactivation, in one embodiment the present invention employs fluorescent sources to achieve results thus far achievable on only expensive equipment.
  • a preferred fluorescent source is a device ("HRI-100") sold commercially by HRI Research Inc. (Berkeley, California, USA) and ULTRA-LUM, INC. (Carson, California, USA). The HRI device is described in U.S. Patent No. 5,184,020, hereby incorporated by reference.
  • fixed relationship is defined as comprising a fixed distance and geometry between the sample and the light source during the sample irradiation.
  • Distance relates to the distance between the source and the sample as it is supported. It is known that light intensity from a point source is inversely related to the square of the distance from the point source. Thus, small changes in the distance from the source can have a drastic impact on intensity. Since changes in intensity can impact photoactivation results, changes in distance are avoided in the devices of the present invention. This provides reproducibility and repeatability.
  • Geometry relates to the positioning of the light source. For example, it can be imagined that light sources could be placed around the sample holder in many ways (on the sides, on the bottom, in a circle, etc.).
  • the geometry used in a preferred embodiment of the present invention allows for uniform light exposure of appropriate intensity for rapid photoactivation.
  • the geometry of a preferred device of the present invention involves multiple sources of linear lamps as opposed to single point sources. In addition, there are several reflective surfaces and several absorptive surfaces. Because of this complicated geometry, changes in the location or number of the lamps relative to the position of the samples to be irradiated are to be avoided in that such changes will result in intensity changes.
  • the light source of the preferred embodiment of the present invention allows for rapid photoactivation.
  • the intensity characteristics of the irradiation device have been selected to be convenient with the anticipation that many sets of multiple samples may need to be processed. With this anticipation, a fifteen minute exposure time or less is a practical goal.
  • one element of the devices of the present invention is a means for supporting a plurality of sample containers, and in particular, commercially available red top vacuum tubes.
  • the supporting means comprises a tube rack placed between two banks of lights.
  • Temperature control is important because the temperature of the sample in the sample at the time of exposure to light can dramatically impact the results. For example, conditions that promote secondary structure in nucleic acids also enhance the affinity constants of many psoralen derivatives for nucleic acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). These conditions are a mix of both solvent composition and temperature. With single stranded 5S ribosomal RNA, irradiation at low temperatures enhances the covalent addition of HMT to 5S rRNA by two fold at 4°C compared to 20°C. Thompson et al., J. Mol. Biol. 147:417 (1981). Even further temperature induced enhancements of psoralen binding have been reported with synthetic polynucleotides. Thompson et al., Biochemistry 21:1363 (1982).
  • E. Inherent Safety Ultraviolet radiation can cause severe burns. Depending on the nature of the exposure, it may also be carcinogenic.
  • the light source of a preferred embodiment of the present invention is shielded from the user. This is in contrast to the commercial hand-held ultraviolet sources as well as the large, high intensity sources.
  • the irradiation source is contained within a housing made of material that obstructs the transmission of radiant energy (i.e. an opaque housing). No irradiation is allowed to pass to the user. This allows for inherent safety for the user.
  • Activation compounds defines a family of compounds that undergo chemical change in response to triggering stimuli.
  • Triggering stimuli include, but are not limited to, thermal stimuli, chemical stimuli and electromagnetic stimuli.
  • Photoreactive, activation compounds (or simply “photoreactive compounds”), defines a genus of compounds in the activation compound family that undergo chemical change in response to electromagnetic radiation (Table 1).
  • furocoumarins One species of photoreactive compounds described herein is commonly referred to as the furocoumarins.
  • the furocoumarins belong to two main categories: 1) psoralens [7H-furo(3,2-g)-(l)-benzopyran-7-one, or ⁇ -lactone of 6- hydroxy-5-benzofuranacrylic acid], which are linear:
  • the present invention also contemplates new and known photoreactive compounds that inactivate pathogens in red blood cells.
  • One such species of compounds is commonly referred to as the group of red absorbing compounds (absorption of light in the range of 580 nm and above).
  • red absorbing compounds are small enough to intercalate in a double helix.
  • Thionine (3,7-diaminophenothiazin-5-ium chloride) is an example of a three membered ring with absorption out in the red (commercially available from Sigma Chemical Co., St Louis, MO). In this compound, not only are there an N and S in the internal ring, there are two aromatic amines attached to the outer two rings. This compound has been used as a nuclear stain in the past.
  • AMT 4'-Aminomethyl-4,5',8-trimethylpsoralen
  • Table 2 shows the extent of 0 2 production by various psoralens and Methylene Blue relative to 8-MOP. Inspection of Table 2 shows that addition of methoxy groups to the 5- and 8- position of the psoralen scaffold significantly reduces the ability of the psoralen to generate 'Oj (e.g. 5,8-dimethoxypsoralen produces 18-fold less 0 2 than unsubstituted psoralen).
  • Aminopsoralens were expected to meet such needs.
  • Aminopsoralens are. defined as psoralens that have amino-substitutions on the 3-, 4-, 5-, 8-, 4'-, or 5'- carbons.
  • the psoralens may or may not have further substitutions, including substitutions on the nitrogen.
  • the first step in synthesizing desirable compounds was to synthesize an intermediate compound from which several compounds of interest could be derived.
  • 4'-Chloromethyl-4,5'-dimethyl-8-methoxypsoralen, Compound 2 of FIG. 24, was designed to be such an intermediate.
  • the synthesis pathway for the compounds of the present invention involves starting with the following compound, Compound 1:
  • This starting compound in the synthesis of Compound 2 is 4,5'-dimethyl-8- methoxypsoralen.
  • the synthesis of this precursor compound has been described previously in the literature. See Bender, et al., J. Org. Chem. 44:2176 (1979).
  • the starting compound may be synthesized by the following method: 8-acetyl-7-(2-chloroallyl)oxy-4-methylcoumarin, described in Bender, et al., J. Org. Chem.
  • FIG. 25 shows chloromethylation of this compound (1), giving 4'- chloromethyl-8-methoxy-4,5'-dimethylpsoralen (2).
  • AMMP 4'- aminomethyl-4,5'-dimethyl-8-methoxypsoralen
  • FIG. 25 4'-Chloromethyl-8-methoxy- 4,5'-dimethylpsoralen (2), is converted via Gabriel synthesis to AMMP (4) in two steps.
  • various N-substituted and N,N'-disubstituted analogs e.g.
  • R and/or R' is alkyl or substituted alkyl
  • R and/or R' which have similar activity
  • 4'-Chloromethyl-8-methoxy-4,5'- dimethylpsoralen can be converted to a secondary or tertiary amine by reaction with the appropriate primary or secondary amine precursor, HNRR' where R and /or R' are linear or branched alkyls (Cl - C20) or alkyls substituted with, for example, hydroxyl, alkoxy, amino, thio or mercapto groups.
  • 4'-Chloromethyl-8- methoxy-4,5'-dimethylpsoralen is stirred with an excess of amine either neat or in the presence of solvent such as ethanol, at 25 - 100°C, to produce a secondary or tertiary amine derivative.
  • the present invention also contemplates methods of synthesizing biotinylated aminopsoralens. While not required, it may be useful to be able to remove the photoreactive nucleic acid binding compounds of the present invention from the decontaminated material once inactivation is complete.
  • the present invention contemplates using biotinilated compounds as photoreactive nucleic acid binding compounds.
  • radiolabelled AMMP can be synthesized as follows: 4'-chloromethyl-4,5'- dimethyl-8-methoxypsoralen is hydrolized to 4'-hydroxymethyl-4,5'-dimethyl-8- methoxypsoralen. Oxidation of 4'-hydroxymethyl-4,5'-dimethyl-8- methoxypsoralen with chromium trioxide gives 4'-formyl-4,5'-dimethyl-8- methoxypsoralen, which is then reduced with tritiated sodium borohydride to produce .
  • the present invention contemplates treating blood with photoreactive activation compound and irradiating to inactivate all contaminating pathogen nucleic acid sequences before using the blood in clinical chemistry tests.
  • inactivation In General As herein defined, something is “inactivated” when it is rendered incapable of replication.
  • activation is here defined as the altering of the nucleic acid of a pathogen so as to render the pathogen incapable of replication.
  • Inactivation sensitivity is an operationally defined term. It is defined only in the context of an “inactivation method” and the particular detection method that is used to measure organisms remaining. Inactivation sensitivity is the number of germination seeds (e.g., viable bacterial cells) that must be present to result in a measurable signal in some defined detection assay following an inactivation procedure.
  • an "inactivation method” may or may not achieve “inactivation,” it is useful to consider a specific example.
  • a bacterial culture is said to be inactivated if an aliquot of the culture, when transferred to a fresh culture plate and permitted to grow, is undetectable after a certain time period.
  • the time period and the growth conditions e.g. temperature
  • This amplification factor along with the limitations of the detection method (e.g. visual inspection of the culture plate for the appearance of a bacterial colony) define the sensitivity of the inactivation method.
  • a minimal number of viable bacteria must be applied to the plate for a signal to be detectable. With the optimum detection method, this minimal number is 1 bacterial cell.
  • the minimal number of bacterial cells applied so that a signal is observed may be much greater than 1.
  • the detection method determines a "threshold” below which the "inactivation method” appears to be completely effective (and above which "inactivation” is, in fact, only partially effective).
  • This interplay between the amplification factor of an assay and the threshold that the detection method defines, can be illustrated.
  • Table 3 bacterial cells are applied to a plate under two different sets of conditions: in one case, the growth conditions and time are such that an overall amplification of IO 4 has occurred; in the other case, the growth conditions and time are such that an overall amplification of IO 8 has occurred.
  • the detection method is arbitrarily chosen to be visual inspection.
  • the detectable signal will be proportional to the number of bacterial cells actually present after amplification.
  • the detection threshold is taken to be IO 6 cells; if fewer than IO 6 cells are present after amplification, no cell colonies are visually detectable and the inactivation method will appear effective, i.e. it would be "substantially inactivated.”
  • the inactivation sensitivity limit would be 100 bacterial cells; if less than 100 viable bacterial cells were present in the original aliquot of the bacterial culture after the inactivation method is performed, the culture would still appear to be inactivated.
  • the inactivation sensitivity limit (assuming the same detection threshold) would be 1 bacterial cell. Under the latter conditions, the inactivation method must be sufficiently stringent that all bacterial cells are, in fact, incapable of replication for inactivation to appear complete (i.e. the inactivation method would need to cause inactivation, not just substantial inactivation).
  • the inactivation method of the present invention renders nucleic acid in pathogens substantially unamplifiable. In one embodiment, the inactivation method renders pathogen nucleic acid in blood preparations unamplifiable. Also, in one embodiment, the inactivation method of the present invention renders pathogen DNA in clinical samples substantially unamplifiable.
  • the inactivation method of the present invention be limited by the nature of the nucleic acid; it is contemplated that the inactivation method render all forms of nucleic acid (whether DNA, mRNA, etc.) substantially unamplifiable.
  • nucleic acid is rendered substantially unamplifiable by the methods and compounds
  • inactivation occurs by either 1) modification of nucleic acid, or 2) inhibition of the amplification enzyme itself.
  • modification of nucleic acid occurs with inactivation compounds, it probably occurs because the compounds react with nucleic acid to create sufficient adducts per base (i.e.
  • the inactivation methods of the present invention are adaptable to the clinical laboratory.
  • the large volume of human serum handled daily could be processed according to the present invention without significantly changing the laboratory practice.
  • the general procedure used in clinical laboratories today is as follows: blood is drawn from a patient into a blood collection tube.
  • a blood collection tube is defined as a tube into which blood is drawn.
  • the specific type of tube generally used is a "clot" tube ("red top” tube).
  • the tube is then centrifuged to separate serum from red blood cells, the tube is reopened by removing the rubber stopper by hand, plastic inserts are manually pushed down into the serum to a level just above the packed cells to maintain the serum/red blood cell separation, serum is then pipetted from the top of the insert into a sample cup which is placed in a processing rack for analysis.
  • the laboratory clinician is exposed to whatever pathogens the blood may carry.
  • An embodiment of the present invention contemplates inactivating any pathogens in the blood before the tube is opened.
  • nucleic acid binding compound could be added to the tube at the point of tube manufacture.
  • this compound is an aminopsoralen, such as AMT or AMMP. The compound could be added dry and then could be resuspended when the blood is drawn into the tube.
  • the present invention contemplates an embodiment where after centrifugation, the tube is irradiated in a photoinactivation device of the present invention.
  • This light device is designed to accommodate the standard commercially available red top vacuum tubes commonly used in clinical laboratories (Available from Terumo Medical, Elkton, MD). Irradiation activates the photoreactive binding compound which then binds nucleic acids, inactivating any pathogen nucleic acid in the blood sample.
  • the tube would then be opened and processed as before, but free from the risk of exposure to hazardous blood born pathogens.
  • the tube could be placed in a photoactivation device of the present invention which exposes only the gel and the serum to UV transmission. The rest of the tube would be protected from UV, thereby avoiding any deleterious effects to the red blood cell fraction from UV exposure.
  • Widespread use of the gell filled tubes proves that the addition of substances to a tube at the point of manufacture is a feasible and necessary approach to combating the spread of infectious diseases.
  • AMT is very efficient in generating active oxygen species, such as singlet oxygen (O 2 ) and superoxide radical.
  • the new psoralen derivative, AMMP, and other aminopsoralens have been designed structurally similar to AMT, to maintain the high reactivity with nucleic acid provided by such a structure. Yet it is modified by the addition of a methoxy group to the 8 position of the central ring, in place of an hydroxy group. Without intending to be limited by any description of the process by which this invention operates, it is presumed that this modification minimizes the generation of active oxygen species. It is expected that hydroxy-groups on the 8 position of AMT are converted into singlet oxygen during radiation with UV.
  • EAA ethyl-acetoacetate
  • DMF N,N-dimethylformamide
  • BUN blood urea nitrogen
  • Creat creatinine
  • phos acid phosphoric acid
  • alk alkaline phosphatase
  • ALT Alanine Aminotransferase
  • AST Aspartate Transaminase
  • LDH lactose dehydrogenase
  • CPK creatinine kinase
  • W watts
  • mW milliwatts
  • ⁇ approximately
  • EXAMPLE 1 contemplates devices and methods for the activation of photoreactive nucleic acid binding compounds.
  • a photoactivation device for decontaminating human serum or plasma samples according to the method of the present invention is described.
  • This device has the following features: 1) an inexpensive source of electromagnetic radiation, 2) temperature control of the sample, 3) a multi-sample holder, 4) a multiple sample irradiation format, and 5) a compact design that requires minimal bench space.
  • FIG. 1 is a side view of one embodiment of the device integrating the above-named features. The figure shows the bottom platform of an opaque housing (100), joined to an opaque cover (101) which is supported in a raised position by four legs (two legs shown, 102 A and 102C).
  • the legs are each jointed at their center, the center joints (103 A and 103C) and at the points the legs attach to the bottom platform (not shown) and to the cover (not shown), permitting the cover to be lowered to rest on the bottom platform.
  • This provides user protection from electromagnetic radiation emitted by a plurality of bulbs within the cover (shown as dashed lines in FIG. 1.)
  • a handle (104), located at one end of the cover, where one pair of legs meets the cover, can be pivoted to raise and lower the cover.
  • the cover fits over a rack (105) resting on the bottom platform, having a plurality of intrusions (not shown) for supporting a plurality of sample vessels (106). It is not intended that the present invention be limited by the nature of the material used to form the rack (105), bottom platform (100), or cover (101). In one embodiment, they are formed with a substance that does not transmit ultraviolet light, thereby protecting the user. In another embodiment, the rack (105) is formed in part by a substance that does not transmit ultraviolet light, and in part by a substance transparent to ultraviolet light. This allows for selective irradiation of only sections of the sample vessels (106).
  • FIG. 2 is a side view of the invention shown in FIG. 1, in the raised position.
  • FIG. 2 shows the legs (102 A and 102B) folding at the center joints (103A and 103B) as the cover (101) is lowered slightly from the raised position.
  • the handle (104) has been rotated approximately 45 degrees from vertical to achieve the lowering of the cover.
  • FIG. 2 also shows how the cover is positioned over the rack (105) and sample vessels (106).
  • FIG. 3 is a side view of the invention shown in FIG. 1, in the lowered position.
  • HG. 3 shows the cover (101) in the fully lowered position resting on the bottom platform (100).
  • the handle (104) is rotated almost 90 degrees from vertical to achieve the fully lowered position.
  • FIG. 4 is a cross sectional view of the invention shown in FIG. 2.
  • FIG. 4 shows the cover (101) having four bulbs (107 - 110) connectable to a power source (not shown).
  • the bulbs serve as a source of electromagnetic radiation and, in one embodiment, ultraviolet radiation. While not limited to the particular bulb type, the embodiment is configured to accept an industry standard, F8T5BL hot cathode dual bipin lamp.
  • the rack (105) and sample vessels (106) are exposed to irradiation from the bulbs (107 - 110) on two sides.
  • the bulbs (107 - 110) are separated from the sample vessels by two chambers (111 and 112). It is not intended that the present invention be limited by the nature of the material used to form the chambers (111 and 112).
  • they are made of glass.
  • they are made of a glass cut-off filter, such as a piece of Cobalt glass.
  • they are made of plastic.
  • the chambers contain a liquid filter solution that transmits only a specific region of the electromagnetic spectrum.
  • the chambers contain an aqueous solution of Co(No 3 ) 2 . This salt solution yields a transmission window of 320-400 nm.
  • the aqueous solution of Co(N0 3 ) 2 is used in combination with NiS0 to remove the 365 nm component of the emission spectrum of the fluorescent or arc source employed.
  • FIG. 4 shows the rack (105) is punctuated with sample holder intrusions (113).
  • Each sample holder intrusion (113) has an adjustable tube holder (114) which allows a sample tube to be placed securely in the rack in a variety of positions.
  • FIG. 5 is a cross sectional view of the cover (101) of the device shown in
  • FIG. 5 shows the cover (101) having four bulbs (107 - 110) connectable to a power source (not shown).
  • FIG. 5 also shows one of the opening joints (115) where the two halves of the cover (101) are linked. The opening joints allow the cover (101) to be opened for cleaning.
  • the opening handle (116) is provided to open the cover (101). When the cover is closed, the latch (117) holds the cover secure.
  • FIG. 6 is a cross sectional view of the cover (101) of the device shown in FIG. 2, in the open position.
  • HG. 6 is a view of the device shown in FIG. 5, in the open position, rotated at the opening joint (115).
  • FIG. 6 shows the clasp (118) onto which the latch (117) is secured when the cover (101) is in the closed position.
  • FIG. 7 is a cross sectional view of the cover (101) of the device shown in FIG. 1.
  • FIG. 7 shows both opening joints (115, 119) connected to the bottom of the cover (101).
  • FIG. 7 also shows two of the bulbs (109, 110) connectable to a power source (not shown).
  • FIG. 8 is a cross sectional view of the bottom of the cover (101) of the device shown in FIG. 1. It shows the opening joints (115, 119) attached to the cover (101). FIG. 8 also shows two of the bulbs (108, 110) connectable to a power source (not shown) which are separated from the sample vessels (not shown) in the rack (105) by two chambers (111 and 112).
  • FIG. 9 is a perspective view of a tube holder device of the present invention, found in the sample holder intrusion (113), as shown in FIG. 4.
  • the tube holder (120) is made of a flexible material that allows a tube (not shown) to be inserted into the sample holder intrusion (113) and provides resistance from tube movement.
  • FIG. 10 is a cross sectional view of the tube holder device shown in FIG. 9.
  • FIG. 11 is a side view of the tube holder device shown in FIG. 9.
  • FIG. 12 is a perspective view of another tube holder device of the present invention, found in the sample holder intrusion (113), as shown in FIG. 4.
  • the tube holder (121) is made of a flexible material that allows a tube (not shown) to be inserted into the sample holder intrusion (113) and provides resistance from tube movement.
  • FIG. 13 is a cross sectional view of the device shown in FIG. 12, exhibiting the flexibility feature of the tube holder (121).
  • FIG. 14 is a perspective view of the tube holder device shown in FIG. 12.
  • FIG. 15 is a perspective view of another tube holder device of the present invention, found in the sample holder intrusion (113), as shown in FIG. 4.
  • the tube holder (122) is made of a flexible material that allows a tube (not shown) to be inserted into the sample holder intrusion (113) and provides resistance from tube movement.
  • FIG. 16 is a cross sectional view of the device shown in FIG. 15, exhibiting the flexibility feature of the tube holder (122).
  • FIG. 17 is a perspective view of the device shown in FIG. 15.
  • FIG. 18 is a cross section of this embodiment.
  • the figure shows the bottom platform of an opaque housing (200), in which rests a removable rack (201).
  • the rack (201) has two tube supports (202, 203) each having a plurality of sample holder intrusions (not shown) for supporting a plurality of sample vessels (204). It is not intended that the present invention be limited by the nature of the material used to form the rack (201) or housing (200). In one embodiment, they are formed with a substance that does not transmit ultraviolet light, thereby protecting the user.
  • the rack is formed in part by a substance that does not transmit ultraviolet light, and in part by a substance transparent to ultraviolet light. This allows for selective irradiation of only sections of the sample vessels (204).
  • the tube supports (202, 203) are constructed of one sheet of flexible material, such as rubber, positioned between two sheets of rigid material, such as plastic, to provide adjustable sample holder intrusions to support sample vessels (204) in various positions in the tube supports (202, 203).
  • the two light source housings (205, only one shown) are situated one in front of the rack (201) and one behind the rack.
  • the light sources (not shown) are fixed within the light source housings (205) and are separated from the rack (201) by two chambers (207, only one shown). Each chamber is situated between one light source housing (205) and the rack (201). It is not intended that the present invention be limited by the nature of the material used to form the chambers (207). In one embodiment, they are made of glass. In another embodiment, they are made of a glass cut-off filter, such as a piece of Cobalt glass. In another embodiment, they are made of plastic.
  • the chambers contain a liquid filter solution that transmits only a specific region of the electromagnetic spectrum.
  • the chambers contain an aqueous solution of Co(No 3 ) 2 . This salt solution yields a transmission window of 320-400 nm.
  • the aqueous solution of Co(N0 3 ) 2 is used in combination with NiS0 4 to remove the 365 nm component of the emission spectrum of the fluorescent or arc source employed.
  • FIG. 19 is a cross sectional view of the device of FIG. 18 from the top of the device of FIG. 18.
  • FIG. 18 shows the rack (201) within the opaque housing (200).
  • the rack (201) has a plurality of sample holder intrusions (209) which hold the sample vessels.
  • the rack (201) is bounded on two sides by the chambers (207, 208) which filter the light from the UV light sources (not shown) within the light source housings (205, 206).
  • the rack (201) is also bounded on two sides by reflective material which serves to increase the light exposure of the sample vessels.
  • the housing (200) has a plurality of ventilation intrusions (212) which allow air to circulate and cool the sample vessels during irradiation.
  • the housing (200) also has a control panel intrusion (213) in which a control panel may be placed.
  • Fig. 20 is a perspective view of one embodiment of the device integrating the above-named features.
  • This device is hereinafter referred to as "the Device of Example 3.”
  • the figure shows an opaque housing (300) with a portion of it removed, containing an array of bulbs (301) above and below a plurality of representative blood product containing means (302) placed between plate assemblies (303, 304).
  • the plate assemblies (303, 304) are described more fully, subsequently.
  • the housing (301) can be opened via a latch (305) so that the blood product can be placed appropriately.
  • the housing (300) when closed, completely contains the irradiation from the bulbs (301).
  • the user can confirm that the device is operating by looking through a safety viewport (306) which does not allow transmission of ultraviolet light to the user.
  • the housing (300) also serves as a mount for several electronic components on a control board (307), including, by way of example, a main power switch, a count down timer, and an hour meter.
  • the power switch can be wired to the count down timer which in turn is wired in parallel to an hour meter and to the source of the electromagnetic radiation.
  • the count down timer permits a user to preset the irradiation time to a desired level of exposure.
  • the hour meter maintains a record of the total number of radiation hours that are provided by the source of electromagnetic radiation. This feature permits the bulbs (301) to be monitored and changed before their output diminishes below a minimum level necessary for rapid photoactivation.
  • FIG. 21 is a cross-sectional view of the device shown in FIG. 20 along the lines of 2—2.
  • FIG. 21 shows the arrangement of the bulbs (301) with the housing (300) opened.
  • a reflector (308A, 308B) completely surrounds each array of bulbs (301).
  • Blood product containing means (302) are placed between upper (303) and lower (304) plate assemblies.
  • Each plate assembly is comprised of an upper (303A, 304A) and lower (303B, 304B) plates.
  • the plate assemblies (303, 304) are connected via a hinge (309) which is designed to accommodate the space created by the blood product containing means (302).
  • the upper plate assembly (303) is brought to rest gently on top of the blood product containing means (302) supported by the lower plate (304B) of the lower plate assembly (304).
  • Detectors may be conveniently placed between the plates (303A, 303B, 304A, 304B) of the plate assemblies (303, 304). They can be wired to a printed circuit board (311) which in turn is wired to the control board (307).
  • FIG. 22 is a cross-sectional view of the device shown in FIG. 20 along the lines of 3—3.
  • Six blood product containing means (302) e.g. teflon platelet unit bags
  • the 5 temperature of the blood product can be controlled via a fan (312) alone or, more preferably, by employing a heat exchanger (313) having cooling inlet (314) and outlet (315) ports connected to a cooling source (not shown).
  • FIG. 23 is a cross-sectional view of the device shown in FIG. 20 along the lines of 4—4.
  • FIG. 23 more clearly shows the temperature control approach of a
  • Upper plate assembly plates (303 A, 303B) and lower plate assembly plates (104A, 104B) each create a temperature control chamber (303C, 304C), respectively.
  • the fan (312) can circulate air within and between the chambers (303C, 304C).
  • the heat exchanger (313) is employed, the circulating air is cooled and passed between the plates (303A,
  • Step 2 8-methoxy-4,5'-dimethyl-4'-(phthalimidomethyl)psoralen (0.2 g) (FIG. 25, 3), 95% EtOH (45 mL) and 85% aqueous hydrazine hydrate (0.6 mL) were placed in a round bottomed flask with a magnetic stirrer and a thermometer. The mixture was heated at 50°C until no starting material remained by TLC (95% CHCl 3 /5% MeOH). The EtOH was removed giving a white solid. CHC1 3 (15 mL) and 1 N NaOH (10 mL) were added and the mixture transferred to a separating funnel.
  • the basic solution was further extracted twice with CHC1 3 , the combined extracts were washed with water, then extracted twice with 0.3 N HCl.
  • the acid extracts were taken to pH 12 with aqueous NaOH solution, then the basic suspension was extracted three times with CHC1 3 .
  • These combined CHC1 3 extracts were washed with water, dried over Na 2 S0 4 , sand the CHC1 3 stripped off to give the amine as the free base (white solid). Absolute EtOH was added, then saturated with anhydrous HCl gas.
  • EXAMPLE 6 Synthesis of a Biotinylated Psoralen This example describes the synthesis of a biotinilated psoralen that may be used in a filtration system which removes the photoreactive compound from the treated material after the material has been decontaminated.
  • Step 1 A mixture of 187 mg (0.64 m ole) of 4'-chloromethyl-8-methoxy- 4,5'-dimethylpsoralen, from Example 4, and 1.69 g of l,2-bis-[(2- methylamino)ethoxy]-ethane was refluxed with stirring in 20 mL of dry toluene overnight. The solvent was then stripped off. The residue was acidified by adding 1 N HCl, then several drops concentrated HCl to pH l and extracted twice with CHC1 3 . The aqueous layer was made basic with NaOH solution and extracted into CHC1 3 , washed with H 2 0, then dried over Na 2 S0 4 .
  • Step 2 8-Methoxy-4,5'-dimethyl-4'-[8-(methylamino)-3,6- dioxaoctylamino)]methylpsoralen, 74 mg, was dissolved in 1.6 mL of DMF. Molecular sieves were added, followed by a solution of biotin-amido caproate N- hydroxy succinimide ester (154 mg) in 2 mL of DMF. The reaction mixture was stirred at room temperature under argon. Reaction progress was followed by TLC in (95% CH 2 Cl 2 /5% MeOH). After the reaction was complete, the solvent was stripped off under reduced pressure. The residue was extracted with MeOH. The MeOH was stripped off.
  • the residue was acidified to pH ⁇ l by adding 1 N HCl and extracted with CHC1 3 about 10 times.
  • the aqueous layer was made basic with NaOH solution pH 12) and extracted into CHCl ⁇ washed with H 2 0, then dried over Na 2 S0 4 .
  • the free base was dissolved in absolute EtOH, then saturated with HCl gas.
  • Step 1 A mixture of 150 mg of 4'-chloromethyl-4,5'-dimethyl-8- methoxypsoralen and water (15 mL) was refluxed with stirring until no starting material was detected by TLC. The resulting suspension was cooled, filtered, rinsed with water and dried to give 4'-hydroxymethyl-8-methoxy-4,5'- dimethylpsoralen (100 mg, 71.5%).
  • Step 2 3,5-Dimethylpyrazole (4g, 43 mmole) was added to a suspension of chromium trioxide (4.25 g, 43 mmole) in methylene chloride (125 mL) and the mixture was stirred at room temperature under argon for 15 minutes. To the resulting dark red solution, 4'-hydroxymethyl-8-methoxy-4,5'-dimethylpsoralen (4.0 g, 16 mmole) was added in one portion and the reaction mixture was then stirred at room temperature for 2 hours. The solvent was removed and the residue was chromatographed on silica gel with CH 2 C1 2 .
  • the borohydride vial was rinsed with 2 x 1 mL EtOH and the rinses were added to the reaction.
  • the reaction was stirred for 3 hrs and monitored by TLC in chloroform : methanol 98:2.
  • the solvent was removed under a vacuum and the solid residue was dissolved in approximately 1 mL chloroform : methanol 98:2. This was loaded onto a 1 cm x 30 cm, 60 - 200 mesh, silica gel column and eluted with 98:2.
  • the product, 8-methoxy-4,5'-dimethyl-4'- hydroxy[ 3 H]methylpsoralen was pooled and solvent removed under vacuum. 23 mg (0.084 mmole) was recovered.
  • Step 4 The 23 mg (0.084 mmole) of 4'-hydroxy[ 3 H]methyl-8-methoxy-4,5'- dimethylpsoralen was dissolved in 2 mL of dry chloroform and stirred under argon. Distilled thionyl chloride (27 ⁇ L, 0.38 mmole) was added to this and the reaction mix was stirred under argon for 3 hours. TLC in chloroform showed complete reaction. The recovery was not determined at this point.
  • Step 5 The 4'-chloro[ 3 H]methyl-8-methoxy-4,5'-dimethylpsoralen (0.042 mmole) was dried and dissolved in 2 mL of distilled DMF. Dry potassium phthalimide (27 mg, 0.147 mmole) was added and the reaction was heated to approximately 40°C with stirring. After several hours TLC in chloroform showed incomplete reaction. Another 33 mg (0.18 mmole) of potassium phthalimide was added and the reaction was stirred overnight at 40°C. The DMF was removed under vacuum and the solid was dissolved in 1 mL of chloroform : methanol 98:2.
  • Step 6 The 4'-phthalimido[ 3 H]methyl-4,5'-dimethyl-8-methoxypsoralen was dissolved in the 4 mL of 95% EtOH. Hydrazine hydrate (40 ul, 0.6 mmole) was added and the reaction was heated to approximately 50°C with stirring overnight. TLC in chloroform : methanol 95:5 showed only about 12% reacted. Another 3mL 95% EtOH was added to try to dissolve everything. A total of 250 ⁇ L additional hydrazine was added and reacted overnight again. TLC the next day showed approximately 80% product. The solvent was removed under vacuum. Chloroform (5mL) and 5mL 0.1M NaOH was added. Next the chloroform was removed by extraction.
  • EXAMPLE 8 The nucleic acid binding affinity of AMMP was measured using radiolabeled AMMP with calf thymus DNA in Tris-EDTA buffer. The formation of psoralen:DNA adducts expressed as numbers of psoralen adducts per 1000 base pairs (bp) was measured under identical (equimolar) conditions along with AMT and 8-MOP (HG. 26).
  • Each tritium labelled psoralen (1.4 uM) was added to a solution of calf thymus DNA (1.0 uM) then irradiated in the Device of Example 3, above, for the indicated time. Aliquots were removed and precipitated 3 times with EtOH. The binding ratio was calculated from the final DNA concentration (determined by optical density) and the amount of covalently bound psoralen determined by scintillation counting. As shown in FIG. 26, AMMP binds approximately twice as well as 8-MOP.
  • AMMP also has a much higher aqueous solubility than 8-MOP, and therefore can provide even higher levels of DNA binding when used at higher concentrations.
  • AMMP also provides significantly higher DNA binding than does 8-MOP at relatively short irradiation times (e.g. 10 minutes), which is an important factor for maintaining platelet function.
  • AMT shows the best binding, forming more than 250 adducts per kilobase pair.
  • This example looks at AMT binding of DNA over time for two different photoactivation devices. The example shows that both devices stimulate adequate binding.
  • the nucleic acid binding affinity of AMT was measured using radiolabeled AMT with calf thymus DNA in Tris-EDTA buffer.
  • Tritium labelled AMT (0.83 ⁇ g/ul) (118 ul) was added to a solution of calf thymus DNA (600 ⁇ g/mL) (2.83 mL) and diluted with 5 M NaCl to reach an AMT concentration of 31 ⁇ g/mL. 100 ul of the solution was placed in each of 8 glass vials and irradiated for 2, 4, 8, or 16 minutes on either the Device of Example 3 or the Device of Example 2. Aliquots were removed and precipitated
  • the binding ratio was calculated from the final DNA concentration (determined by optical density) and the amount of covalently bound psoralen determined by scintillation counting.
  • H9 cells infected with HIV were added to standard human platelet concentrates (2.5 x IO 7 cells per concentrate), final concentration 5x10 s cells per concentrate.
  • Aliquots of HIV contaminated platelet concentrate (5 mL) were placed in Pyrex chambers. The chambers had previously been coated on the inside with silicon.
  • the platelet concentrates were treated with either AMMP (100 ⁇ g/mL), 8-MOP (300 ⁇ g/mL), or AMT (20 ⁇ g/mL) and irradiated with 320- 400 nm (20mW/cm 2 ) for up to 12 min. on a device similar to the Device of Example 3.
  • the photoactivation device used here was previously tested and found to result in light exposure comparable to the Device of Example 3. (Data not shown).
  • AMMP is substantially more efficient than 8-MOP.
  • EXAMPLE 11 A major concern is whether the decontamination process interferes with the chemistry tests. Clearly, one does not want to inactivate pathogens and render the serum or plasma unusable for testing.
  • a total sample volume of 0.3 mL was selected on the basis of the volume requirements of the Kodak Ectachem Analyzer instrument, Kodak Corp., for the 19 clinical tests.
  • a volume of stock concentrate of each compound was added to the necessary amount of serum in a 1 mL Eppendorf tube.
  • 8-MOP 0.015 mL of concentrate was added to 0.285 mL of serum.
  • AMT 0.030 mL of concentrate was added to 0.270 mL of serum.
  • 5B-AMMP 0.006 mL was added to 0.294 mL of serum.
  • Irradiation was performed on the HRI-100 device. A single 15 minute irradiation time was used. Given the short irradiation time, the irradiations were performed without cooling of the reaction chamber.
  • the results are shown in Tables 4-8.
  • the data for each of the five patients is in two sets of tables (A and B): the first shows the results of AMT and 5B- AMMP with relevant controls; the second shows the results with 8-MOP along with the 8-MOP controls. No adjustments in the test values have been made to reflect the dilutions.
  • Example 11 indicated that the most significant issue is presented by the irradiation process. A second experiment was therefore performed without any compounds to evaluate this question. Again, stored patient serum was used. Two patient samples were selected for testing Uric Acid and Total Bilirubin. One sample was taken that, upon visual inspection, appeared to have a slightly elevated bilirubin. The other sample appeared to have a normal bilirubin.
  • HG. 29 is a bar graph showing the clinical chemistry testing results for Total Bilirubin
  • HG. 30 is a bar graph showing the clinical chemistry testing results for Uric Acid. The values for both patients are shown side by side.
  • EXAMPLE 13 The reduction in Total Bilirubin and Uric Acid values in Example 12 may be due to absorption by these analytes of higher wavelengths than those needed to activate psoralens. This offers the opportunity to filter out these wavelengths without impairing the inactivation significantly.
  • experiments were performed using wavelength filters in the HRI-100 device. Specifically, an aqueous solution of Co(No 3 ) 2 was used in combination with NiS0 4 to substantially remove the 365 nm component of the emission spectrum of the light source employed.
  • the Co-Ni solution can be conveniently used in place of water as a coolant during the irradiation.
  • Total Bilirubin could be avoided under conditions where certain wavelengths were filtered. In this example, it is demonstrated that this filtering does not impair the inactivation process.
  • the binding of AMT to calf thymus DNA was studied using the HRI-100 and a liquid filter solution that transmits only a specific region of the electromagnetic spectrum.
  • Example 14 examined the effect of filtering on binding. The results demonstrated that although filtering the UV light lowers intensity 40 fold, it only reduces binding of AMT 6 fold. This example addresses the issue of how filtering the UV light effects the inactivation process when using AMT and AMMP.
  • This example presents data on AMT and AMMP inactivation of cell-free HIV in serum and in culture media.
  • HIV-infected H9 cells were seeded into human serum or synthetic medium. Aliquots of medium were placed in Pyrex chambers, described in Example 10, and AMT was added to a concentration of 20 ⁇ g/mL AMT. Some samples were irradiated unfiltered with 320-400 nm (20mW/cm 2 ) using the irradiation device used in Example 10, for 5, 10 or 15 minutes. Others were irradiated for 15, 30, or 60 minutes with UV filtered with a liquid solution that transmits only a specific region of the electromagnetic spectrum. An aqueous solution of Co(N0 3 ) 2 and NiS0 4 removed the 365 nm component of the emission spectrum of the fluorescent light source.
  • EXAMPLE 16 Examples 14 and 15 demonstrated that a reduction in AMT binding with nucleic acid results when the source of irradiation is filtered. This experiment demonstrates that an increase in the concentration of AMT will increase nucleic acid binding to compensate for the drop resulting from the filter.
  • HIV inactivation efficiency was evaluated for AMT.
  • concentrations of AMT were compared for ability to inactivate cell-associated virus (HIV).
  • HIV-associated virus HIV was performed as follows. HIV-infected H9 cells were seeded into human serum or synthetic media.
  • Control containers of serum and of culture media were treated with UVA light alone. Controls for each concentration of AMT were not irradiated. Residual HIV infectivity was assayed using the MT-2 infectivity assay. Hanson, C.V., J. Clin. Micro 28:2030 (1990).
  • EXAMPLE 17 As shown in Example 13, the damage to bilirubin and uric acid from light alone is controlled by filtering out certain wavelengths. In this example, it is determined whether, in combination, irradiation and photoreactive compounds effect common clinical tests. In this example, AMT and AMMP, both in the presence of ultraviolet light, are compared with regard to their effect on nineteen common clinical chemistry tests. As in Example 13, theorizing that any reduction in Uric Acid and Total Bilirubin might be do to wavelengths other than those needed to activate psoralens, experiments were also performed under conditions to filter out these wavelengths. As with prior experiments, stored patient serum was used. Two patient samples (both approximately one week old) were selected for testing. One sample appeared to have a low to normal bilirubin upon visual inspection ("Patient 1"). The other appeared to have a normal to high bilirubin level (“Patient 2").
  • a total sample volume of 0.4 mL was selected for convenience. Both AMT and AMMP were used at high concentration (200 ⁇ g/mL). 20 minute irradiations were performed on the HRI-100 device with and without the liquid filter in the sample trough. The irradiations were performed without cooling of the reaction chamber.
  • Example 17 shows that AMMP is much less destructive than either AMT OP. Without intending to be limited to any mechanism, it is postulated that the reduction of bilirubin and uric acid assay values result from oxygen radicals produced by photoreactive compounds during and after irradiation. To determine the generation of active oxygen species by AMMP relative to 8-MOP and AMT, the production of superoxide radical was measured.
  • AMMP produced significantly less superoxide radical than AMT (HG. 35). This supports the above postulation that oxygen radicals produced by some photoreactive compounds during and after irradiation cause damage to some clinical analytes, such as bilirubin and uric acid, making them less compatible with chemistry tests. On the other hand, AMMP, which produced less oxygen radicals during and after irradiation, may be more compatible with chemistry tests.
  • EXAMPLE 19 In the previous examples, the compatibility of the present invention with chemistry tests was examined. This example examines whether the methods of the present invention have adverse effects upon antibody based clinical tests. This experiment looks at the effects of AMMP and ultraviolet light on a clinical Rubella IgG antibody assay.
  • a total sample volume of 1 mL was selected for convenience.
  • AMMP was added to a concentration of 90 ⁇ g/mL.
  • Irradiations were performed for 10, 20, or 30 minutes on the Device of Example 2. The irradiations were performed without cooling of the reaction chamber.
  • the serum was treated as follows: i) no treatment, ii) compound but no light, and iii) compound and varied times of irradiation.
  • the serum was then tested for the presence of Rubella IgG by SmithKline Beecham Laboratories, CA, using an enzyme linked immunosorbant assay.
  • Rubella antigen was attached to a solid phase surface such as wells in a plastic strip. The Rubella antigen, if present in the serum, bound to the attached antigen. Unbound antibody was then removed by washing.
  • the present invention provides methods for inactivation of contaminants in material intended for in vitro use, and in particular the inactivation of pathogens in human fluids prior to clinical testing.
  • the methods are effective against a wide range of pathogens.
  • the methods do not unduly interfere with laboratory operations or clinical analysis.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Procédé de décontamination de liquides humains avant leur traitement en laboratoire. Les techniques utilisées permettent de traiter d'importantes quantités de sérum humain sans affecter le résultat des analyses. Des nouveaux composés pour la photodécontamination de produits biologiques, compatibles avec les analyses cliniques en ce sens qu'ils n'interfèrent pas avec les analytes des sérums, sont également décrits.
PCT/US1994/002607 1993-03-11 1994-03-10 Decontamination de prelevements cliniques WO1994020090A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64024/94A AU6402494A (en) 1993-03-11 1994-03-10 Decontaminating clinical samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3249093A 1993-03-17 1993-03-17
US08/032,490 1993-03-17

Publications (1)

Publication Number Publication Date
WO1994020090A1 true WO1994020090A1 (fr) 1994-09-15

Family

ID=21865207

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/002607 WO1994020090A1 (fr) 1993-03-11 1994-03-10 Decontamination de prelevements cliniques

Country Status (2)

Country Link
AU (1) AU6402494A (fr)
WO (1) WO1994020090A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0707476A1 (fr) * 1993-06-28 1996-04-24 Steritech, Inc Composes de photodecontamination de pathogenes contenus dans le sang
WO1998030545A1 (fr) * 1997-01-06 1998-07-16 Cerus Corporation Composes frangibles pour inactivation pathogene
US5798238A (en) * 1990-04-16 1998-08-25 Baxter International Inc. Method of inactivation of viral and bacterial blood contaminants with quinolines as photosensitizer
US6093725A (en) * 1997-01-06 2000-07-25 Cerus Corporation Frangible compounds for pathogen inactivation
WO2002032875A1 (fr) * 2000-10-18 2002-04-25 Fresenius Hemocare Gmbh Agent inactivant des agents pathogenes, comprenant un element qui se lie aux acides nucleiques, et son utilisation
US6469052B2 (en) 1993-06-28 2002-10-22 Cerus Corporation Compounds for the photodecontamination of pathogens in blood
US6514987B1 (en) 1997-01-06 2003-02-04 Cerus Corporation Frangible compounds for pathogen inactivation
EP1364944A1 (fr) * 1997-01-06 2003-11-26 Cerus Corporation Composés frangibles pour inactivation pathogène
US6686480B2 (en) 1993-06-28 2004-02-03 Cerus Corporation Compounds for the photodecontamination of pathogens in blood
CN109996603A (zh) * 2016-11-22 2019-07-09 肝化学股份有限公司 光化学装置
US11883544B2 (en) 2019-06-28 2024-01-30 Cerus Corporation System and methods for implementing a biological fluid treatment device
US12011510B2 (en) 2019-06-22 2024-06-18 Cerus Corporation Biological fluid treatment systems
US12214092B2 (en) 2017-12-29 2025-02-04 Cerus Corporation Systems and methods for treating biological fluids

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4294822A (en) * 1980-07-29 1981-10-13 Thomas C. Elder, Inc. 5-Aminoalkyl-4,4,8-trialkylpsoralens
US4370344A (en) * 1979-09-10 1983-01-25 Elder Pharmaceuticals, Inc. 5'-Aminoalkyl-4'-alkylpsoralens
US4727027A (en) * 1983-05-02 1988-02-23 Diamond Scientific Co. Photochemical decontamination treatment of whole blood or blood components

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4370344A (en) * 1979-09-10 1983-01-25 Elder Pharmaceuticals, Inc. 5'-Aminoalkyl-4'-alkylpsoralens
US4294822A (en) * 1980-07-29 1981-10-13 Thomas C. Elder, Inc. 5-Aminoalkyl-4,4,8-trialkylpsoralens
US4727027A (en) * 1983-05-02 1988-02-23 Diamond Scientific Co. Photochemical decontamination treatment of whole blood or blood components

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANNUAL REVIEW OF BIOCHEMISTRY, Volume 54, issued 1985, G.D. CIMINO et al., "Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry", pages 1151-1193. *
BIOCHEMISTRY, Volume 16, issued 1977, S.T. ISAACS et al., "Synthesis and Characterization of New Psoralen Derivatives with Superior Photoreactivity with DNA and RNA", pages 1058-1064. *
BLOOD CELLS, Volume 18, issued 1992, P. MOREL et al., "Photochemical Inactivation of Viruses and Bacteriophage in Plasma and Plasma Fractions", pages 27-42. *
NUCLEIC ACIDS RESEARCH, Volume 16, Number 22, issued 1988, B.K. LEE et al., "Interaction of Psoralen-Derivatized Oligodeoxyribonucleoside Methylphosphonates with Synthetic DNA Containing a Promoter for T7 RNA Polymerase", pages 10681-10697. *
QUARTERLY REVIEW OF BIOPHYSICS, Volume 17, issued 1984, J.E. HEARST et al., "The Reaction of Psoralens with Deoxyribonucleic Acid", pages 1-44. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5798238A (en) * 1990-04-16 1998-08-25 Baxter International Inc. Method of inactivation of viral and bacterial blood contaminants with quinolines as photosensitizer
US6686480B2 (en) 1993-06-28 2004-02-03 Cerus Corporation Compounds for the photodecontamination of pathogens in blood
EP1776952A3 (fr) * 1993-06-28 2007-05-02 Cerus Corporation Photodécontamination de pathogènes contenus dans le sang
EP0707476A4 (fr) * 1993-06-28 1998-12-23 Steritech Inc Composes de photodecontamination de pathogenes contenus dans le sang
EP0707476A1 (fr) * 1993-06-28 1996-04-24 Steritech, Inc Composes de photodecontamination de pathogenes contenus dans le sang
EP1776952A2 (fr) * 1993-06-28 2007-04-25 Cerus Corporation Photodécontamination de pathogènes contenus dans le sang
US6469052B2 (en) 1993-06-28 2002-10-22 Cerus Corporation Compounds for the photodecontamination of pathogens in blood
US6503699B1 (en) 1993-06-28 2003-01-07 Cerus Corporation Method for photodecontamination of pathogens in blood using 5'-primary aminopsoralens
US6093725A (en) * 1997-01-06 2000-07-25 Cerus Corporation Frangible compounds for pathogen inactivation
EP1364944A1 (fr) * 1997-01-06 2003-11-26 Cerus Corporation Composés frangibles pour inactivation pathogène
US6514987B1 (en) 1997-01-06 2003-02-04 Cerus Corporation Frangible compounds for pathogen inactivation
WO1998030545A1 (fr) * 1997-01-06 1998-07-16 Cerus Corporation Composes frangibles pour inactivation pathogene
CN101676268B (zh) * 1997-01-06 2013-09-25 塞鲁斯公司 用于病原体灭活的脆性化合物
WO2002032875A1 (fr) * 2000-10-18 2002-04-25 Fresenius Hemocare Gmbh Agent inactivant des agents pathogenes, comprenant un element qui se lie aux acides nucleiques, et son utilisation
CN109996603A (zh) * 2016-11-22 2019-07-09 肝化学股份有限公司 光化学装置
US12214092B2 (en) 2017-12-29 2025-02-04 Cerus Corporation Systems and methods for treating biological fluids
US12011510B2 (en) 2019-06-22 2024-06-18 Cerus Corporation Biological fluid treatment systems
US11883544B2 (en) 2019-06-28 2024-01-30 Cerus Corporation System and methods for implementing a biological fluid treatment device

Also Published As

Publication number Publication date
AU6402494A (en) 1994-09-26

Similar Documents

Publication Publication Date Title
US5556958A (en) Inactivation of pathogens in clinical samples
US5399719A (en) Compounds for the photodecontamination of pathogens in blood
Matthews et al. Photodynamic therapy of viral contaminants with potential for blood banking applications
US6469052B2 (en) Compounds for the photodecontamination of pathogens in blood
US6455286B1 (en) Psoralens for pathogen inactivation
US5871900A (en) Method of inactivating pathogens in biological fluids using photoactivated 5-primaryamino psoralens
US5593823A (en) Method for inactivating pathogens in blood using photoactivation of 4'-primary amino-substituted psoralens
Prince et al. Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin
EP0368902B1 (fr) Procede d'eradication d'agents contaminants infectieux presents dans les tissus
US5854967A (en) Device and method for photoactivation
US6420570B1 (en) Psoralen compounds
WO1994020090A1 (fr) Decontamination de prelevements cliniques
PT633721E (pt) Descontaminacao de plaquetas com 8-metoxipsoraleno
Williams ASSESSMENT OF THE PHOTOCHEMISTRY OF

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT.BUL.21/94 UNDER INID (30)"PRIORITY DATA",REPLACE "930311" BY "930317"

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA