Device and method for photoactivation

ES 2 201 093 T3

DESCRIPTION

Device and procedure for photoactivation.

Field of the invention

The present invention relates generally to a device and method for the treatment of blood components in blood bags.

Background

All blood collected from volunteer donors to transfusion recipients is normally separated into its components: red blood cells, platelets, and plasma. Each of these fractions is individually stored and used to treat a multiplicity of specific ailments and disease states. For example, the red blood cell component is used to treat anemia; the concentrated component of platelets is used to control bleeding; and the plasma component is frequently used as a source of coagulation factor VIII for the treatment of hemophilia.

Ideally, all blood cell preparations should come from freshly drawn blood and then immediately transfuse the recipient. However, the logistics of a blood donor center prevent this possibility in the vast majority of cases. Transfusions are needed day and night and it is difficult, if not impossible, to organize donor recruitment at unusual hours. Consequently, modern blood donor centers must use stored blood products.

In the United States, blood storage procedures are subject to government regulation. The maximum storage periods for blood components collected in these systems are specifically prescribed. For example, all blood components collected in an “open” (eg, non-sterile) system must, according to government regulations, be transfused within 24 hours and in most cases within 6 to 8 hours. In contrast, when all blood components are collected in a 'closed' (eg sterile) system, red blood cells can be stored for up to 42 days (depending on the type of anticoagulant and storage medium used) and plasma is You can freeze and store for even longer periods.

Murphy and Gardner, New Eng. J. Med. 280; 1,094 (1,969), demonstrated that platelets stored as platelet-rich plasma (PRP) at 22 ° C had a longer half-life in vivo than those stored at 4 ° C. Thus, more acceptable platelet concentrates could be transfused after storage at room temperature. Until recently, standards allowed the storage of platelet concentrates at room temperature for up to seven days (depending on the type of storage container). However, it was recognized that the incidence of bacterial growth and consequent transfusion reactions were increased in the recipient to unacceptable levels with a seven-day platelet concentrate. Platelet concentrates cannot now be stored for more than five days.

The blood bags used for the platelet concentrate preparations are sterile, as are the connected satellite bags. One might therefore think that keeping the blood preparation sterile during the manipulations necessary to concentrate platelets is a relatively straightforward fact. However, bacteria can be introduced by at least two different means. First, if the donor is suffering from mild bacteremia, the blood will be contaminated, regardless of the collection or storage procedure. Donors with proper histories and physics will decrease but will not eliminate this problem. See BJ Grossman et al., Transfusion 31; 500 (1991). A second, more persistent source of contamination is venepuncture. Even when using "sterile" skin preparation procedures, it is extremely difficult to sterilize the crypts surrounding the sweat glands and hair follicles. During venepuncture, this contaminated skin is often cut into a small "core" using a fine needle. This nucleus can serve to "seed" the blood bag with bacteria that can grow and become a risk to the recipient.

In fact, many patients requiring platelet transfusions lack host defense mechanisms for normal compensation and destruction of the bacteria due to either chemotherapy or basic hematologic disease. The growth of apparently innocuous organisms on stored platelets can, after transfusion, cause reaction and death of the recipient. See, for example, BA Myhre JAMA 244;

I. 333 (1980), JM Heal et al., Transfusion 27; 2 (1987).

The reports that determine the degree of contamination in platelets differ in their procedures, sample size, and bacterial detection systems. DH Buchholz, et al., Transfusion 13; 268 (1973) reported a total platelet contamination level of 2.4% when a larger sample (> 1000 bags) was examined and more extensive measures were taken for bacterial culture. While some units were highly contaminated after only 24 hours of storage, the total incidence varied according to the age of the concentrate and increased with the widespread practice of pooling individual units; about 30% of the funds were contaminated within 3 days. See also DH Buchholz, et al., New Eng.

J. Med. 285; 429 (1971). While other clinical studies suggest lower numbers, recent studies indicate

ES 2 201 093 T3 septic platelet transfusions are significantly unaccounted for. See, for example, JF Morrow et al., JAMA 266; 555 (1991).

Precultured platelets are not a solution to the problem of bacterial contamination. The culture test takes 48 hours to detect growth. Maintaining the platelet units for two more days to await assay results would, ironically, create a smaller margin of safety. See Table 2 in JF Morrow et al., JAMA 266; 555 (1991). While highly contaminated units would be detected on exit, lightly contaminated units would be allowed to grow for two days. The oldest and potentially most contaminated units would eventually be transfused.

Washing blood cells (for example with saline solutions) or filtering bacteria are also not practical solutions. These techniques are time consuming and ineffective as they can reduce the number of viable blood cells available for transfusion. More importantly, they typically involve an "entry" into the storage system. Once an entry has been made in a previously closed system, the system is considered “open” and the transfusion must be performed quickly, regardless of how the blood was collected and processed in the first place.

Nor are antibiotics a reasonable solution. Pollution is produced by a wide spectrum of organisms. Antibiotics would be necessary to cover this spectrum. Many recipients are allergic to antibiotics. In addition, there is a growing number of drug resistant strains of bacteria that would not be inactivated.

Recently there has been interest in the inactivation of pathogens in blood using photoreactive compounds, such as psoralens. Psoralens are tricyclic compounds formed by the linear condensation of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts with the pyrimidine bases by absorption of long-wavelength ultraviolet light (UVA). GD Cimino et al., Ann. Rev. Biochem. 54; 1,151 (1985). Hearst et al., Quart. Rev. Biophys. 17; 1 (1984). If there is a second pyrimidine adjacent to a psoralenpyrimidine monoadduct and on the opposite chain, the absorption of a second photon can lead to the formation of a diaduct that functions as an interchain crosslinking. ST Isaacs et al., Biochemistry 16, 1058 (1977). ST Isaacs et al, Trends in Photobiology (Plenum) pp. 279-294 (1982). J. Tessman et al., Biochemistry 24; 1,669 (1985). Hearst et al., US Patent Nos. 4,124,589, 4,169,204, and 4,196,281.

Psoralens have been shown to inactivate viruses in some blood products. See HJ Alter et al., The Lancet (II, 446) (1988). L. Lin et al., Blood 74; 517 (1989). GP Weisehahn et al., US Patent Nos. 4,727,027 and 4,748,120 describe the use of a combination of 8-methoxypsoralen (8-MOP) and irradiation.

They show that 300 µg / ml of 8-MOP in conjunction with an hour or more of UV irradiation can effectively inactivate viruses. However, these treatment conditions cause damage to the blood product due to energy transfer. Their approach is only feasible if cell damage is specifically suppressed by limiting the concentration of molecular oxygen, a difficult and expensive procedure.

Isopsoralens, like psoralens, are tricyclic compounds formed by the fusion of a furan ring with a coumarin. See Baccichetti et al., US Patent No. 4,312,883. F. Bordin et al., Experientia 35; 1,567 (1,979). F. Dall'Acqua et al., Medecine Biologie Envir. 9; 303 (1981). S. Caffieri et al., Medecine Biologie Envir. eleven; 386 (1983). F. Dall'Acqua et al., Photochem Photobio. 37; 373 (1983). G. Guiotto et al., Eur. J. Med. Chew-Chim. Ther. 16; 489 (1981). F. Dall'Acqua ycolaboradores, J. Med. Chem. 24; 178 (1984). Unlike the psoralens, the isopsoralen rings are not linearly bound. While they are capable of intercalating between the base pairs of double-stranded nucleic acids and form covalent adducts with the bases of nucleic acids by absorption of long-wavelength ultraviolet light, isopsoralens, due to their angular geometry, normally they cannot form crossovers with DNA. See generally, GD Cimino et al., Ann. Rev. Biochem., 54, 1,151 (1985).

There are devices currently used that emit ultraviolet radiation to activate psoralens and other photoactive compounds. US Patent No. 5,184,020, by Hearst et al., Describes a device for the photoactivation of psoralens. However, the device described is designed for the irradiation of samples in tubes such as beakers. It does not describe a device for use on blood bags. Furthermore, although the patent describes a cooling system for irradiated samples, this system would not work for blood bags because it depends on the flow of fluid around the sample vessels.

Documents WO-A-93 / 17,553 and WO-A-94 / 03,054 describe a device that emits ultraviolet radiation for the activation of psoralens. This device comprises: a) means for providing the appropriate wavelength of electromagnetic radiation to produce the activation of at least one photoreactive compound; b) means for supporting a plurality of blood products in a fixed relationship with the means providing radiation during activation; and c) means for maintaining the temperature of the blood products within a desired temperature range during activation such as a fan or a heat exchanger.

Other devices are not suitable for activating psoralens, but can be used for other purposes with blood bags. For example, US Patent Nos. 4,726,949 and 4,866,282 to Miripol describe an irradiation device for use in the prevention of alloimmunization. This device is not practical for

ES 2 201 093 T3 for use in laboratories that will process large amounts of blood for sterilization. The device only supports one blood container, which would be a bottleneck in blood processing. (See figure 1, ref. No. 10, of any Miripol patent). In addition, it provides radiation of a wavelength of 280 to 320 nanometers, including band 313, (see claim 1 of the patent 4,866,282) to which nucleic acids absorb radiation and could be damaged. The UVB interval can also destroy platelet function. The Miripol patents state that UV-A range sources "do not provide a good reduction in the effect of alloimmunization of lymphocytes." Column 2, lines 61-64, of 4,726,949. Finally, the Miripol patents describe the use of only one means for cooling the system during irradiation, an extractor fan. The goal in those patents is to keep the temperature at or below 31 degrees Celsius. Column 3, lines 44-46. However, platelets are currently stored at 22-24 degrees. CG Stack and L. Snyder, "Storage of Platelet Concentrate," Blood Separation and Platelet Fractionation, pp. 9-125 (1991 Wiley-Liss, Inc.).

Lastly, there are devices described that would not be appropriate either for the activation of psoralens or for other uses in blood products. US Patent No. 4,421,987, by Herold, describes an apparatus for irradiating dental objects that employs radiation in the spectrum range of 400 to 500 nanometers, for whitening treatments of teeth. The device is fitted with a selective reflector that reflects only the spectral fraction that falls in the desired spectral range (approximately 400 to 500 nm) of the total radiation emitted by the lamp while transmitting or passing the fraction of radiation that falls outside the desired spectral range. The device also has a temperature control system, employing the combination of a blower with an absorption filter that, like the reflector, removes radiation outside the desired spectral range. This apparatus does not fulfill the present purpose of a photodecontamination treatment, because it is designed for use with wavelengths of light that are harmful to some blood components, while eliminating wavelengths necessary to activate certain photoreactive compounds. Furthermore, it is not equipped with a temperature maintenance system that would keep the temperature of the blood samples low enough to prevent damage.

In sum, there is a need for a means of inactivating bacteria in blood components prior to storage and transfusion so as to lend itself to use in a closed system, such as a blood bag system. This approach must be capable of handling a large volume of blood and a variety of organisms while effectively controlling temperature and avoiding damage to the blood product or the recipient of the transfusion.

Summary of the invention

The present invention relates to a device and a method for the treatment of blood components in blood bags as defined in the claims. Specifically, the present invention contemplates a device for the photoactivation of new compounds and known compounds in a manner that binds and inactivates pathogens present in the blood. In accordance with the present invention, a nucleic acid binding compound is selectively employed to treat contamination by microorganisms.

In one embodiment, the present invention contemplates: a photoactivation device for the inactivation of pathogens in blood products, comprising:

a) a housing; b) means for providing electromagnetic radiation comprising a plurality of tubular bulbs; c) means for supporting a plurality of blood bags at a fixed distance from said radiation providing means; d) means for maintaining the temperature of blood products within a desired temperature range characterized in that an edge is wrapped around the ends of the tubular bulbs to obstruct these ends from the source of electromagnetic radiation from the blood bags of irradiation on the device. The device may comprise a bottom plate assembly transparent to ultraviolet light within said housing, on which said blood bags can rest; and an upper plate assembly transparent to ultraviolet light, positioned on top of said lower plate assembly, said upper and lower plate assemblies defining a channel, isolated from significant exchanges with air originating outside said housing during irradiation, in the which air can be circulated to cool these blood bags. In another embodiment, said lower plate assembly comprises a plate at the top and at the bottom and an air circulation chamber between said plates at the top and at the bottom, open to said channel to allow the air exchange between said air circulation chamber and said channel.

In another embodiment, the device further comprises: first means for maintaining the temperature, comprising: means for pumping air from outside, through said casing, between said means providing irradiation and said plate assemblies, positioned within and adjacent to said housing, to cool said means providing the irradiation; and second means for maintaining the temperature, placed inside said casing, for the circulation of cold air through said cold air circulation chamber through said air circulation chamber and said channel, comprising: an exchanger heat, between said plate assemblies, to remove heat from the air present in said housing, and; means for air circulation, placed in a fixed relationship to said heat exchanger. In one embodiment, said heat exchanger comprises a conduit having an inlet port and an outlet port so that the temperature control liquid can enter and exit. In one embodiment, said upper and lower plate assemblies are approximately 1 to 10 cm apart. However, it is preferred that when said

ES 2 201 093 T3 blood bags rest on said lower plate assembly, said upper plate assembly does not contact said blood bags.

Due to the benefits of rapid processing, in a contemplated embodiment said bottom plate assembly has sufficient dimensions to support six of said blood bags. Said blood bag support means may further comprise means for placing a plurality of accessories connected to said blood bags, such that said accessories do not significantly reduce the intensity of radiation on said blood bags, including tubes for transferring a blood product in or out. of said blood bags and blood product storage bags. The device may further comprise means for agitating said blood bag support means, which may comprise a plurality of detectors, positioned adjacent to said blood bag support means, for mixing a sample in a blood bag during irradiation. The present invention contemplates that said lower plate assembly has a ridged upper surface to maintain the position of said blood bags during shaking.

In a preferred embodiment, said casing comprises materials that obstruct said electromagnetic radiation so that users are protected from said electromagnetic radiation during said activation. A means for controlling said radiation providing means is also contemplated, which may comprise a plurality of detectors, positioned around said radiation providing means, to measure said electromagnetic radiation; and a feedback control, connected to said detectors, that cuts off said means providing radiation at a desired output of radiation detected by said detectors. Preferably, the intensity of the radiation provided by said radiation providing means is at least 15 mW / cm² , and said means that provide radiation have the maximum wavelength limit above 400 nanometers. Additionally, it is contemplated that the upper and lower plate assemblies are composed of a material that filters out said electromagnetic radiation to provide the minimum wavelength limit below 320 nanometers.

The radiation providing means may further comprise an upper bank and a lower bank of light sources, said upper bank is located above said upper plate assembly, and said lower bank is located below said lower plate assembly. Reflecting means, adjacent to said upper bank and said lower bank of light sources, are also contemplated to reflect electromagnetic radiation from said light sources towards said blood bag support means.

The present invention contemplates a photoactivation device comprising means for controlling said radiation providing means. The means for controlling said radiation providing means may comprise: a plurality of detectors positioned around said radiation providing means, for measuring said electromagnetic radiation; and a feedback control connected to said detectors, which cuts off said means providing radiation at a desired output of radiation detected by said detectors. In a preferred embodiment, the intensity of radiation provided by said radiation providing means is at least 15 mW / cm² and said means that provide radiation have the maximum wavelength limit above 400 nanometers.

In one embodiment, said plate assemblies are composed of a material that removes radiation of wavelengths that damage blood products from said electromagnetic radiation. Specifically, it is contemplated that said materials filter out said electromagnetic radiation to provide the minimum wavelength limit below 320 nanometers. It is also contemplated that said radiation providing means comprise an upper bank and a lower bank of light sources, said upper bank is located above said upper plate assembly, and said lower bank is located below said lower plate assembly. . The reflective means may be positioned adjacent to said upper bank and said lower bank of light sources, which reflect electromagnetic radiation from said light sources towards said blood bag support means.

The present invention also contemplates a process for the photoactivation of photoreactive compounds, comprising:

a) providing: i) a blood bag containing one or more photoreactive compounds and a blood component suspected of containing a pathogen and ii) a device according to any one of Claims 1 to 4; b) placing said blood bag in said device; and c) irradiating said blood bag with said source of electromagnetic radiation to activate at least one of said photoreactive compounds and inactivate said pathogen.

Preferably, the fluorescent source of electromagnetic radiation delivers an intensity of electromagnetic radiation greater than 1 mW / cm² to these blood bags.

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Description of the figures

Figure 1 is a perspective view of an embodiment of the device of the present invention in the close position.

Figure 2 is a cross-sectional view of the device shown in Figure 1, in the open position, from the beginning of 2-2.

Figure 3 is a cross-sectional view of the device shown in Figure 1 from the beginning of 3-3.

Figure 4 is a cross-sectional view of the device shown in Figure 1 from the beginning of 4-4.

Figure 5 schematically shows the decontamination approach of the present invention applied specifically to blood products.

Figure 6 is a graph showing photoactivation of 8-methoxypsoralen to nucleic acid.

Figure 7 is a graph showing the degradation of 8-methoxypsoralen (8-MOP) compared to 4'-aminomethyl-4,5 ', 8-trimethylpsoralen (AMT), as obtained by HPLC.

Description of the invention

The present invention relates to a device and a method for the treatment of blood components in blood bags as defined in the claims.

As previously observed, all blood is collected and usually separated into red blood cells, platelets, and plasma. Each of these fractions is stored individually under specific conditions prior to their use in vivo. In many cases, the degree of contamination is related to the storage time due to growth. A procedure that inactivates microorganisms at the time of blood collection would be expected to prevent growth during storage.

TABLE 1

<td>Photoreactive Compounds</td>

<td>Actinomycins</td>

<td>Anthracyclinones</td>

<td>Anthramycin</td>

<td>Benzodipironas</td>

<td>Fluorenes and fluorenones</td>

<td>Furocoumarins</td>

<td>Mitomycin</td>

<td>Monostral Rapid Blue</td>

<td>Norfilin A</td>

<td>Many organic dyes not specifically listed</td>

<td>Phenanthridines</td>

<td>Fenazationio salts</td>

<td>Phenazines</td>

<td>Phenothiazines</td>

ES 2 201 093 T3

TABLE 1 (continued)

Photoreactive Compounds

Phenylazides

Quinolines

Thiaxanthenones "Photoactivation compounds" (or "photoreactive compounds") define a family of compounds that undergo chemical changes in response to electromagnetic radiation (Table 1). One species of photoreactive compounds described herein is commonly referred to as the furocoumarins. Furocoumarins belong to two main categories: 1) psoralens [7H-furo (3,2-g) - (1) -benzopyran-7-one, or 6-hydroxy-5-benzofuranacrylic acid δ-lactone], which are linear;

<img file="ES2201093T3_D0001.tif" />

and in which the two oxygen residues added to the central aromatic fraction have a 1,3 orientation and also in which the fraction of the furan ring is attached to the 6 position of the two rings of the coumarin system, and 2) the isopsoralens [ 2H-furo (2,3-h) - (1) -benzopyran-2-one, or 4-hydroxy-5-benzofuranacrylic acid δ-lactone], which are angular;

<img file="ES2201093T3_D0002.tif" />

in which the two oxygen residues added to the central aromatic fraction have a 1,3 orientation and also in which the fraction of the furan ring is attached to the 8 position of the two rings of the coumarin system. The derivatives of the psoralens are derived from the substitution of linear furocoumarin in the positions 3, 4, 5, 8, 4 ', or 5', while the derivatives of isopsoralens are derived from the substitution of the angular furocoumarin in the positions 3, 4, 5, 6, 4 'or 5'.

In one embodiment, the present invention contemplates the inactivation of blood products after separation but before storage. In this embodiment, a nucleic acid binding compound is selectively employed to treat contamination by microorganisms.

In one embodiment, the nucleic acid binding compound is selected from the group consisting of furocoumarins. In a preferred embodiment, the furocoumarin is a psoralen or an isopsoralen.

The inactivation method of the present invention provides a method of inactivation of unicellular and multicellular organisms, and in particular, bacteria, fungi, mycoplasmas and protozoa. In contrast to previous approaches, the method of the present invention does not harm blood products. There is no significant damage to the cells and therefore it is not necessary to limit the concentration of molecular oxygen.

The present invention contemplates the use of much lower concentrations of nucleic acid binding compounds than previously employed. For example, the present invention contemplates the use of 8-MOP at concentrations of 30 µg / ml or less. In fact, a preferred concentration of 8-mOp for bacterial decontamination in platelet concentrates is 3 µg / ml or less, for example a concentration one hundred times less than that employed by GP Wiesehahn et al., Supra.

The present invention also contemplates the use of lower irradiation doses than those previously described. This is accomplished with lower intensity irradiation sources, with filters that limit wavelengths (see below), and / or shorter irradiation periods. In a preferred embodiment, the irradiation time is variable and is controlled from 1 second to 99 minutes, in one second increments.

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While the present invention is not intended to be limited by the inactivation theory, the use of lower compound concentrations and irradiation doses is understood to be where the present invention applies to the decontamination of a unicellular or multicellular organism (on the contrary than a virus), a lower level of nucleic acid binding will achieve inactivation. Furthermore, it is recognized that it is not essential that the inactivation be complete. That is, partial inactivation will be adequate as long as the viable portion is unable, within the storage period, to grow to levels sufficient to cause disease.

To appreciate that, in any given case, an inactivation procedure may or may not achieve complete inactivation, it is useful to consider a specific example. A bacterial culture is said to be sterilized if an aliquot of the culture, when transferred to a fresh culture plate and allowed to grow, is undetectable after a period of time. Time period and growth conditions (eg temperature) define an "amplification factor". This amplification factor together with the limitations of the detection procedure (eg, visual inspection of the culture plate for the appearance of a bacterial colony) defines the sensitivity of the inactivation procedure. A minimum number of viable bacteria must be applied to the plate as a signal to be detectable. With the optimal detection procedure, this minimum number is 1 bacterial cell. With a suboptimal detection procedure, the minimum number of bacterial cells applied such that a signal is observed can be much greater than 1. The detection procedure determines a "threshold" below which the procedure appears to be completely effective (and therefore above which the procedure is, in fact, only partially effective).

This interaction between the amplification factor of an assay and the threshold that defines the detection procedure can be illustrated. For example, bacterial cells can be applied to a plate: the arbitrarily chosen detection procedure is visual inspection. Assume growth conditions and time are such that a total amplification of 10⁴ . The detectable signal will be proportional to the number of bacterial cells present at that time after amplification. For calculation purposes, a detection threshold of 10 is taken⁶ cells, if fewer than 10 are present⁶ cells after amplification, there are no visually detectable cell colonies and the inactivation procedure will be effective. Given the amplification factor of 10⁴ and the detection threshold of 10⁶ , the limit sensitivity would be 100 bacterial cells; if fewer than 100 viable bacterial cells were present in the original aliquot of the bacterial culture after the sterilization procedure is performed, the culture would still appear sterile.

A situation like this is common for bacterial growth assays. The sensitivity of the assay is such that viable bacterial cells are present but the assay is unable to detect them. This may explain, at least in part, the variability of the results obtained by researchers in their attempt to determine the degree of bacterial contamination in blood products. See DH Buchholz et al., Transfusion 13; 268 (1973), in which such variability is discussed.

It should be noted that, in many countries, contamination of blood products by cellular organisms is more persistent and therefore more serious than viral contamination. For example, in South America, the most important organism present in the blood is T. cruzi, which is the etiological agent of Chagas disease. Approximately 16-18 million people are infected in the Americas (including 11% of Chile's population). It is contemplated that the decontamination process of the present invention is well suited for the inactivation of these protozoa.

The present invention contemplates devices and methods for photoactivation and specifically, for the activation of photoreactive nucleic acid binding compounds. The present invention contemplates devices that have a cheap source of electromagnetic radiation that is built into the unit.

The present invention contemplates devices and methods for photoactivation and specifically, for the inactivation of pathogens that contaminate blood products by activating photoreactive compounds. The main characteristics of an embodiment of the device of the present invention involves: A) a cheap source of ultraviolet radiation at a fixed distance from the medium that supports the sample vessels, B) rapid photoactivation, C) processing of large numbers of samples , D) temperature control of irradiated samples, E) inherent safety and F) sample containers.

A. Source of electromagnetic radiation

A preferred photoactivation device of the present invention has an inexpensive source of ultraviolet radiation at a fixed distance from the medium that supports the sample vessels. Ultraviolet radiation is a form of energy that occupies a portion of the electromagnetic radiation spectrum (the spectrum of electromagnetic radiation ranges from cosmic rays to radio waves). Ultraviolet radiation can come from many natural and man-made sources. Depending on the source of ultraviolet radiation, it may be accompanied by other (non-ultraviolet) types of electromagnetic radiation (for example, visible light).

Here particular types of ultraviolet radiation are described in terms of wavelength. Wavelength is described here in terms of nanometers ("nm"; 10^-9 meters). For purposes herein, ultraviolet radiation ranges from about 180 nm to 400 nm. When a radiation source, by virtue of filters or other means, does not pass radiation of wavelength shorter than a length of

ES 2 201 093 T3 particular wave (eg 320 nm) is said to have a "limit" lower than that wavelength (eg "a short wavelength with a limit of 320 nanometers"). Similarly, when a radiation source passes only radiation with wavelengths longer than a particular wavelength (eg 360 nm), it is said to have a "limit" higher than that wavelength (eg , "A long wavelength with a limit of 360 nanometers").

For any photochemical reaction it is desired to eliminate or at 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 process. In a system where only nucleic acid and psoralen are present, endogenous chromophores are the nucleic acid bases themselves. Restricting the activation procedure to wavelengths greater than 320 nm minimizes direct damage to nucleic acids since there is very little absorption by nucleic acids at wavelengths longer than 313 nm.

In blood products, nucleic acid is usually present together with additional biological chromophores. If the biological fluid is only protein, the short wavelength limit at 320 nm will be adequate to minimize side reactions (aromatic amino acids do not absorb at wavelengths shorter than 320 nm). If the biological fluid includes cells and / or cell constituents, there will be many other chromophores including heme groups and flavins.

Heme groups are abundant in blood products where they arise from the lysis of red blood cells. Flavins, like heme groups, are required for metabolic respiration. Both of these endogenous chromophores will cause damage to cells if excited by photoradiation.

Heme groups have three main absorption bands: two are in the red region of the visible spectrum; the other is centered at approximately 400 nm. Flavins have two main absorption peaks: one at 450 nm and the other at 370 nm.

In view of the presence of these endogenous chromophores in blood products, it is envisaged that in an embodiment of the device of the present invention the device is designed to take into account irradiation within a small range of specific and desirable wavelengths. , and thus avoid damage to cells caused by energy transfer. The preferred range of desirable wavelengths is between 320 and 350 nm.

Some selectivity can be achieved by choosing commercial irradiation sources. For example, while typical fluorescent tubes emit wavelengths ranging from 300 nm to above 400 nm (with a broad peak centered around 360 nm), BLB-type fluorescent lamps are designed to eliminate wavelengths. longer than 400 nm. This, however, only provides a long wavelength limit.

In a preferred embodiment, the device of the present invention comprises an additional filtration medium. In one embodiment, the filter medium comprises a glass boundary filter, such as a piece of cobalt glass. In another embodiment, the filter medium comprises a liquid filter solution that transmits only a specific region of the electromagnetic spectrum, such as an aqueous solution of Co (NO3) 2. This saline solution provides a transmission window of 320-400 nm. In a preferred embodiment, the aqueous Co (NO3) 2 solution is used together with NiSO4 to eliminate the 365 nm component of the emission spectrum of the fluorescent or arc source used. The Co-Ni solution preserves its initial transmission remarkably well even after ten hours of exposure to direct light from high energy sources.

The present invention is not intended to be limited by the particular filter employed. Numerous inorganic salts and crystals satisfy the necessary requirements. For example, cupric sulfate is a more useful general filter for removing infrared, when only ultraviolet is isolated. Offers stability in strong sources. Other salts are known to someone skilled in the art. Aperture or reflector lamps can also be used to achieve specific wavelengths and intensities.

When ultraviolet radiation is described here in terms of irradiance, it is expressed in terms of flux intensity (milliwatts per square centimeter or “mW / cm²”). "Output" is defined here to encompass both the radiation emission (yes or no; on or off) as well as the irradiance level. In a preferred embodiment, intensity is monitored at at least 4 locations; with at least 2 for each side of the irradiation plane. In one embodiment, the monitors are photodiodes, each positioned to measure the output of one or more radiation sources.

A preferred source of ultraviolet radiation is a fluorescent source. Fluorescence is a special case of luminescence. Luminescence involves the absorption by a substance of electromagnetic radiation and the conversion of the energy into radiation of a different wavelength. With fluorescence, the substance that is excited by electromagnetic radiation returns to its ground state by emission of a quantum of electromagnetic radiation. While it was heretofore thought that fluorescence sources had to be of very low intensity to be useful for photoactivation, in one embodiment of the present invention fluorescent sources are employed to achieve hitherto achievable results only with expensive equipment.

ES 2 201 093 T3

As used herein, "fixed distance" is defined as a constant distance between a point in the plane defining the medium that supports a plurality of blood bags and a point within the light source. Thus, small changes in distance from the source can have a drastic impact on intensity. Since changes in intensity can affect the results of photoactivation, the present invention contemplates the use of an extensible lamp bar for a radiation source. Extendable bar lamps minimize the effect of small changes in distance on radiation intensity, providing reproducibility and repeatability.

The geometry is related to the position of the light source. For example, you can imagine that the light sources could be placed around the specimen holder in many ways (to the sides, to the bottom, in circles, etc.) The geometry used in a preferred embodiment of the present invention allows uniform light exposure, from more than one sample, of appropriate intensity for rapid photoactivation. The geometry of a preferred device of the present invention involves multiple linear lamp sources compared to point light sources. In addition, there are numerous reflective surfaces and numerous absorbent surfaces. Reflective surfaces can help equalize the light exposure to each plurality of samples. Due to this complicated geometry, changes in the position or number of lamps relative to the position of the samples to be irradiated should be avoided in that such changes will result in changes in intensity and variability in exposure to intensity. multiple samples.

Another consideration in obtaining uniform light exposure is provision for accessories to the sample containers during irradiation. The present invention contemplates accessories such as tubes, valves, blood product storage bags, and any other apparatus commonly attached to bags containing blood products. This avoids obstruction of the light by accessories. In one embodiment, the means supporting the blood bags has means for locating a plurality of accessories connected to said blood bags, such that said accessories do not significantly reduce the intensity of radiation to said blood bags.

It is useful for an irradiation device to deliver the same intensity of radiation to the sample whether there are numerous samples or a single sample is being irradiated. The present invention contemplates the use of parabolic reflective meshes that can be placed between the light sources and the sample to be irradiated. These meshes direct the light that passes through them, to reduce light scattering and avoid dips in light that affects samples when more than one sample is irradiated at the same time.

In another embodiment, the present invention contemplates the use of agitation means, such as a shaker or shaker, to mix the samples during irradiation. This mixing can have an effect that averages the radiation received by the sample material in different parts of the bag. Without being limited to any mechanism by which agitation causes predictability of irradiation, it is contemplated that the sample material is moved through the bag during irradiation by agitation, in such a way as to expose every part of the sample. shown at many different positions to receive radiation. If there are variations in the intensity of radiation in different areas of the bag, the movement would act to reduce the variation in intensity within the sample.

The present invention further contemplates that the delivery of light from the light sources will be approximately uniform along the length of the light source. Some light sources, particularly long tubular bulbs, show a dropout at the ends of the bulbs. The ends also tend to give off the most heat. To ensure uniform illumination and a controlled temperature, the device of the present invention has an edge that wraps around the ends of the light sources to obstruct approximately 2-6 cm of the light source at each end of the irradiation of samples on the device.

B. Rapid photoactivation

The light source of the preferred embodiment of the present invention allows rapid photoactivation. The intensity characteristics of the irradiation device have been selected to be convenient in anticipation that many multi-sample systems may be required to be processed. With this anticipation, an exposure time of fifteen minutes or less is a practical goal. Because ultraviolet light sources can vary their flux over a set amount of time, in a preferred embodiment of the present invention, numerous light output detectors are located throughout the device to measure the output of the light sources light. In one embodiment, the detectors are connected to a feedback control, which can be adjusted to turn off the light source when a certain output level has been reached. This ensures repeatability, which is preferable when pathogens are inactivated in blood products. By controlling the light exposure a blood product receives, one can also ensure sufficient exposure to inactivate pathogens, without having to expose the sample to excess light, which could be harmful.

In the design of the devices of the present invention, the relative positions of the elements of the preferred device have been optimized to allow a fifteen minute irradiation time, so that it is provided, when measuring the wavelength between 320 and 350 nanometers, a flux intensity greater than about 1 mWcm^-2, and preferably 15 mWcm^-2 to the sample beakers. In a preferred embodiment, the device irradiates both sides of the bag.

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C. Processing a large number of samples

As can be seen, another important characteristic of the photoactivation devices of the present invention is that they are intended for the processing of a large number of samples. In this regard, an element of the devices of the present invention is a medium that supports a plurality of blood products, and in particular, blood bags. In the preferred embodiment of the present invention the support means comprise glass plates between two banks of lights with a capacity of six 50 ml bags (equivalent to a Dupont Stericell ™ bag) plus connectors and tubes, at one time. Accepting the common use of commercially available blood bags, the device of the present invention allows the proper processing of a large number of samples.

In a preferred embodiment, the plate has a means for placing accessories connected to said blood bags, such as tubes and satellite storage bags, such that said accessories do not significantly reduce the intensity of radiation to said blood bags.

D. Temperature control

As can be seen, one of the important characteristics of the photoactivation devices of the present invention is the temperature control. Temperature control is important because the temperature of the sample during the time of exposure to light can dramatically affect the results. For example, conditions that promote nucleic acid secondary structure also increase the affinity constants of many psoralen derivatives for nucleic acids. Hyde and Hearst, Biochemistry, 17, 1,251 (1978). These conditions are a mixture of both the solvent composition and the temperature. With single stranded 5S ribosomal RNA, low temperature irradiation increases the covalent addition of HMT to 5S rRNA by 2 times at 4 ° C compared to 20 ° C. Thompson et al., J. Mol. Biol. 147; 417 (1981). Even higher temperature has been reported to induce increased binding of psoralens to synthetic polynucleotides. Thompson et al., Biochemistry 21; 1,363 (1982).

With regard to bacteria, it should be noted that repair of the crossovers occurs during irradiation. However, where a lower temperature is used during irradiation, the bacterial repair process is suppressed. Thus, irradiation at 15 ° C has a significant effect on the level of inactivation that is observed.

Additionally, certain blood preparations can be damaged by small changes in temperature. For example, platelets are better preserved if they are kept at 22 ± 2 ° C. Thus it is preferred that a device for the photoactivation of platelets keeps platelets within or near this range during radiation or the clinical efficacy of platelets can be reduce.

Without being limited to any particular means of controlling the temperature of blood products during irradiation in the device, in one embodiment the device employs two means of controlling the temperature. A first temperature control means is a means for pumping air from outside the housing of the device, through the means that provides the irradiation and back out, to cool them and prevent heat transfer to the irradiated samples. .

A second temperature control means operates in a closed system, cooling and circulating the air within the system only, to avoid recycling of the heat introduced by the air escaping from the first temperature control means. The second means is for the cool air to circulate through the air circulation chamber and into the channel on which the blood bags rest. This second temperature control means uses a heat exchanger and a means to circulate the cold air. Preferably, the heat exchanger is a conduit, having an inlet port and an outlet port for the circulation of the temperature control liquid. The duct can be covered with a corrugated material with high thermal conductivity, which serves to increase the heat exchange surface between the duct and the air. In one embodiment, the means for circulating the air is powered by a direct current motor, which produces less heat than an alternating current motor. Alternatively, the means for circulating the air can be propelled from outside the housing. Either alternative controls the amount of heat produced within the shell.

Cold air is circulated through the duct, through the blood bags that contain the blood products, and through the chambers and channels that surround the blood bags. The chambers and channels are also isolated from significant exchange with air originating outside the device housing or from air that passes through the means that provide electromagnetic radiation, in such a way as to create a "closed system" that recirculates the air. In an alternative embodiment, the present invention contemplates that the second temperature control means comprises a refrigeration unit installed within the housing of the device for photoactivation.

The circulating air in the second temperature control means does not mix with the air pumped through the first temperature control means. This separation from temperature control allows the samples to cool properly.

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In one embodiment, the device of the present invention comprises a stirring means, such as a shaker or a shaker, giving the samples a sinusoidal and unidirectional horizontal movement of variable frequency and amplitude. The use of a means of agitation during irradiation with the device is contemplated for maintaining a uniform temperature in all samples within the blood bags by mixing a sample in the blood bags during irradiation. Additionally, the use of a shaker for platelet samples reduces platelet activation during storage. In one embodiment, a shaker is placed within the casing of the irradiation device, moving the samples by direct contact of the means supporting the blood bags. It is contemplated that the top plate of the bottom plate assembly cannot be fixed relative to the rest of the bottom plate assembly, thus allowing a shaker to contact the top plate directly to achieve agitation. Movement of the entire bottom plate assembly by a shaker is also contemplated. Alternatively a shaker can be placed outside the housing, moving the samples by movement of the entire device housing. The present invention contemplates the use of a roughened surface on the blood bag supporting means that provides sufficient friction to maintain the position of the sample blood bags during agitation.

In another embodiment, the heat from the lamps, ballasts, and other sources is kept away from the blood bags by one or more partitions between the various heat sources and the bags. This also helps in maintaining a biologically acceptable temperature in the samples.

E. Inherent security

Ultraviolet radiation can cause severe burns. Depending on the nature of the exposure, it can also be carcinogenic. The light source of a preferred embodiment of the present invention is shielded from the user. This is in contrast to commercial handheld UV sources as well as large, high intensity sources. In a preferred embodiment the irradiation source is contained within a housing made of material that obstructs the transmission of radiant energy (eg, an opaque housing). No radiation is allowed to pass to the user. This allows inherent security for the user.

F. Sample containers

The material of the container that houses the sample that is irradiated in the irradiation device can interfere with the proper functioning of the irradiation device. The material used can cause radiation penetration of the sample and the amount of radiation scattering affecting the container. The sample container of one embodiment of the present invention is a blood bag made of an ultraviolet light transparent plastic, preferably Teflon (available from American Fluroseal, Silver Spring, MD). Some other acceptable plastic components are ethyl vinyl acetate (bags available from Terumo, Japan); polyvinyl chloride (PVC) (bags available from Baxter Travenol or Cutter, Covina, CA), which can be combined with plasticizers; or polyolefins (bags available from the Fenwal Division of Baxter Travenol Laboratories, Inc., Dearfield, Illinois). For PVC, contemplated plasticizers are di (2-ethylhexyl) phthalate (DEHP), tri (2-ethylhexyl) trimellitate (TEHTM). The present invention, however, is not intended to be limited to any blood bag composition, but contemplates the use of any bag that is somewhat transparent to ultraviolet light. The material can also interfere with the concentration of the components in the samples to be irradiated. In a preferred embodiment, the sample is irradiated on the irradiation device in a bag that does not bind a significant percentage of photoreactive compound contained in the sample.

The container parameters also control to a certain degree how the sample is affected during irradiation. For example, a blood bag for platelet storage better preserves platelets if their walls are thin enough to allow sufficient oxygen transfer to prevent an increase in the rate of lactate production that results in a decrease in platelet viability. . Carmen, R., "The Selection of Plastic Materials for Blood Bags", Transfusion Med. Rev. 7; 1 (1993).

The nature of the blood products can have an impact on efficacy. Red blood cells, for example, absorb different wavelengths of light than platelets do. Red blood cells can reduce the effectiveness of irradiation due to light obstruction. Therefore, platelets contaminated with red blood cells may receive a lower intensity of light than platelet preparations that do not contain red blood cells.

As noted above, changes in intensity can affect the results of photoactivation, and these changes can result from changes in the distance within the sample through which radiation must travel. The thickness of the sample, defined by the walls of the blood bag, and the volume of the blood product, also affects how much light the sample can reach. In a preferred embodiment of the present invention, when the blood bags containing the sample for irradiation are supported within the radiation device, a film of blood product is formed having a "central path length" of between about 0 , 1 and 4 cm. A "central path length" is defined herein as the shortest distance between two walls of a blood bag that passes through the center of the bag. In one embodiment, the "center path length" of the sample is a fixed value for all bags used. This confers playability and repeatability. In a preferred embodiment, a shaker is employed to provide movement, or agitation, of the sample material so that each part of the sample reaches the surface of the blood bag during irradiation. This can allow variations in the central path lengths of the bags, while

ES 2 201 093 T3 that preserves the reproducibility and repeatability, because the agitation can circulate the sample to the surface of the blood bag. Thus, it is ensured that sufficient light can reach the samples regardless of the central path length. In another preferred embodiment, no pressure is required to be exerted on the bag by the radiation device, or any other source, other than the force of gravity, to obtain the preferred "center path length". In one embodiment, the upper and lower plate assemblies are spaced approximately 1 to 10 cm apart, to accommodate bags that have a central path length within that range.

Experimental

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope of these.

In the experimental description that follows, the following abbreviations apply: eq (equivalents); M (Molar); µM (micromolar); N (Normal); mole (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); µm (microns); nm (nanometers); ° C (degrees Centigrade); HPLC (High Pressure Liquid Chromatography).

Example 1

As noted above, the present invention contemplates devices and methods for the activation of photoreactive nucleic acid binding compounds. In this example, a photoactivation device for decontaminating blood products according to the method of the present invention is described. This device comprises: a) means for providing suitable wavelengths of electromagnetic radiation that causes the activation of at least one photoreactive compound; b) means for supporting a plurality of blood products at a fixed distance from the means providing the radiation during activation; and c) means for maintaining the temperature of the blood products within a desired temperature range during activation.

Figure 1 is a perspective view of an embodiment of the device that integrates the aforementioned characteristics. The figure shows an opaque housing (100) with a portion of it removed, containing a series of bulbs (101) above and below a plurality of representative blood product-containing media (102) positioned between plate assemblies (103). , 104). The plate assemblies (103, 104) are described in more detail below.

The bulbs (101), which are connectable to a power source (not shown), serve as a source of electromagnetic radiation. While not limited to the particular bulb type, the embodiment is configured to accept an industry standard, the dual two-pin lamp.

The housing (100) can be opened by a closure (105) so that the blood product can be properly placed. As shown in Figure 1, the housing (100), when closed, completely contains the irradiation of the bulbs (101). During irradiation, the user can confirm that the device is working by looking through a safe viewing port (106) that does not allow the transmission of ultraviolet light to the user.

The housing (100) also serves as a mount for numerous electronic components in a control panel (107), including, by way of example, a main current switch, a timer, and an hour meter. For convenience, the current switch can be connected to the timer which is instead connected in parallel to an hour meter and a source of electromagnetic radiation. The timer allows a user to preset the irradiation time to a desired exposure level. The hour meter keeps a record of the total number of hours of radiation provided by the source of electromagnetic radiation. This feature allows the bulbs (101) to be monitored and changed before their output drops below a minimum level necessary for rapid photoactivation.

Figure 2 is a cross-sectional view of the device shown in Figure 1 from the beginning of 2-2. Figure 2 shows the organization of the bulbs (101) with the housing (100) open. A reflective medium (108A, 108B) completely surrounds each series of bulbs (101). The means containing the blood products (102) is located between the upper (103) and lower (104) ultraviolet transparent plate assemblies. When the upper plate assembly (103) is lowered onto the lower plate assembly (104), the upper (103) and lower plate assemblies (104) define a channel (116 - not shown in this figure) through which air can be circulated to cool media containing blood products. Each plate assembly is composed of plates at the top (103A, 104A) and at the bottom (103B, 104B). The plate assemblies (103, 104) are connected by a hinge (109) that is designed to accommodate the space created by the media containing the blood products (102). The top plate assembly (103) is brought to rest just above the top of the blood product containing media (102) held by the bottom plate (104B) of the bottom plate assembly (104). In an alternate embodiment, the top plate assembly (103) may be in a fixed relationship with the housing (100) and the entire housing cover, including the top plate assembly (103) that can be brought to bear. rest just above the top of the media containing the blood products (102).

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The detectors (110A, 110B, 110C, 110D) can be conveniently located between the plates (103A, 103B, 104A, 104B) of the plate assemblies (103, 104). They can be connected to a printed circuit board (111) which is instead connected to the control panel (107).

Figure 3 is a cross-sectional view of the device shown in Figure 1 from the beginning of 33. Six media containing blood products (102) (eg, bags of Teflon ™ platelet units) are placed in a fixed relationship above a series of bulbs (101). The temperature of the blood products can be controlled by a fan (112) alone or, more preferably, by employing a heat exchanger (113) having a cooling inlet (114) and outlet ports (115) connected to a power source. cooling (not shown).

Figure 4 is a cross-sectional view of the device shown in Figure 1 from the beginning of 4-4. Figure 4 more clearly shows the temperature control approach of a preferred embodiment of the device. When the top plate assembly (103) is lowered onto the bottom plate assembly (104), the top (103) and bottom (104) plate assemblies define a channel (116) bordered by the bottom plate ( 103B) of the upper assembly (103) and the upper part plate (104A) of the lower assembly. The upper plate assembly plates (103A, 103B) and the lower plate assembly plates (104A, 104B) each define an air circulation chamber (103C, 104C), respectively. The fan (112) can circulate air within the chambers (103C, 104C). When the heat exchanger (113) is used, the circulating air is cooled and passed between the plates (103A, 103B, 104A, 104B) inside the air circulation chambers (103C, 104C), by the fan (112) and it is brought back to the heat exchanger (113) through the channel (116) between the upper (103) and lower (104) plate assemblies, thereby cooling the media containing the blood products. The circulating air is kept within a closed system when the housing (100) is in the closed position, comprising the channel (116), the air circulation chambers (103C, 104C) and the surface of the heat exchanger (113) and the fan (112). Air within the closed system does not mix or exchange with air outside the housing or inside the housing that is not part of the closed system, such as the area surrounding the bulbs (101).

Example 2

Figure 5 shows an embodiment in which platelets are treated by the method of the present invention. Following fractionation, platelets are transferred to a bag containing a nucleic acid binding compound (shown in Figure 1 as a shaded bag). This bag, which has transmission properties and other characteristics suitable for the present invention, is then placed in an irradiation device (as described in Example 1, above) and irradiated. The free compound can be collected or "captured" as desired by a capture device. In such a case, the bag would contain only the compound that is contained in the cells; the bag would have no free compound (this bag is indicated in Figure 1 as unshaded).

Example 3

In this example, the decontamination procedures of the present invention are applied to inactivate Yersinia enterocolitica, wild type, serotype 3, biotype 4. This organism is found in blood products. See generally RY Dodd, In: Transfusion Medicine in the 1990's (American Assoc. Blood Banks 1990) (SJ Nance, ed.). See also BJ Grossman et al., Transfusion 31; 500 (1991).

The organism was cultured overnight by inoculating 10 ml of Brain Heart Infusion Broth (BHI) from a motility puncture. This was kept at 35 ° C and 0.1 ml of this was used to inoculate 20 ml of BHI broth for use in the experiment. After incubating overnight at 35 ° C, the stationary culture was centrifuged for 15 minutes at 1,900 g, the supernatant was discarded, and the bacterial pellet was resuspended in a 1 ml pool of heat-inactivated normal serum. This was infused into a unit of dead human platelets recently obtained from the Blood Bank of Alameda-Contra Costa Medical Association. Aliquots of 5 ml of bacteria containing platelet concentrate were removed from the bag and received specific amounts of 8-MOP and UVA irradiation, except for controls, who were irradiated without psoralens, or received no treatment (see Table 2). The temperature was maintained at 25 ° C during irradiation by placing the platelet concentrate in water-lined chambers with stoppered glass attached to a circulating water bath. The irradiation device (Derma Control, Dolton; Ill .; Model No. 1,224-Special) employed two series (six lamps / series separated 6.35 cm (2.5 inches), one series above the sample and one bench below the sample (the sample is thus about 7.62 cm (3 inches) from the lamps)). Each series is separated from the others by approximately six inches, has a polished metal reflector behind it, and is covered by a sheet of acrylic plastic that transmits UVA rays. The sample to be processed (eg bag of platelets) sits on the bottom sheet.

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TABLE 2

<td></td><td>Drug</td><td>8-MOP / ml</td><td>Ir Time (min)</td><td>Log / ml</td><td>Qualification</td>

<td> 1</td><td>Not</td><td></td><td> 0</td><td> 9,1</td><td></td>

<td> 2</td><td>Not</td><td></td><td> 10</td><td> 9,3</td><td> 0,2</td>

<td> 3</td><td>8-MOP</td><td>30 μg</td><td> 10</td><td> &lt; 0</td><td> &gt; -9,1</td>

<td> 4</td><td>8-MOP</td><td> 10</td><td> 10</td><td> &lt; 0</td><td> &gt; -9,1</td>

<td> 5</td><td>8-MOP</td><td> 3</td><td> 10</td><td> 3,4</td><td> -5,7</td>

<td> 6</td><td>8-MOP</td><td> 0,2</td><td> 10</td><td> 6,8</td><td> -2,3</td>

<td> 7</td><td>8-MOP</td><td> 0,06</td><td> 10</td><td> 9,0</td><td> -0,1</td>

Derma Control F587T12-BL-HO type bulbs were used. These are "black light" tubes (designed to emit specific wavelengths through an inner layer of phosphor) that are 24 inches long. The maximum wavelength is below 360 nm, unlike simple mercury lamps or common “BLB” fluorescent bulbs. Total intensity is less than 20mW / cm².

Bacteria were quantified by plating 0.1 ml of 10-fold serial dilutions in BHI broth in 100 mm petri dishes containing BHI agar. After 24 hours of incubation at 35 ° C, the colonies were counted and the bacterial concentration per ml was calculated. The results (Table 2) show that as little as 3 µg / ml of 8-MOP is capable of inactivating almost six bacterial logs. At 10 μg / ml, ten minutes provides more than sufficient irradiation. In fact, at 10 μg / ml, five minutes of irradiation appears to be adequate.

Example 4

Artuc et al. Examined the solubility of 8-MOP in human and bovine serum proteins, and showed that at concentration ranges of 8-MOP from 100 to 1000 ng / ml concentrations similar to these were observed in patients treated with ultraviolet A psoralen therapy ( PUVA) for psoriasis. 75% to 80% was bound to albumin. M. Artuc et al., Brit. J. Derm. 101; 659 (1979).

In this example, the binding of 8-MOP to calf thymus DNA is compared using plasma and protein-free medium in order to validate the efficacy of psoralen-nucleic acid interactions under the decontamination procedures of the present invention. Although this measurement used eukaryotic nucleic acid rather than bacterial nucleic acid, it is a useful indicator of the degree of adduct formation for the bacteria.

Was prepared ³H-8-MOP at a concentration of 115 μg / ml in ethanol at a specific activity of 4.7x10⁶ CPM / microgram (hereinafter "stock 8-MOP"). After that, 130.5 or 22 μl of 8-MOP stock (2 each) were exhausted for samples containing DNA (“DNA +”) and 52.2 or 8.7 μl for samples that did not contain DNA (“DNA - ”). To the DNA + samples, 40 µl of DNA stock (7.7 mg / ml) was added as well as either 460 µl of plasma (frozen of the day) or 450 µl of Tris-EDTA buffer ("TE"). Finally, 10 µl of 5m NaCl was also added. For DNA samples (eg controls), 184 µl of plasma and 16 µl of water were added.

The samples were gently vortexed for approximately one hour and counts were counted to confirm that the 8-MOP had dissolved.

Each sample (100 μ ^ was irradiated on a HRI-100 (HRI Research Inc., Concord, CA) at 25 ° C for 0, 2, 4, 6, 8, 7 16 minutes. Samples were kept at 4 ° C overnight after irradiation. After that the samples were extracted. First, a phenol solution was prepared at pH 8 equilibrating with 0.1 M Tris pH 8. Each sample was then extracted with 100 µl phenol. Each sample it was centrifuged for 5 minutes to remove the aqueous phase to a new tube. A second extraction was carried out with 100 μl of phenol: chloroform 1: 1. A final extraction was carried out with 100 µl of chloroform.

The final aqueous phase was precipitated by adding 50 µl of NaCl adjusted to give a final NaCl concentration of 0.2 M and then adding 250 µl of ethanol. The samples were centrifuged again (10 minutes). The supernatant was removed and the pellets were dried. The pellets were resuspended in 100 µl TE and re-precipitated. This was repeated for a total of 3 precipitations. The final sediments were dissolved in 600 µl of water and 100 µί were counted. Each sample was tested for DNA by measuring absorbance (260 nm). 8-MOP levels were represented as adducts per 1000 base pairs ("8-MOP: kBP").

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The results (Figure 6) show that plasma significantly changes the addition kinetics of 8-MOP to DNA. Nucleic acid addition is much better in a protein-free medium.

The frequency of formation of the 8-MOP-DNA adduct in protein-free media predicts a high multiplicity of modification of the bacterial genome. Furthermore, this type of biochemical measurement has the potential to provide a means of monitoring the effectiveness of the photochemical inactivation procedure.

Example 5

Photoactivation of psoralens and isopsoralens can result in a variety of photo products. "Photoproducts" is best understood by considering the possible reactions of photoreactive compounds when exposed to wavelengths of activating electromagnetic radiation. While not limited to any precise mechanism, it is believed that the reaction of photoreactive compounds in their ground state ("C") with activating electromagnetic radiation wavelengths creates a short lived excited species "C *":

C -> C *

What happens next is largely a function of what potential reagents are available to the excited species. Since it is short lived, it is believed that a reaction of these species with nucleic acid ("NA") is only possible if the nucleic acid is present at the time the excited species are generated. Thus, the reaction must, in operational terms, be in the presence of activating electromagnetic radiation wavelengths, for example it is "photobinding"; It is not a dark union. The reaction can be represented as follows:

C * + NA -> NA: C

The product of this reaction will hereinafter be referred to as "Photoaddition Product" and is to be distinguished from "Photoproduct".

With this reaction described, one can now consider the situation where nucleic acid is not available to bind at the time the compound is exposed to activating electromagnetic radiation wavelengths. Since excited species are short-lived and have no nucleic acid to react with, excited species can simply revert to ground state:

C * -> C

On the other hand, excited species can react with themselves (eg, a ground state or excited species) to create a ground state complex ("C: C"). The product of these self-reactions where two compounds react is referred to as a "photodimer" or simply a "dimer". Self-reactions, however, are not limited to two compounds: a variety of multidimers (trimers, etc.) can be formed.

Excited species are not limited to reacting with themselves, they can react with their environment, such as solvent elements ("E") (for example, ions, gases, etc.) to produce other products:

C * + E -> E: C

This is the type of reaction that is believed to cause cell damage (for example, reacting with oxygen to create singlet oxygen species). Furthermore, it can simply be internally rearranged ("isomerized") to a derivative of the ground state ("["):

C * -> [

Finally, excited species may undergo reactions other than those described here.

The present invention and the understanding of "photo products" are not dependent on which (if any) of these reactions actually occurs. "Photoproducts" - whatever their nature - are considered to exist if, following the reaction of a compound and activating wavelengths of an activating electromagnetic radiation, there is a resulting product formed that can interact with other components of the reaction environment .

With psoralens such as 4'-hydroxymethyl-4,5 ', 8-trimethylpsoralen (HMT), there are a number of resulting products produced when HMT is exposed to activating electromagnetic radiation wavelengths. The main products resulting from HMT are two cyclobutyl photodimers. In one of the dimers, the two pyrone rings

ES 2 201 093 T3 are linked in a cis-sin configuration, while in the other dimer, the union occurs between the furan end of one molecule and the pyrone end of the other, again in a cis-sin configuration. A third product resulting from HMT is a monomeric HMT photoisomer. In this isomer, the oxygens in the central ring assume a 1.4 orientation instead of the normal 1.3 orientation. While the two photodimers would not be expected to have intercalating activity due to geometric considerations, the photoisomer remains flat, and consequently is contemplated to have a positive intercalating association with the double-stranded nucleic acid and thus could be a mutagen. .

In this example, the photochemical analysis of 8-MOP is compared to AMT. Samples were analyzed by reverse phase HPLC using a Rainen Dynamax 300A column. The elution gradient was performed with 0.1M ammonium acetate / acetonitrile (0-70% acetonitrile in 42 minutes). AMT elutes as a single peak at approximately 24 minutes under these conditions. Detection was by absorption at either 260 or 330 nm. The last wavelength was used for the plasma containing the samples.

Standard solutions of each compound were prepared at various concentrations. These solutions were then diluted 1:10 in water, then 300 µΐ was injected for analysis. All samples were monitored at 300 nm. Peaks were analyzed by measuring either the height of the peak or by measuring the area of ​​the peak, then converted to a gh / ml value using the standard plot. The area of ​​the peak was determined by photocopying the trace, clipping the copy of the peak, then weighing the resulting trace. The two procedures gave essentially the same result.

The results are shown in Figure 7. Clearly, AMT degrades more rapidly than 8-MOP. It would be expected, therefore, that more photoproducts would be generated - which would eventually end up in the transfusion container. In contrast, 8-MOP was not expected to generate a significant amount of photoproducts. This is important when one considers that the weight of authority has concluded that unactivated 8-MOP is not mutagenic.

Example 6

When platelets are activated, an alpha-granule membrane glycoprotein called GMP140 is expressed on the surface of platelets. Less than (5%) of fresh, unstimulated normal platelets express detectable levels of GMP 140 by flow cytometry. See generally MJ Metzeiaar, Studies on the Expression of Activation-Markers on Human Platelets (Thesis 1991).

To measure GMP140, a small aliquot of platelet rich plasma is placed in a HEPES buffer containing a GMP140 binding antibody or mouse IgG control. CD62 is a commercially available monoclonal antibody that binds to GMP 140 (available from Sanbio, Uden, The Netherlands; Caltag Labs, So, San Francisco, CA, and Becton Dickinson, Mountain View, CA). After a fifteen minute incubation, FITC-conjugated goat anti-mouse IgG is added to the tube in saturating amounts. Finally, cells are diluted in isotonic saline, fixed with paraformaldehyde, and analyzed on a FACSCAN ™ (Becton Dickinson, Mountain View, CA). The positive control is made by adding phorbol myristate acetate (PMA) to the test system at a final concentration of 10^-7 M.

In this example, CD62 was used to measure the impact, if any, of irradiation alone on platelet activation. The antibody was stored in small aliquots (0.01 mg / ml) at -40 ° C prior to use. A mouse IgG control (0.05 mg / ml) (Becton Dickinson, Mountain View, CA # 9040) concentrated 5x was used. At the time of use, it was diluted 1: 5 in HEPES buffer. The secondary antibody was FITC-conjugated goat anti-mouse IgG (TAGO, Burlingame, CA # 3506). This was stored in small aliquots at -20 ° C. Phorbol myristate acetate (PMA) (Sigma, St. Louis, MO) was stored at -40 ° C. At the time of use, it was dissolved in DMSO (the working concentration was 1.62x10^-5 M).

16% paraformaldehyde (PFA) (Sigma, St. Louis, MO) was prepared by adding 16 grams of paraformaldehyde in 100 ml of deionized water. This was heated to 70 ° C, whereupon 3M NaOH was added dropwise until the solution was clear. The solution was cooled and the pH was adjusted to 7.4 with 1N HCl. This was filtered and stored. A commercially available isotonic buffer was used; Hematall Isotonic Diluent (Fisher # CS 606-20).

To measure platelet activation of platelet concentrates, a human platelet unit was obtained from the Blood Bank of Alameda-Contra Costa Medical Association. Aliquots of 5 ml were removed from the bag and received specific amounts of UVA irradiation, except for the control, which received no other treatment than placing it in an irradiation chamber. The temperature was maintained at 25 ° C during irradiation by placing the platelet concentrate in water lined chambers with stoppered glass attached to a circulating water bath. The irradiation device (Derma Control, Dolton; Ill .; Model No. 1,224-Special) was as described in Example 3, above.

After irradiation, platelets were stored for 5 days. At specific point in time, aliquots were taken and processed.

Processing included the addition of an aliquot (eg 5 microliters) of platelet concentrate to each microfuge tube containing the antibody and appropriate reagents and this was mixed very gently on the vortex. The samples were incubated for 16 minutes at room temperature.

ES 2 201 093 T3

Goat anti-mouse IgG-FITC (diluted 1:10 in HEPES buffer) (5 microliters) was added to each tube and the solution was gently vortexed. Samples were incubated for a further 15 minutes at room temperature.

Isoton 11 (1 ml) was added to each tube and mixed gently with a disposable polypropylene pipette. 8% PFA in HEPES (150 microliters) was added to each diluted sample to a final concentration of 1%. Platelets were analyzed on the FACSCAN ™. The results are shown in Table 3.

TABLE 3

<td></td><td colspan="2">Day 3</td><td colspan="2">Day 5</td>

<td>Terms</td><td>Inactivated</td><td>PMA activated</td><td>Inactivated</td><td>PMA activated</td>

<td>Control</td><td> 17</td><td> 85</td><td> 25</td><td> 89</td>

<td>UV 5 '</td><td> 17</td><td> 87</td><td> 24</td><td> 86</td>

<td>UV 10 '</td><td> 51</td><td> 84</td><td> 77</td><td> 79</td>

Activation is expressed as a percentage. Clearly, irradiation for 10 minutes (10 'UV) resulted in a significant negative impact on stored platelets; platelets were highly activated. In contrast, irradiation for five minutes (5 'UV) did not result in significant activation over the control that received no irradiation.

Example 7

Given the results of Example 6, it is clear that either a shorter irradiation period or the use of filters is necessary to avoid damage to cells by UV irradiation. In this example, CD62 is used to measure the impact of irradiation in the presence of psoralens on platelet activation. Shorter irradiation periods and wavelength filters are used separately.

Shorter irradiation periods

A unit of human platelets was again obtained from the Blood Bank of Alameda-Contra Costa Medical Association. Aliquots of 5 ml were removed from the bag to receive five minutes (5 ') of UVA irradiation in the presence of 10 μg / ml of 8-MOP, except for the control, which received no other treatment than to place it in the chamber for irradiation. . The temperature was maintained at 25 ° C during irradiation by placing the platelet concentrate in water-lined chambers with stoppered glass attached to a circulating water bath. The irradiation device (Derma Control, Dolton; Ill .; Model No. 1,224-Special) was as described in Example 3, above.

After irradiation, platelets were stored again for 5 days as in Example 6. At specific time points, aliquots were taken and assayed with the CD62 antibody and analyzed on the FACSCAN ™ to show that, under these conditions, the Platelets can be inactivated without damaging cells and stored for five days prior to transfusion.

Wavelength filters

An aqueous solution of Co (NO3) 2 was used together with NiSO4 to significantly eliminate the 365 nm component of the emission spectrum of the light source used. The Co-Ni solution can conveniently be used in place of water as a coolant during irradiation.

After ten minute irradiation with the filter, platelets were stored and tested with the CD62 antibody on the FACSCAN ™ to show that, under these conditions, platelets can be inactivated without damaging the cells and stored for the previous five days. to transfusion.