NO300911B1 - Method of inactivating viruses in blood and blood products - Google Patents

Abstract

The invention relates to a process for the selective removal of photodynamic substances from blood and blood products, where the purification takes place on adsorbents. An arrangement which has proved particularly advantageous for this purpose is one in which the blood or the relevant blood fraction is passed over a separating column which contains the adsorbent.

Description

The present invention relates to a method for inactivating viruses in blood and blood products in that the solutions, respectively the suspensions to be treated are exposed to phenothiazine dyes and are finally irradiated with visible light in the maximum area for absorption to the dyes and then the blood, respectively. the blood products, passed through adsorbent to remove the dyes.

It is known that photodynamic compounds in connection with visible light or UV light can have an inactivating effect on viruses. The reason for this is the affinity that these substances have for external viral structures or viral nucleic acids. Both apply to phenothiazine dyes. They react with membrane structure-enveloped viruses and damage these irreversibly under the influence of light, so that the virus loses its infectivity (cf. Snipes, W. et al., 1979, Photochem. and Photobiol. 29, 785-790). Furthermore, they react with viral RNA or DNA, especially with the guanine residues. Photodynamic compounds also react with viral RNA or DNA, particularly with the guanine residues of these nucleic acids. After the formation of a dye-nucleic acid complex, these are affected by light energy, so that the nucleic acids are denatured and eventually the strands are broken up. Furthermore, the phenothiazine dyes cause the conversion of molecular oxygen into oxygen radicals which are highly reactive and which can be virulent in various ways (cf. Hiatt, C.W., 1972, in: Concepts in Radiation Cell Biology, pp. 57-89, Academic Press, New York ; OH Uigin et al., 1987, Nucl. Acid. Res. 15, 7411-7427).

In relation to other photodynamic dyes for virus inactivation, phenothiazine dyes such as methylene blue, neutral red and toluidine blue are therefore of importance, due to the fact that already in connection with visible light they can inactivate a number of viruses, including, under certain conditions, those that do not contain lipid coatings , like for example. Adenovirus.

Here it can be mentioned that e.g. methylene blue (MB) and toluidine blue

(TB) itself has a therapeutic application, i.a. as an antidote for carbon monoxide poisoning and for long-term therapy-psychotic illnesses. Here negligible next to effective amounts of MB or TB for use (1-2 mg/kg body weight), which is much higher than those required for virus inactivation. The low toxicities of MB and TB are also shown by data from animal experiments.

Since 1955, it has been assumed that dye concentrations, especially for toluidine blue, below 2.5 µM are insufficiently virus inactivating (cf. F. Heinmets et al., 1955, Joint Report with the Naval Medical Research Institute, Walter Reed Army Institute of Research , USA).

In the previously described investigations for virus inactivation with phenothiazine dyes, the dye concentrations are between 10 uM and 100 uM. (Chang and Weinstein, 1975, Photodynamic Inactivation of Herpes-virus Hominis by Methylene Blue

(38524), Proceedings of the Society for Experimental Biology and Medicine, 148:291-293; Yen and Simon, 1978, Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red, J. gen. Virol., 41:273-281). At these concentrations, the disadvantage is that not only does virus inactivation take place, but also inactivation of plasma proteins such as e.g. coagulation factors. This is one of the reasons why phenothiazine dyes have long failed to achieve a role in virus inactivation in blood and blood products.

The object of the present invention is a method for virus inactivation, where viruses and different types are killed by the addition of phenothiazine dyes without functional impact on the plasma proteins. The object of the invention is further to make the method so simple that blood, or blood products, are immediately processed in purchased blood bags and so that the added dyes can be removed again after the treatment.

The present invention therefore relates to a method for inactivating viruses in blood and blood products where the solution, or the suspension to be inactivated is added to phenothiazine dyes and irradiated with visible light in the area at the absorption maximum of the dyes, and then the blood or the blood products possibly passed over an adsorption agent to remove the dyes, characterized by the fact that the phenothiazine dyes are added in a concentration of 0.1 to 2 jjM, and that the irradiation is carried out immediately in transparent containers such as blood bags, which are used for blood collection and storage.

The irradiation takes place with daylight of sufficient strength or with monochromatic light, preferably a cold light source, which has a wavelength in the region of the absorption maximum of the dye in question. Furthermore, the following conditions for virus inactivation in blood plasma, resp. in plasma protein solutions, are used. The working temperature should be in the range 0 to 37°C, preferably in the range 4 to 20°C. The inactivation duration is in particular 5 minutes to 5 hours, preferably 10 minutes to 3 hours, and the pH value should lie between pH 5 and pH 9, preferably between pH 6 and pH 8.

The essential advantage of the method according to the invention is that it is simple. F. Heinmet et al. (as stated above) describes an intricate apparatus, through which e.g. blood plasma must be led. Problems with attention and also apacity problems occur here. It was now surprisingly established that significantly smaller amounts of dyes can be used, and labor-intensive technical equipment for photoinactivation is not necessary.

It was also unexpected that a non-enveloped virus such as adeno, which cannot be inactivated under physiological conditions in plasma, allows itself through a freeze/thaw step to become photosensitized and thus inactivated. In this way, the inactivation, regardless of the sequence of the work steps freeze/thaw and addition of dye, could be determined. By freezing is understood a deep-freezing treatment with liquefied gas as a refrigerant at temperatures of approximately -20°C to approximately 80 K. As a rule, it is deep-frozen below -30°C.

Virus inactivation can be carried out immediately in blood or plasma sacs, despite the fact that these are only partially light penetrating. The dye must be added. The bag with contents is then illuminated, and then the product can be further processed.

The procedure can thus be carried out without major technical problems, and can be integrated into blood banks when processing individual blood donors. The small amount of added dye may remain in the treated liquid or be removed by adsorbent.

As blood, respectively blood products, among others the following applies:

- thoroughbred

erythrocyte concentrate

thrombocyte concentrate

plasma

serum

cryoprecipitate

concentrate of coagulation factors

inhibitors

fibronectin

albumen.

For use in the present method according to the invention, phenothiazines with the following structural formula are suitable.

Example 1

With methylene blue (MB), the dependence on photoinactivation of vesicular stomatitis virus (VVS) in human plasma is shown below.

About. 5 x IO<7> plaque-forming units (PFU) per ml VSV was suspended in human plasma and reacted with different concentrations of MB. No dye was added to the control sample. The sample volume was 0.5 ml. A control sample and part of the MB-containing sample were irradiated for 4 hours at room temperature with visible light; and the others were stored for the same length of time in the dark. A slide projector that had been replaced with a 150 W halogen bulb (Osram Xenophot) serves as the light source. The distance between the lens of the slide projector, i.e. the light outlet opening and the sample, was 30 cm in this and all further tests. (With the exception of virus inactivation in blood bags).

After the end of irradiation, virus titers were determined in all samples using a plaque analysis. BHK cells were used as indicator cells. The test results are listed in table 1. Tab. 1: Inactivation of VSV in human plasma with and without irradiation.

Irradiation duration: 4 hours.

The results from tab. 1 shows that from a concentration of MB of 0.5 µM, the infectious titer of VSV was reduced by more than 6 titer powers. Significantly higher concentrations of the dye, from approximately 50 uM, lead without irradiation to a significant reduction of VSV titers.

Example 2

By means of the following experiment, the virus inactivation at low dye concentrations was determined.

VSV in the presence of plasma and different amounts of methylene blue, in aliquots of 500 µl volume overnight in a cold room, were irradiated from a distance of 30 cm with the slide projector. Sample A

- F was irradiated, sample G was not irradiated.

The results of these experiments are listed in tab. 2. They show that under the above conditions added VSV was inactivated by more than 4 tier powers. Here, 0.5 jjM methylene blue was necessary.

Titers to VSV are likely to have been reduced only by overnight incubation at 4°C by 1-2 titers, and this explains the relatively low starting titer. However, this was not taken into account in the experiment.

Comparison of A (irradiated) and G (dark) shows that light alone obviously does not exert a large influence on the infectivity of the virus.

Example 3

The photoinactivation of viruses in the presence of phenothiazine dyes is dependent on the duration of the irradiation. To investigate which irradiation times are sufficient for photoinactivation of VSV, 10^ plaque-forming units (PFU) per ml suspended in plasma, and irradiated as described for different times at 22°C. Table 3 shows the results obtained. It appears that, under the stated experimental conditions, one hour of irradiation is sufficient to reduce the infectious VSV titer by more than 6 orders of magnitude.

Example 4

A similar experiment was carried out instead with MB in the presence of 1 µM of another phenothiazine precursor, TB. The results listed in Table 4 show that VSV can also be effectively inactivated with the help of TB.

The inactivating action of the phenothiazine dye was also shown in Herpex-Simplex virus (HSV) as well as human immunodeficiency virus type 1 (HIV-1).

Example 5

In the presence of methylene blue (1 uM), HSV is also inactivated. Tab.

5 shows the kinetics of MB-mediated photoinactivation of HSV.

Example 6

A similar experiment was conducted with the AIDS-causing HIV-1. Virus titers were 6 x IO<2> PFXJ/ml. MT4 cells are used as indicator cells. Table 6 shows that HIV-1 is particularly susceptible to photoinactivation; already within 10 min. virus titers were reduced by more than 600-fold.

Example 7

In the attempt to inactivate non-enveloped viruses under normal physiological conditions in the presence of 80% plasma, no result could be obtained. Adenovirus, as a model for a non-enveloped virus, was pre-incubated for a longer time (4°C, dark) in the presence of the dye methylene blue (MB), 1 µM. This was followed by a 30-minute irradiation with a halogen beam (150,000 Lux). The infectivity of adenovirus was unchanged here.

Titers were determined as TCID50 (calculation method "Tissue Culture Infectious Dose" according to Spearman and Kaeber). The virus was titrated on FL cells (defined cell line for virus titration).

When using toluidine blue under the same experimental conditions, no reduction in virus titers could likewise be determined.

In order to achieve an inactivation of adenovirus, a freeze/dry (F/T) step during deep freezing at -30°C was introduced during the course of the experiment. Thus, the order of F/T and addition of the dye (1 uM MB) only plays a minor role. The samples are irradiated using halogen rays. 120,000 Lux was measured.

Tab. 8: Photosensitization of adenovirus on the basis of an introduced F/T step.

Carrying out the virus titration took place as described in tab. 7.

Example 8

The particular problem with the use of higher dye concentrations lies in the immediate action of these substances on the plasma proteins. In a further experiment, different dye concentrations were then examined with a view to their effect on the activity of the coagulation factors.

Human plasma (2 ml aliquots) was reacted with different amounts of MB. Immediately afterwards, the activity of coagulation factors V, VIII and IX was measured. As can be seen from table 7, in all three cases they were inhibited dose-dependently, those of factors VIII and V from around 10 pM and of factors IX already from 2.5 µM. Then, at higher concentrations, MB acts directly on the proteins without the need for light to intervene.

Example 9

It is not only the added dye concentration that has an impact on the activity of the coagulation factor, but also the irradiation time. This time dependence was determined at different methylene blue concentrations.

Human plasma (2 ml aliquots) was reacted with various amounts of MB and irradiated for 1 to 4 hours (as described in Example 1). The control samples were not phototreated. As can be seen from Table 8, the activity of the three coagulation factors V, VIII and IX was inhibited in a time- and dose-dependent manner. Especially with factors VIII and IX, it turns out that higher MB concentrations and the light exposure time from 2 h cause an increase in thrombolytic activities.

Tab. 10: Effect of light and MB on the activity of coagulation factors: Time and dose dependence.

Example 10

According to a preferred embodiment of the present invention, the photoinactivation of viruses can occur immediately in plasma bags. Blood or blood products are added with dye in the required quantities, and the bag can then be irradiated with light. In this simple way, it is possible to process the blood products from individual donors.

In one experiment, three samples of fresh human plasma were thawed and transferred into plasma bags with 1.5 x 106 PFU VSV each. For two samples, MB was added in concentrations of 1 and 10 uM added. A sample was taken from the MB-free plasma and stored as a positive control in the dark at 4°C. The three bags were then fixed between two flexiglass plates to enable a layer thickness that was as good as possible equal to approx. 2.5 cm. They were then irradiated from a distance of approx. 90 cm using a slide projector. After 4 hours, samples were taken to determine virus titers, and these were measured using plaque analysis on FL cells. The results in tab. 9 shows that

1 >jm MB is sufficient to reduce the infectious titer of VSV by more than three orders of magnitude in plasma pouches during four hours of irradiation. Even in the absence of genetic material fat, the irradiation led to a reduction in virus titers, but only by approximately 50$.

The phenothiazine dyes added for virus activation can remain in the concentrations used here of up to 1 uM in blood or blood products without side reactions occurring. They can also be subsequently removed via dialysis, gel filtration or adsorption.

Of the aforementioned methods, the adsorbent is particularly of interest, because they are the least labor-intensive in terms of time and technique, and the relevant plasma protein solutions are not diluted.

Certain adsorbents, on the other hand, are obviously unsuitable, such as e.g. those of Hiatt (Consepts in Radiation Cell Biology, pp. 57-89, Academic Press, New York, 1972) mentioned ion exchangers, due to the fact that, in addition to the dye, they also bind the plasma proteins very strongly, i.a. the coagulation factors.

It could now surprisingly be established that MB and other phenothiazine dyes are bound very strongly on different commercially available separation gels, including those that have either no or only weak protein binding. Such absorbents are also suitable for the subsequent removal of photo-oxidation images. Of the adsorbents tested, these apply to the separation of MB and other phenothiazine dyes.

In most cases, at an addition concentration of 10 pM MB, 2 g of the applicable adsorbent was required to extract the dye completely, in hatch, from a plasma protein solution.

As an adsorption type, the following have proven to be particularly suitable: 1. Silk gels, respectively. silica gels with pore sizes, which are so small

(40 to approx. 100 Å diameter), that plasma proteins cannot penetrate the gel matrix, but instead only low molecular weight dye molecules which, due to ionic, electrostatic and hydrophobic interactions, are thus bound.

Examples of commercially available adsorbents of this type are Matrex silica gel (Amicon, Witten), Daltosil (Serva, Haidelbeg) and Kiesel-Gel (Merck, Darmstadt). 2. Gels based on polystyrene-divinylbenzene or acrylic ester polymer. They are also produced with suitable pore sizes.

Examples of commercially available gels of this type are Amberlite (among others Rohm and Haas, Frankfurt) and Bio Beads (Bio Rad, Munich). They mainly serve to remove from aqueous solutions non-polar compounds, resp. surfactants, e.g. detergents. Either they are not polar, or they are weakly polar.

Example 11

Fresh plasma was reacted with methylene blue (10 µM). 5 ml aliquots were reacted with different amounts of Saltosil (pore width 75 A) or Bio Beads SM 16 (pore width 144 A) and mixed for 30 minutes. The gel is then allowed to settle. In plasma, factor VIII- or the factor V content, the extinction at 660 nm and for individual samples the protein content was measured. It appears from the extinction values that, apart from the dye, additional compounds are extracted from the plasma. This is not about plasma proteins. The extinction values of the plasma treated with 100 to 250 mg of adsorbents per 5 ml, i.e. with 2-5 weight #

{% v/v), hardly differ from those extracted with 10% v/v adsorbents. It therefore appears that at an MB concentration of 10 µM in both cases, 2-556 v/v adsorbent is sufficient to remove the dye from the plasma in batch-wise treatment. Correspondingly less absorbents are required when the added concentration of the dye is lower.

Example 12

In a further experiment, a 5% human serum albumin solution (5% HSA) was added instead of blood plasma. The MB concentration was further 10 uM. 5 ml aliquots were batch extracted, each with 100 mg, i.e. 2% v/v of the following adsorbents at different times: Daltosil

(pore size 75 A, Kieselgel (pore size 40 A) and Bio Beads SM16 (pore size 144 A).

As Abb. 1 shows, in all three cases the distinction at 660 nm returns within 20-30 minutes to a constant value, i.e. this time is sufficient to remove photo-oxidizing agents batch-wise processing from the plasma protein solution. As Abb. 1 also shows, Bio Beads SM16 and kieselgel 40 in the specified case are obviously a better adsorbent than Daltosil with 75 A pore width.

Example 13

Column chromatographic removal of MB from plasma protein solutions.

From this experiment, it must be determined whether the adsorptive removal of photo-oxidizing agents can also take place chromatographically. The background for this is the idea that the virus inactivation is carried out using a dye in combination with light in a container, for example in a blood bag, and then transfers the plasma protein solution over a small separation column adapted in between, which contains the adsorptive agent in a second "container, e.g. a If the unit first bag - adsorption column - second bag was prefabricated, then a closed system is available, and virus-inactivated plasma protein preparations, including those originating from individual donors, could be produced in a simple way with little risk of contamination.

Here, 250 ml of 5% albumin solution was passed at different speeds through a separation column containing 5 ml of silica gel (pore size 40 Å). 10 ml fractions were collected and the extinction values measured at 660 nm.

As can be seen from table 10, it is possible to pass total volumes of the albumin solution with flow rates of 5 or 7.5 ml/min. through the column, without it being possible to detect remaining MB in the flow-through fractions. The time it takes to remove the dye from 250 ml of solution is 30 to 35 minutes at most.

The result of the experiment shows that the chromatographic separation of the photo-oxidizing agent does not pose a problem, and secondly, it is proved that the above-described production of virus-inactivated plasma protein preparations from single donors is possible.

Claims (7)

1. Procedure for inactivating viruses in blood and blood products where the solution, respectively the suspension to be inactivated is added to phenothiazine dyes and irradiated with visible light in the area at the absorption maximum of the dye f one, and then the blood or the blood products possibly passed over an adsorption agent to remove the dyes, characterized in that the phenothiazine dyes are added in a concentration of 0.1 to 2 uM, and that the irradiation is carried out immediately in transparent containers such as blood bags, which are used for blood collection and storage. 2. Method according to claim 1, characterized in that the phenothiazine dyes toluidine blue or methylene blue are used. 3. Method according to one of claims 1 or 2, characterized in that the solution or the suspension to be inactivated is first deep-frozen, and then further thawed before irradiation. 4. Method according to claim 3, characterized in that the dye is added before the freezing process. 5. Method according to claim 3, characterized in that the dye is added after thawing and before irradiation. 6. Method according to one of claims 1 to 5, characterized in that this is carried out over two suitable containers for blood collection such as a blood bag with a separation column connected between them, which contains the adsorbent for the phenothiazine dye. 7. Method according to claim 6, characterized in that silica gel or those based on polystyrene-divinylbenzene or acrylic ester polymers are added as adsorbent.