AU635068B2 - Process for 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

1 PROCESS FOR INACTIVATING VIRUSES IN BLOOD AND BLOOD PRODUCTS Specification: The invention is directed to a process for inactivating viruses in blood and blood products, comprising: adding phenothiazine dyes to the solutions or suspensions to be treated and subsequently irradiating said phenothiazine dye-containing solutions or suspensions with visible light in the range of the absorption peak of the respective dye, whereafter the blood or blood products may be passed over adsorbing agents for removal of the dyes.

It is known that photodynamic substances in combination with visible light or UV-light may have a virus inactivating effect.

This is due to the affinity of these substances to external virus structures or to viral nucleic acid. Both facts apply to phenothiazine dyes. They react with the membrane structures of enveloped viruses and damage the same irreversibly under the action of light, whereby the virus loses its infectiousness (cf. Snipes, W. et al., 1979, Photochem. and Photobiol. 29, 785-790).

However, .photodynamic substances also interact 'with viral RNA or DNA, especially with the guanine residues of these nucleic acids. When a dye/nucleic acid-complex has been formed it is stimulated by light energy so that denaturation of the nucleic acid and finally strand breakages result. Also, phenothiazine dyes induce the conversion of molecular oxygen to oxygen radicals which are highly reactive and may have various virucidal effects (cf. Hiatt, 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).

2 1 In contrast to other photodynamic dyes for virus inactivation, phenothiazine dyes such as methylene blue, neutral red and toluidine blue are of special interest because they can inactivate a number of viruses already in combination with visible light and, under certain conditions, even viruses that do not possess a lipid envelope, such as adenovirus.

In addition to that, methylene blue (MB) and toluidine blue (TB) for instance are themselves being used therapeutically, among other uses also as antidotes to carbon-monoxide poisoning and in long-term therapy of psychotic diseases. In this connection quantities of MB or TB much higher than those required for virus inactivation are used (1 to 2 mg/kg body weight) without any significant side effects. The low toxicities of MB and TB are also substantiated by data obtained from animal experiments.

However, since 1955 those of skill in the art have assumed that dye concentrations, especially in the case of toluidine blue, of less than 2.5 pM have only an insufficient virus inactivating effect (cf. F. Heinmets et al. 1955, Joint Report with the Naval Medical Research Institute, Walter Reed Army Institute of Research, In the previously described investigations of virus inactivation with phenothiazine dyes the dye concentrations are between pM and 100 pM (Chang and Weinstein, 1975, Photodynamic Inactivation of Herpes-virus Hominis by Methylene Blue (38524), Proceedings of the Society for Experimental Biology and Medi- 30 cine, 148:291-293; Yen and Simon, 1978, Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red, J. gen. Virol.

41:273-281). But at these concentrations there arises the drawback that not only viruses may be inactivated but also plasma proteins, such-as'the coagulation factors. This'is one of the reasons why phenothiazine dyes have so far not achieved any significance in the inactivation of viruses in blood and blood p- products.

3 1 It is the object of the subject invention to provide a process for inactivating viruses in which various kinds of viruses are killed by the use of phenothiazine dyes without any functionally detrimental effects on the plasma proteins. It is a further object of the invention that said process be of simple design, such that blood or blood products may be subjected to direct treatment in commercially available blood bags and the added dyes may be removed after processing if so desired.

The specified object is accomplished in accordance with the invention in that the phenothiazine dyes are used at a concentration of from 0.1 to 2 uM and irradiation is effected directly in transparent containers, such as blood bags, of the kind used for the collection and storage of blood.

The irradiation is performed either with daylight of sufficient intensity or with monochromatic light, preferably from a cold light source at a wavelength in the range of the absorption peak of the respective dye. Also, the following conditions should be observed for virus inactivation in blood plasma or plasma protein solutions: The operating temperature should be in the range of from 0 to 37 0 C, if possible from 4 to The inactivating time ranges especially from 5 minutes to hours, preferably from 10 minutes to 3 hours, and pH should be between pH 5 and pH 9, preferably between pH 6 and pH 8.

An essential advantage of the process according to the invention lies in its simplicity.. F. Heinmets et al. (as specified above) describes a highly complex apparatus through which, for instance, blood plasma must be passed. Here, problems of maintenance and above all capacity arise. Surprisingly, it has now been found that substantially smaller quantities of dye are sufficient and that no complex technical apparatus is required for photoinactivation.

Unexpectedly, it has also been found that a non-enveloped virus, such as adenovirus, which could not be inactivated under _T L-Ij physiological conditions in plasma, could be photosensitized by 3A 2. a freezing/thawing step and could thus be inactivated. In this connection inactivation has been ascertained irrespective of the employed order of the freezing/thawing steps and the addition of the dye. Freezing here means a deep-freezing operation at temperatures of from approximately -20 0 C to approximately Normally, deep-freezing is carried out at temperatures below -30 0

C.

Virus inactivation may be carried out directly in blood or plasma bags although these are transparent only to a limited extent. It is mer,,ly necessary to add the dye. Then the bag inclusive of its contents is exposed to light, whereafter the respective product can be further processed.

Thus, the process can be carried out without any major technical effort and is excellently suited for integration in the processing flow of individual blood donations. The small quantity of the dye used may either remain in the treated fluid or nay be removed by adsorbing agents.

X; V 'K 4 1 Hence, the method can be carried out without any technical effort and is excellently suited ntegration in the processing flow of indivi ood donations. The small quantity of the d ed may either remain in the treated fluid or m removed by adsorbing agents.

Suitable blood or blood products include: whole blood red cell concentrates platelet concentrates plasma serum cryoprecipitate concentrates of coagulation factors inhibitors cold insoluble globulin albumin.

Phenothiazines having the following structural formula are suitable for use in the method according to the invention:

H

I

N R2 R7 X Ra X R R R 2 .3 7 t A- neutral red N CH NH N(CH 3 2 3 2 toluidine blue S CH NH N(CH 3 2 N 32 methylene blue S H N(CH N(CH h2 2 phenothiazine S H H H 1 Example 1 Below, the dependence of photoinactivation on methylene ve 5 l cL'(r S torm -fitS Vir-Is blue (MB) is shown for(VSV)in human plasma.

Varying concentrations of MB were added to human plasma con- 7 taining approximately 5 x 10 Plaque Forming Units (PFU) per ml of VSV. Control samples did not contain any dye. The sample volume was 0.5 ml. One control sample and a portion of the MB containing samples were irradiated with visible light for 4 h at room temperature; the others were stored in the dark for the same.length of time. The light source used was a slide projector equipped with a halogen bulb of 150 W (Osram Xenophot). The distance between the slide projector lens, i.e. the light outlet, and the samples was 30 cm in these and all further tests (with the exception of blood bag virus inactivation).

Following completion of irradiation, the virus titer was determined in all samples by means of a plaque assay. The indicator cells used were BHK cells. The test results are listed in Table 1.

6- 1 Samples MB concentr. Light Virus Inactiva- (pM) tion Factor contr.1 0 4.8 contr.2 0 1 1 0.01 11.8 2 0.1 28.5 6 3 0.5 6 4 1 6 5 10 6 6 50 6 7 100 8 1 1 9 10 10 50 11.8 11 100 Table 1: Inactivation of VSV in human plasma with and without illumination.

Exposure time: 4 h The results of Table 1 show that the infectious titer of VSV was reduced by a factor of more than 61og at a minimal MB concentration of about 0.55 pM. Significantly higher concentrations of the dye, from about 50 pM and up, resulted in a significant reduction in the VSV titer even without exposure to light.

Example 2 The following test confirmed virus inactivation at low dye concentrations.

?jAL In the presence of plasma and varying amounts of mcthylene blue S/ in aliquots of 500 pl, VSV was irradiated overnight in a cold- I storage room with the slide projector from a distance of 30 cm.

Samples A to F were illuminated, sample G was not.

7 1 The results of this tes,t are presented in Table 2. They show that under the above-mentioned conditions the VSV used was inactivated by a factor of more than 41og This required 0.5 pM of methylene blue.

It is probable that the VSV titer had already been reduced by 1 to 2 logs by the overnight incubation at 4 0 C, which would explain the relatively low initial titer. However, this was not simultaneously test in our experiment.

A comparison of A (exposed) and G (dark) shows that light alone evidently does not influence the infectiousness of the virus to any great extent.

Sample .final MB Titer/200 pl Inactivation concentr. M factor 4 A 0 2 x 10 2.2 4 B 0.01 2.4 x 10 1.8 4 C 0.05 2 x 10 2.2 2 D 0.25 3 x 10 147 4 E 0.5 <1 >4.4x10 4 F 1.0 <1 >4-.-4x10 4 G 0 4.4 x 10 1 Table 2: Virus inactivation at low dye concentrations ExamDle 3 The photoinactivation of viruses in the presence of phenothiazine dyes depends on the exposure time. To find out what exposure times would be sufficient for photoinactivation of VSV, ^LIA 106 Plaque Forming Units (PFU) per ml were suspended in plasma Sand illuminated as described for different times at 22°C. The S results are listed in Table 3. It is evident that under the 8 1 specified test conditions an exposure time of one hour was sufficient to reduce the ipfectious VSV titer by a factor of more than 61og10 Sample 'Exposure time (min) Inactivation factor control 0 1 1 5 2 30 1666 6 3 60 Table 3: Kinetics of the photoinactivation of VSV by MB Example 4 A similar test was carried out in the presence of 1 pM of another phenothiazine dye, TB, instead of MB. The results listed in Table 4 show that effective inactivation of VSV can also be achieved by using TB.

Sample Exposure time (min) Inactivation factor control 0 1 1 10 3 2 60 >4 x Table 4: Kinetics of the photoinactivation of VSV by TB The inactivating effect of the phenothiazine dyes was also shown for herpes simplex virus (HSV) and for type 1 human immunodeficiency virus (HIV-1).

9- 1 Examnle HSV is also inactivated in the presence of methylene blue (1 pM). Table 5 shows the kinetics of photoinactivation of HSV by MB.

Sample Exposure time (min) Inactivation factor control 0 1 1 20 2 60 1500 4 3 180 >3 x Table 5: Kinetics of the photoinactivation of HSV by MB Examle 6 A similar test was conducted with the AIDS virus HIV-1. The vi- 2 rus titer was 6 x 10 PFU/ml. MT4-cells were used as indicator cells. Table 6 shows that HIV-1 is apparently especially sensitive to photoinactivation: the virus titer was already reduced by a factor of more than 600 within the first 10 minutes.

Sample Exposure time (min) Inactivation factor control 0 1 1 10 >600 2 60 >600 3 120 >600 Table 6: Kinetics of the photoinactivation of HIV-1 by MB

I.

2' *P :1 10 1 Examole 7 There was no success in an attempt to inactivate non-enveloped viruses under the usual physiological conditions.in the presence of 80% plasma. As an example of a non-enveloped virus, adenovirus was pre-incubated for a prolonged period of time (4 0 C, dark) in the presence of methylene blue (MB) dye, 1 pM.

Then, irradiation was effected for 30 minutes with halogen bulbs (150,000 lx). There was no change in the infectiousness of adenovirus.

Sample Pre-Incubation Dye Virus time titer (loglO) control 0 h 1 Oh MB 2 1 h MB 3 4 h MB 4 24 h MB Table 7: Influence of the pre-incubation time on the photosensitization of adenovirus.

The virus titer was determined as TCID50 (calculation method "Tissue Culture Infectious Dosis" by Spearman and Kaerber). The virus was titrated on FL cells (defined cell line suitable for virus titration).

When toluidine blue was used under the same experimental condi- :ions, there was also no reduction of the virus titer that could be detected.

To achieve inactivation of adenovirus, a freeze/thaw step (F/T) with deep-freezing to -30 0 C was incorporated in the test run.

3 Here, the order of F/T and the addition of the dye (1 uM MB) was of secondary importance only. The samples were again irradiated using halogen bulbs, as described above. 120,000 lx were measured.

11 Sample Preparation of Sample Virus Titer (loglO) control

A

B

C

D

E

F

G

F/T

F/T 60 min irradiation F/T MB 60 min pre-incubation min irradiation MB F/T MB F/T 10 min irradiation MB F/T 30 min irradiation MB F/T 60 min irradiation Table 8: Photosensitization of adenovirus due to an incorporated F/T step.

Virus titration was carried out as described in Table 7.

Example 8 The special problem when using high dye concentrations is in the immediate effect of these substances on plasma proteins.

Therefore, the influence of different dye concentrations on the activities of-coagulation factors was investigated in a further test.

Varying amounts of MB were added to human plasma (2-ml aliquots). The activities of the coagulation factors V, VIII and IX were measured immediately thereafter. As is evident from Table 9, said factors are inhibited in all three cases in dependence on the concentration of the dye, whereby the activities of the factors VIII and V are inhibited from about 10 pM and those of factor IX already from 2.5 pM. Consequently, at higher concentrations MB has a direct effect on the proteins, without S/need of the action of light.

12 1 Methylene Blue (pM/1) Factor V E/ml Factor VIII E/ml Factor IX E/ml 0 0.80 0.38 1 0.76 0.41 1.9 0.78 0.41 1.6 0.74 0.38 1.45 0.54 0.35 1.20 0.44 0.28 1.10 Table 9: Influence of MB on the activities of coagulation factors Example 9 However, it is not only the dye concentration used but also the exposure time which influences the activities of coagulation factors. This time-dependence has been determined for varying concentrations of methylene blue.

Human plasma (aliquots of 2 ml) received varying amounts of MB and was then exposed to light for 1 to 4 hours (as described in Example Control samples were not subjected to photo-treatment. As is evident from Table 10, the activities of the three coagulation factors V, VIII and IX are inhibited in dependence on time and the concentration of the dye. Especially in the cases of factors VIII and IX higher MB concentrations and exposure times from 2 hours upwards cause an apparent increase in their thrombolytic activities.

13 Exposure time MB Concentration pM/1 Factor V E/ml Factor VIII Factor IX E/ml E/ml 0 h 0 1 2.5 0 1 0 1 0 1 0.86 0.86 0.82 0.72 0.84 0.72 0.68 0.47 0.82 0.64 .0.68 0.60 0.76 0.55 0.49 0.42 0.33 0.45 0.33 0.30 0.40 0.24 0.24 0.16 0.44 0.23 0 .22 0.15 0.38 0.16 0.29 0.27 1.20 1 0.46 0.44 0.76 0,92 0.82 0.68 0.10 0.90 0.72 0.74 0.98 0.94 0.82 0.64 Table 10: Influence of light and MB on the activities of coagulation factors: dependence on time and MB-concentration Example In accordance with a preferred embodiment of the subject invention the photoinactivation of viruses may be effected directly in the plasma bag. The dye at the required concentration is merely added to the blood or the blood products and then the bag is exposed to light. In this simple way it is possible at any time to treat blood products from individual donors.

14 1 In a test three samples of fresh human plasma were thawed. Each sample was then inoculated with 1.5 x 106 PFU VSV within the respective plasma bags. MB at concentrations of 1 and 10 1IM, respectively, was added to two samples. A sample Was taken from the MB-free plasma and stored in the dark at 4 0 C as a positive control. Then, the three bags were mounted between two Plexiglas plates to ensure a highly uniform layer thickness of approx. 2.5 cm. In turn, said samples were irradiated by means of a slide projector from a distance of approx. 90 cm. After 4 hours, samples were taken to determine the virus titer and the same was measured by plaque assay on FL-cells. The results listed in Table show that 1 uM MB is already sufficient to reduce the infectious titer of VSV by a factor of more than 31og0 by means of a four-hour exposure in the plasma bag. Even in the absence of the dye the exposure resulted in a reduction of the virus titer, although only by about Sample Exposure MB Concentra- Weight of VSV Titer time tion Bag '(PFU/ml) 3 control 0 0 323 5x10 3 1 4 0 323 2.5x10 2 4 1 289 0 3 4 10 257 0 Table 11: Photoinactivation of VSV in plasma bag The phenothiazine dyes used for virus inactivation may remain in the blood or the blood products, particularly at the concentrations used here, without side effects occurring. However, they may be removed later by means of dialysis, gel filtration or adsorption.

Of the specified methods the adsorptive ones are of main interest because they require the least effort as to time and tech-

S\

nical apparatus, and the respective plasma protein solutions are not diluted.

15 However, some adsorbing agents are obviously unsuitable, such as the ion exchangers mentioned by Hiatt (Concepts in Radiation Cell Biology, pp. 57-89, Academic Press, New York, 1972) because in addition to the dye they also strongly bind plasma proteins, such as coagulation factors.

Surprisingly, it has now been found that MB and other phenothiazine dyes bind very strongly to a various commercially available separation gels, including those which either do not or only weakly bind Droteins. Such adsorbing agents are therefore especially suitable for the later removal of the photo-oxidant.

Of the adsorbing agents tested, the following ones may be used for the removal of MB and other phcnothiazine dyes.

Adsorbino Agent Mate r i a Manufacturer or Supplier Daltosil 75 Si 100-Polyol RP 18 Kieselgel 40 0 Nucleosil 50 A pore size 0 Nucleosil 100 A pore size Vydac SC-201 RP Modified Silica Gel Derivatized Silica Gel containing C 18 -groups Silica Gel Silica Gel Silical Gel Glass beads coated with Silica Gel bearing

C

18 groups Controlled pore glass (porous glass beads) Polystyrene DVB (Divinylbenzene), Polyacrylester Serva, Heidelberg (FRG) Serva, Heidelberg (FRG) Merck, Darmstadt (FRG) Macherey Nagel, Diren (FRG) Macherey Nagel, DOren (FRG) Macherey Nagel, Duren (FRG) Pierce Europe (FRG) Bio Rad, MUnchen (FRG) Rbhm Haas, Frankfurt

(FRG)

CPG 40 Bio beads, Amberlite adsorbent resins In most cases 2 g of the respective adsorbing agent, used as a batch, were sufficient at a feed concentration of 10 pM to completely extract the dye from a plasma protein solution.

16 1 Two types of adsorbing agents proved to be particularly suitable: 1. Silica gels having pores of so small a size (40 to approx.

100 A diameter) that plasma proteins cannot penetrate the gel matrix while the low molecular weight dye molecules can do so and are thus bonded thereto due to ionic, electrostatic and hydrophobic interaction.

Examples of commercially available adsorbing agents of this type are Matrex Silica Gel (Amicon, Witten), Daltosil (Serva, Heidelberg and Kiesel-Gel (Merck, Darmstadt).

2. Gels of the type based on polystyrene divinyl benzene and acrylic ester polymer, respectively. They, too, are manufactured with suitable pore sizes.

Examples of commercially available gels of these types are Amberlite (R6hm Haas, Frankfurt, among others) and Bio Beads (Bio Rad, Manchen). They are mainly used to remove non-polar substances or surface-active agents such as detergents from aqueous solutions. They are either non-polar or only slightly polar.

ExamDle 11 Methylene blue(10 pM) was added to fresh plasma. 5-ml aliquots 30 received varying amounts of Daltosil (pore size 75 A) and Bio Beads SM16 (pore size 144 respectively, and were then stirred for 30 minutes. Then the gel was left to settle. In the plasma the factor VIII and factor V contents, extinction at 660 nm and, for some samples, the protein contents were measured.

17 E (660 nm) Fresh plasma Fresh plasma MB Daltosil 50 mg 100 mr 250 mg 500 mg 0 .909 1.450 0.576 0.571 0.491 0.477 0.666 0.571 0.571 0.530 Protein (Rg/ml) 66.8 -65.6 Factor VIII (U/ml) 1.10 0.42 0.60 1.10 1.10 1.25 Factor V (U/mi) 1.20 0.96 1.05 1.10 1.20 1.20 66.8 Bio Beads SM 16 50 mg 100 mg 250 mg 500 mg 0.82 1.05 1.05 0.80 1.05 1.10 1.10 1.15 72.5 Table 12: Extraction of methylene blue the. boj It is evident from the extinction values that apparently further substances in addition to the dye are extracted from the plasma. But these substances are not plasma proteins. The extinction values of the plasma which had been treated with 100 to 250 mg of adsorbing agent per 5 ml, i.e. with 2 to 5 weight percent hardly differ from those which had been extracted with 10% w/v adsorbing agent. Hence, at an MB concentration of 10 pM 2 to 5% w/v of adsorbing agent are sufficient in both cases for removing the dye from the plasma in a batchwise operation. If the feed concentration of the dye is lower, the amount of adsorbing agent required is correspondingly lower.

Example 12 In a further test a 5% human serum albumin solution HSA) was used instead of blood plasma. Again, the MB concentration So was 10 M. Aliquots of 5 ml were extracted batchwise with 100 mg, respectively, i.e. 2% w/v, of the following adsorbing 18 1 agents for varying periods of time: Daltosil (pore size 75 A Kiesel-Gel (pore size 40 A) and Bio Beads SM16 (pore size 144 A).

As Figure 1 shows, the extinction at 660 nm decreases to a constant value in all three cases within a period of 20 to 30 minutes, i.e. 'this time period is sufficient to remove the photooxidant in batches from a plasma protein solution. As is further evident from Figure 1, Bio Beads SM16 and Kiesel-Gel appear to be somewhat better adsorbing agents in the subject case than Dalto sil with a pore size of 75 A.

E660nm 0 10 20 30 40 t (min) SM 16 Dallosil 75 Kieselgel 40 -HSA Fig. 1: Adsorbtion kinetics of methylene blue (10 pM) at RT with HSA 5% 100 mg gel/5 ml HSA 19 ExamDle 13 Removal of MB from plasma protein solutions by column chromatography The aim of this test was to find out whether or not the adsorptive removal of the photo-oxidant can also be effected by chromatography. This was based on the idea of carrying out the virus inactivation by means of a dye in combination with light in a container, such as a blood bag, and in turn transfering the plasma protein solution to another container, such as a second blood bag, via a small separating column interposed between said containers, and containing the adsorbing agent. If the assembly, comprising the first bag, the adsorbing column and the second bag were prefabricated so that a closed system were available, it would be possible in a very simple way and at the minimum risk of contamination to produce virus-inactivated plasma protein preparations, including from single donor units.

To this end 250 ml of 5% albumin solution were passed at varying flow rates through a separating column containing 5 ml of Kiesel-Gel (pore size 40 A) Fractions of 10 ml each were collected and their extinction was measured at 660 nm.

As can be seen from Table 13, the overall volume of the albumin solution could be passed through the column at flow rates of and 7.5 ml/min, respectively, and no MB residues could be detected in the fractions coming off the column. Hence, the time required for removal of the dye from 250 ml of solution is only to 35 minutes at most.

The test result shows that the removal of the photo-oxidant by chromatography may be effected without any problems, and also proves that the above-mentioned production of virus-inactivated plasma protein preparations from single donor units is indeed possible.

20 Starting Material MB3 extinction.- (660 ma): 0. 067 fraction No. 1 3 7 9 13 Flow' Pate (ml/min) extinction 0.002 0.000 0.000 0,002 0.001 0.000 0.000 0.002 (660 mm) .0.001 0,001 0.002 0.003 0.001 0.001 0.001 0.001 Table 13: Chromiatographic separation of MB from a 5% albumin solution (1 paM MB concentration) '.1

A

1/ I, i Ak

Claims (6)

1. A process for inactivating viruses in blood and blood pro- ducts, comprising: adding phenothiazine dyes to the solutions or suspensions to be treated and subsequently irradiating said phenothiazine-dye containing solutions or suspensions with vis- ible light in the range of the absorption peak of the respec- tive dye, whereafter the blood or blood products may be passed over adsorbing agents for removal of the dyes, characterized in that the phenothiazine dyes are used at a concentration of from 0.1 to 2 uM and irradiation is effected directly in transparent containers, such as blood bags, used for collecting and storing blood.

2. The process as claimed in claim 1, characterized in that toluidine blue or methylene blue is used as the phenothiazine dye.

3. The process as claimed in.any one of the claims 1 or 2', characterized in that the solutions or suspensions to be treated are initially subjected to deep-freezing and are 'hen thawed prior to irradiation. o' 4. The process as claimed in claim 3, characterized in that the dye is added prior to the deep-freezing step. as claimed-in claim 3, characterized in that the dye is added after thawing and prior to irradiation. 1 6. The process as claimed in any one of the claims 1 to characterized in that said process is carried out using two containers suitable for collecting blood, such as blood bags, with a separating column interposed between said containers, and containing the adsorbing agent for the phenothiazine dyes.

7. The process as claimed in claim 6, characterized in that the adsorbing agents used are silica gels or such agents based on polystyrene divinylbenzene or acrylic ester polymers. i ,i4 7 INTERNATIONAL SEARCH REPORT Iniernational Apolication No PCT/DE 90/00691 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols applv. Indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC 5 A 01 N 1/02 II FIELDS SEARCHED Minimum Documentation Searched Classi t cation System Classification Symbols Int.Cl.: A 01 N; C 12 N Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched Ill. DOCUMENTS CONSIDERED TO RE RELEVANT Category Citation of Document, with indication, where appropriate, of the relevant oassages I Relevant to Claim No. X Proceedings of the Society for E>perimental Biology 1-10 and Medicine, vol. 148, 1975, pages 291-293, Te-Wen Chang et al.: "Photodynamic Inactivation of Herpes- virus Hominis by Methylene Blue (38524)", see the whole document X Corceptsin Radiation Call Biology, chapter 2, 1972, 1-10 pages 57-89,.-C.W. Hiatt, "Methods for Photo- inactivation of Viruses", see pages 79-83 X Photochemistry and Photobiology, vol. 29, 1979 1-10 Wallace Snipes et al.: "Inactivation of lipid-- containing viruses by hydrophobic photosensitizers and near-ultraviolet radiation", see page 785 page 790 and the whole document X J. gen. Virol., vol. 41, 1978 Grace S.L. Yen et al.: 1-10 "Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red", see page 273 page 281 and the whole document P,X WO, Al, 9007876 (NEW YORK UNIVERSITY) 26 July 1990, 1 see claims 11-13: page 3, lines 20-23 Special categories of cited documents i' later document oublished after the international filing date or document defining the general stall of the art which is not priority date and not in contlict with the application but cited to considered to be of particular relevance understand the princolle or theory underlying the invention earlier document out published on or alter the international document of oarticular relevance: the claimed invention cannot filing date De considered novel or cannot be considered to involve an Inventive sted document which may throw doubts on onorlty claim(s) or which is cited to establish the Dublication date of another "Y document ol oarticular relevance: the claimed invention cannot citation or other special reason las sDecifiedi De considered to involve an inventive step when the document is combined with one or more other such documents. such document referring to an oral disclosure, use exhibition or combination being obvious to a person skilled in the art other means document member of the same patent family document published onor to the international filing date but later than the oriority date claimed IV. CERTIFICATION Date of the Actual Completion of the International Search I Date of Mailing of this International Search Report 29 November 1990 (29.11.90) 19 December 1990 (19.12.90 International Searching Authority I Signature of Authorized Officer European Patent Office Form PCT ISA'210 (second sneet) (January 1985S Internationales Alkienzeichen PCT/DE 90/0 0691 III FINSCHLAGIGE VEROFFENTLICHUNGEN (Fortsetzung von Blatt 2) A rt Kennzeichnunq der Verbffentlichung, soweit erforderlich unter Angabe der ma~geblichen Teile Betr. Anspruch Nr. X J. gen. Virol., Band. 41, 1978 Grace S.L. Yen et 1-10 al.: "Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red", siehe Seite 273 Seite 281 und das ganze Dokument P,X WO, Al, 9007876 (NEW YORK UNIVERSITY) 1 26 Juli 1990, Siehe Anspriiche 11-13; Seite 3, Zeilen 2 0-23 Formtlat PCT/ISAI21'n (Zusatzbogen) (Januar 198S) INTERNATIONALER RECHERCHENBERICHT Internationales Aktenzeichen PCT/DE 90/00691 1. KLASSIFIKATION DES ANMELDUNGSGENSTANDS (bei mehreren Klassi fi kations symbol en sind 2lle 2nzugeben) Nach der Internationalen Patentklassifikation (IPC) Oder nach der nationalen Klasssifikation und der IPC lnI.CI.S A 01 N 1/02

11. RECHERCHIERTE SACHGEBIETE Recherchierter Mindestpr~fstof1 7 Klass ifi kati ons system KI ass iti ka ions sym bole I nL CI A 0 1 N; C 12 N Recherchierte nicht zum Mindestpriifstolf gehbrende Verbfentlichungeni, soweit diese unter die recherchierten Sachgebiete fallen 9 11l._EINSCHL.AGiGE VEROFFENTLICHUNGEN' Art Kennzeichnung der Verbflentlichung" ,soweit erforderlich tinter Angabe der rna~geblichen Teile' 2 Betr. Anspruch Hr 1 X Proceedings of the Society for Experimental Biology 1-10 and Medicine,Band 148, 1975, Seiten 291-293, Te-Wen Chang et al: "Photodynamic Inactivation of Herpesvirus Hominis by Methylene Blue (38524)", Siehe das ganze Dokument X Concepts in Radiation Cell Biology, Kapitel 2, 1-10 1972, Seiten 57-89, C.W. Hiatt, "Methods for Photo- inactivation of Viruses", Siehe Seiten 79-83 X Photochemistry and Photobiology, Band. 29, 1979 1-10 Wallace Snipes et al.: "Inactivation of .lipid-containing viruses by hydrophobic photosensitizers and near-ul* traviolet radiation", siehe Seite 785 Seite 790 und das ganze Dokument Besondere Kategorien von angegebenen Veroffentlichungeni ^T ptrVebf.nlchgdeahdmitraiolnA- A. Veroflentlichung, die den aligemeinen Stand der Techni'k-- TStreVrfntihgdeahdeitrninlnA- defiier, abr ncht ls esodersbedutsa anusehn it meldedatum Oder dam Priorititsdatum ver~tlentlicht worden defiier, aer nchtalsbesnder beeulam asushenist st und mit der Anmeldung nicht kollidiert, sondern nur sum 51teres Dokument, das jedoch erst am Oder nach dem interna- Versthndnis des der Erfindung zugrundeliegenden Prinzips tionalen Anmeldedatum verbffentlicht warden ist Oder der ihr zugrundetiegenden Theorie angegeben ist Verbtfenttichung, die geeignet ist, einen Priorit5tsanspruch Veroffentlichung von besondarer Bedeutung, die beanspruch- zweifelhaft erscheinen zu lassen, Oder durch die das Verbf- te Erfindung kann nicht als neu Oder auf erfinderischer fentlichungsdatum einer anderen im Recherchenbericht ge- keit beruhend betrachtet werden nannten Verbffentlichung belegt werden soil Oder die aus amn- emn anderen besonderen Grund angegeben ist (wie ausgetiihr) Verbtfentlichung von besonderer Bedeutung, die beanspruch- te Erfindung kann nicht als auf erfinderischer Titigkeit be- Verbtfentlichung, die sich auf eine mi~ndliche Otfenbarung, ruhend betrachtet werden, wenn die Veroffentlichung mit eine Benutzung, emse Aussteltung Oder andere Maflnahmen eitner Oder mehreren anderen Veroffentlichungen dieser Kate- bezieht gonie in Verbindung gebracht wird und diese Verbindung (Ur IP Veroffentlichung, die vor dem internationalen Anmeldada- einen Fachmann nah .eliegend ist turn, aber nach dem beanspruchten Priorit~tsdalum veroffent- Verbtfenttichung, die Mitgtlied dersetben PatentlAmitie ust ttcht worden 1st IV. BEScHEINIGUNG Datum des Abschlusses der internationalen Recherche Absendedatum des internationafen Recherchenberichts

29. November 1990 19. 12. Internationale Recherchenbehbrde Unterschrift des bevollmichtigten Bediensteten Europ~isches Patentamt F.W. HECK 4 Formbtstt PCTIISAJ210 (Blatt 2) (Januar 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO.PCT/DE 90/00691 SA 39764 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 01/11/90 The European Patent office is in no way liable for theseparticulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date WO-Al- 9007876 26/07/90 AU-D- 5085190 13/0b/90 For more details about this annex see Official Journal of the European patent Office, No. 12182 EPO FORM P0479 ANHANG ZUM INTERNATIONALEN RECHERCHENBERICHT OBER DIE INTERNATIONALE PATENTANMELDUNG NR .PCT/OE 90/00691 SA 39764 In diesem Anhang sind die Mitglieder der Patentfamilien der im obengenannien internationalen Recherchenbericht angefiihi-en Pa7tentdokumente angegeben. 01/11/90 Die Angaben Ober die Familienmitglieder entsprechen dem Stand der Datei des Europ~ischen Patentanits am Diese Angaben dienen nur zur Unterrichtung und erfolgen ohne Gew~hr. IWO-Al- 9007876 26/07/90 AU-D- 5085190 13/08/90 Fi~r nihere Einzelheiten zu diesem Anhang :siehe Amtsbiatt des Europiischen Patentnts, Nr.12/82 EPO FORM P0473