WO1996037242A1 - Apparatuses and methods for treatment of blood - Google Patents

Abstract

Apparatuses and methods for inactivating lipid containing envelopped viruses such as HIV and hepatitis virus after these cells have been separated from the plasma of infected blood. In treating the blood, it is separated into uninfected components which are returned to the patient, and infected components which are treated with organic agents in liquid form including ethers and then returned to the patient. In treating red cells and platelets, the viral load of the cells is reduced and then the virus is inactivated by concentrations of the organic agents. After treatment is completed, the ether is removed and its residues are measured to assure that they are within an acceptable range. The apparatuses include centrifugal separators (50), an air-tight chamber (13), distillators (38), and a gas chromatograph (58).

Description

APPARATUSES AND METHODS FOR TREATMENT OF BLOOD

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-In-Part of U.S. Patent Application Serial No. 08/451,531 filed May 26, 1995, pending, which is a Continuation-In-Part of U.S. Patent Application Serial No. 08/068,510 filed May 27, 1993, now U.S. Patent 5,419,759, which is a Continuation-In-Part of U.S. Patent Application Serial No. 07/272,535 filed November 17, 1988, now U.S. Patent 5,484,396.

TECHNICAL FIELD OF THE INVENTION

This invention relates to apparatus and methods for treating infections of blood and blood components caused by enveloped viruses having lipids in their envelope, such as viruses which cause HIV infections and AIDS disease, hepatitis type B virus, non-A, non-B virus (hepatitis C) , and cytomegalovirus. This invention also relates to apparatus and methods for inactivating enveloped viruses associated with red cells and platelets separated from the plasma of donated blood without jeopardizing the viability of the cells.

BACKGROUND OF THE INVENTION

THE BIOLOGY OF HIV: The virus is an RNA virus and it has the enzyme reverse transcriptase which enables it to make a DNA copy of the viral RNA. The virus has an outer envelope and a dense cylindrical core containing RNA genome. The envelope consists of a lipid membrane with protruding spikes which are coated with glycoproteins. The glycoproteins contain different sugar molecules, some of which appear to be constant, but some may be variable. It is thought that it is the variability of these sugar molecules that eludes the body's immune system in detecting the virus particles.

The virus exists in the blood circulation of the patient in two forms: As cell-free virus or mature virion having a lipid envelope, and as cell-associated virus or replicating virus in the infected cells (T-4 lymphocytes, - 2 -

monocyte-macrophages) . The virus is essentially an intracellular parasite and in order to survive and perpetuate itself it has to penetrate and infect the host cells. The lipid envelope with its glycoprotein spikes provide the means for penetrating and infecting the white cells. The virus will replicate inside the infected cells and will produce mature virions with lipid envelope and glycoprotein spikes, budding from the membrane of the infected cell. These mature virions in turn will penetrate and infect the new and healthy cells as they are released from the hematopoietic system, and the vicious cycle will go on.

LABORATORY STUDIES: A number of laboratory tests have been developed to detect the serum antibodies to HIV antigens. Some of these tests are done at special clinical laboratories and some are mainly used for research.

Enzyme-linked immunosorbent assays (ELISA) are used to detect antibody to Human Immunodeficiency Virus (HIV) for screening blood products, for clinical purposes and for epidemiologic studies. Confirmation is accomplished by a second assay such as the Western blot test.

In Western blot, HIV antigens are separated electro- phoretically and the ELISA-reactive serum is tested against all of these separated antigens. Clear reactivity to certain virus antigens demonstrates true infection with HIV.

Seroconversion or the presence of detectable antibody levels is seen at 4 to 12 weeks after infection occurs. However, prolonged seronegativity up to a year or even longer has been reported.

BACKGROUND ART Background Art Related To Treating The Blood

EFFECTS OF ANTIVIRAL AGENTS ON AIDS VIRUS (HIV) : Antibiotics are not effective on viruses. Prior art does not offer a drug or a method to kill the AIDS virus. Prior art uses mainly nucleoside analogues, e.g. zidovudine or ribavirin, to treat HIV infections and AIDS. However, these antiviral drugs have been found to be effective only to a limited extent. These drugs are virustatic and not virucidal; they can inhibit the viral replication but they can not kill the virus.

Azidothymidine (zidovudine) has been offered in the market under the trade name of Retrovir. Zidovudine is a thymidine analogue; it is phosphorylated by cellular kinases to zidovudine triphosphate. The viral-enzyme reverse transcriptase incorporates zidovudine triphosphate (which competes with thymidine triphosphate) into viral DNA. Once incorporated into viral DNA, zidovudine triphosphate prematurely terminates the viral DNA, thus it inhibits the viral replication.

Ribavirin, which is another nucleoside analogue, can inhibit the viral replication by a similar mechanism. It has been reported that ribavirin delays the onset of AIDS in patients with lymphodenopathy.

There has been a number of complications reported to be associated with Retrovir therapy. However, the most serious and life threatening complications are hematologic toxicity including severe anemia and granulocytopenia requiring repeated blood transfusions and/or interruption of therapy. Another complication is hepatotoxicity.

The main disappointment with zidovudine, in addition to the hematologic toxicity, has been that it has failed to re-establish or at least improve the status of the immune system which is shattered in AIDS patients.

In the inventor's opinion this problem is inherent not only in the nucleoside analogues but in any antiviral drug or any method of treatment which may only be able to inhibit the viral replication in cell-associated virus, and which is not able to kill the cell-free virus. The cell-free virus which is thus not killed keeps penetrating and infecting the fresh and uninfected nucleated blood cells, i.e. T-4 lymphocytes, monocytes and macrophages, as they are released from the hematopoietic system. Another problem with the prior art is the emergence of drug-resistant viral mutants. Toxic effects of the antiviral drugs on host cells is also a problem since viral replication is an intracellular process.

In the U.S. Patent 4,381,004 titled "Extracorporeal Systems For Treatment Of Infections And Parasitic Diseases", a method for treatment for viral diseases in general has been provided. However, that method of prior art has a major problem: it uses antiviral drugs for treatment of viral infections in blood or plasma. Whereas it is well known to the art that antiviral drugs have no effects whatsoever on the mature viruses or virions (cell-free viruses) . The effects of antiviral drugs are only on the replicating viruses inside the infected cells. Thus by using this method of the prior art the cell-free virus or virion which is not affected by the antiviral drugs will keep penetrating and infecting the fresh white blood cells, particularly the T-4 helper lymphocytes. Consequently, the biological cycle of the virus will still go on. In other words, as I have explained before, it is the inherent limitation of the effects of the antiviral drugs used in prior art which is the problem, regardless of whether these drugs are administered to the patient directly or used extracorporeally. Further, this problem is not dose related, so using antiviral drugs extracorporeally in the hope that larger amounts may give better results would be futile.

Cham, U.S. Patent 4,895,558 discloses a method for delipidation of autologous plasma wherein the blood is drawn from a patient, separated into red cells and plasma and the plasma is treated with a lipid solvent. The delipidated plasma is remixed with the red cells and reintroduced to the same patient from whom it was removed. The red blood cells are not treated.

Babb, U.S. Patent 4,381,104 discloses an extra¬ corporeal method of treatment for diseases caused by pathogenic microorganisms in the blood stream of a patient or in the blood of a blood donor. In one embodiment, the blood is separated into plasma and red cells. The plasma is treated with a microorganism inactivator such as an antibiotic or an antiviral. In another embodiment, whole blood is treated. Babb does not provide a method for inactivating microorganisms which might be associated with the red blood cells or platelets.

Eibl et al, U.S. Patent 4,904,641 discloses a method of inactivating reproducible filterable pathogens in the blood plasma products by applying elevated temperature. The inactivation is done in a closed container in the presence of organic compounds in gaseous state and in the presence of water which is bound to the plasma products. Eibl et al does not suggest treating the red blood cells or platelets to inactivate viruses which may be present in them.

Background Art Related To Treating

Blood Compositions Containing Viable Cells

The prior art does not provide a method for inactivating the enveloped viruses which may be present with the red blood cells and platelets that have been separated from plasma of infected donated blood.

The prior art, however, does provide a variety of serological testings to detect viral contamination of the cells, which testings are usually accomplished by detecting antibodies against viruses, notably against HIV and hepatitis type B. However, there are problems. For example, a seriological screening test known as emzyme-linked immunosorbent assays (ELISA) to detect antibodies against HIV in blood products fails in about 1 in 200 cases (0.5%). There is no reliable way to exclude hepatitis B carriers by seriological testing because the carriers fail to develop antibodies. As a result, there is always a possibility that a recipient may receive HIV or hepatitis B virus with red cells (packed cells) or platelets donated by a person who is a carrier for hepatitis type B, or may be a seropositive for HIV but in whose case the antibody testing has failed to demonstrate his infectivity. In blood centers the donated blood is separated into red cells (packed cells) , platelets, and plasma. The plasma is submitted to fractionators which fractionate it into various fractions such as factor II, factor III, factor VII, factor IX, human globuline, etc. for various pharmaceutical use. The fractionators use the combination of non-ionic detergents and organic solvents for inactivating the enveloped viruses in plasma. The concentrations of organic solvents used for this purpose is high, around 20%, which would be detrimental to the cells.

However, the technology for inactivating enveloped viruses associated with red cells and platelets, which may originate from an HIV infected donor or hepatitis carrier, has not been developed. The problem with developing such technology has been the fragile viability of the red cells and platelets when they come into contact with detergents and the high concentrations of solvents, which lyse the cells.

The blood banking industry, in order to reduce the possibility of infected cells getting transfused to a recipient, runs a battery of seriological testings to detect the possible presence of antibodies to HIV, hepatitis virus, etc. Based on these testings, the units of cells that show positive reactions are discarded. However, as noted above, the serological testings are not 100% reliable. Consequently, the industry relies in part on careful screening of the potential donors. Of course the combination of screening and serological testing does reduce the risk of spreading HIV or hepatitis infections, but it does not totally eliminate those possibilities.

In the case of treating the plasma, however, the prior art offers a variety of methods to inactivate the enveloped viruses or other microorganisms.

Hrinda et al, U.S. Patent 5,300,433 discloses the use of sodium thiocyanate in combination with a physical process, preferably ultrafiltration for treating blood products containing a labile protein, to inactivate the virus. The blood products treated are prothrombin, factor II, factor VII, factor IX, factor X, protein C, and protein S.

Neurath et al, U.S. Patent 4,540,523 discloses the use of di- or trialkylphosphate or mixtures thereof with a nonionic detergent, to inactivate lipid containing viruses in a labile protein composition.

Neurath et al, U.S. Patent 4,764,369 discloses the use of di- or trialkylphosphate and a nonionic detergent, to inactivate at least 4 logs of virus in protein containing compositions of blood.

Neurath et al, U.S. Patent 4,820,805 discloses the use of di- or trialkylphosphate in the presence of a nonionic detergent and another inactivating agent such as alcohol or ether, to inactivate lipid containing viruses in a composition of protein which is the product of cancerous cells or gene splicing.

None of the Neurath et al patents teach a method for treating virus-contaminated red blood cells or platelets.

Shandrom, U.S. Patent 5,186,945 discloses the use of glycyrrhic triterpenoid compounds and a detergent to inactivate cytomegalovirus in human plasma.

Prince, U.S. Patent 4,481,189 discloses a process for inactivating hepatitis type B virus in blood plasma, wherein the plasma is contacted with a mixture of ether and a nonionic detergent.

Prince, U.S. Patent 4,459,505 discloses a process for inactivating hepatitis type B virus in blood plasma using alcohol, nonionic detergent, and ether.

Prince does not suggest inactivating hepatitis virus associated with red blood cells or platelets in either one of these patents.

My U.S. Patent 5,419,759, incorporated herein by reference, discloses an apparatus and methods for treatment of blood infections due to HIV, hepatitis type B, and other enveloped viruses. The treatment is effected by a blood treatment apparatus wherein the blood is circulated and separated into a healthy component including red cells and platelets and an infected component including plasma and white cells. The infected component is treated with organic agents such as ethrs, halogenated ethers, etc. After the completion of treatment, the organic agents are removed, and the treated and tested plasma is returned to the patient. The red cells and platelets are not treated.

Horowitz et al, U.S. Patent 5,232,844 is the only patent found by the inventor which deals with inactivation of enveloped viruses in a biological composition containing cells. Horowitz et al utilizes a phthalocyanine compound in combination with light and a quencher to inactivate viruses. It is not known whether this method will have practical and commercial utility or not.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates schematically the basic components of the apparatus used for treatment of HIV infections, AIDS, and other infections caused by viruses.

Fig. 2 illustrates schematically the basic components of the apparatus used for treatment of viruses in blood compositions containing viable cells.

DESCRIPTION OF THE INVENTION This invention is directed toward apparatus and methods for treating infections of blood and blood components caused by enveloped viruses having lipids in their envelope, such as viruses which cause HIV infections and AIDS disease, hepatitis type B virus, Non-A, Non-B (hepatitis C) , and cytomegalovirus. This invention is also directed toward apparatus and methods for inactivating enveloped viruses associated with red cells and platelets separated from the plasma of donated blood without jeopardizing the viability of the cells. Method for Treating The Blood

Using HIV infections as an example, the premise of the invention is that in order to break the vicious cycle by which the virus perpetuates itself, it is imperative that we do two things: a) Inactivate the cell-free virus, b) Stop or substantially reduce the replication of virus inside the infected cells (T-4 lymphocytes and monocyte-macrophages) , by killing these cells or removing them from the blood. The virus is essentially an intracellular parasite and in order to survive and perpetuate itself it has to penetrate and get inside the host white cells (particularly T-4 lymphocytes) .

INACTIVATING THE CELL-FREE VIRUS AND REPLICATING VIRUS: As it has been described above, the AIDS virus (HIV) is an enveloped virus having lipids in its outer envelope. It is this outer envelope with its glycoprotein spikes which provides the means for penetrating and infecting the healthy T-4 lymphocytes, monocytes and macrophages. Since organic solvents, particularly highly volatile ethers such as diethyl ether and ethyl vinyl ether can dissolve lipids, I sought to develop a clinical method which would be technically feasible to dissolve or destroy the lipid envelope of HIV by adding diethyl ether, in an extracorporeal setting, to the patient's blood containing cell-free virus; thereby destroying the glycoprotein spikes and rendering the virus unable to penetrate and infect the healthy cells.

However, it was not known as to what would be the effects of the organic solvents such as ether: a) on the blood factors, enzymes, complement, antibodies, etc. that the patient either naturally possesses or has acquired during his life-time and which are vital to the physiological well-being of the patient; b) on the healthy cellular elements such as platelets which are needed for blood coagulation, without which after surgical incisions or injuries the patient may bleed excessively or possibly may bleed to death; c) on the red cells which are needed for blood oxygenation transporting the oxygenated blood to various organs; d) on the granulocytes which are needed for defense against offensive micro-organisms; and e) on the replicating virus inside the infected T-4 lymphocytes, monocytes and macrophages.

To elucidate these points, three series of experiments were conducted:

I. To study the effects of ether on the complement, enzymes, antibodies, blood factors, etc., various amounts of ether were added to human plasma in the test tubes (from 10% to 50% by volume) . The mixture was incubated for varying periods of time (from 5 minutes to 30 minutes) at room temperature (22° +/- 2° C) . After incubation, the ether was removed by placing the test tubes in a water bath at temperatures below 56° C (complement and some other components of plasma get destroyed at 56° C or higher temperatures) followed by centrifugation. The test tubes were then sent to a reliable clinical laboratory for testing. Recommended tests for evaluation of the immune system, serum antibodies, various proteins and enzymes were requested. The results were compared with the results from base-line tests without adding ether. All the tests showed values within the normal range indicating that ether did not damage or destroy complement, enzymes, antibodies, blood factors, etc.

II. The studies on the effects of ether on HIV and on the white cells infected with HIV were done at Bionetics Research, Inc., now named Advanced Bioscience Laboratories, Inc. , Kensington Maryland.

This study was done to determine the concentration of diethyl ether needed to kill the cell-free virus in plasma. In this study we ran into a problem but we also discovered that low concentrations of ether would also kill the living cells. After exposing the cell-free virus to varying dilutions of ether over varying amounts of time, then removing the ether, apparently there had been a residual amount of ether which had not been removed. After the first week of incubation we found that this residual ether had been toxic to the Target Cells (H-9 lymphocytes) , leaving no viable Target Cells to which the virus could be transmitted (if there were any viruses that had survived the exposure to ether) . Consequently the cultures related to all the dilutions of ether showed no viral growth. It was not clear as to whether the negative growth could be attributed to the effect of ether on the virus, or to the death of the cells.

We repeated the study with an improvement in the technique. By dilution, suction, and ultracentrifugation, the residual ether was removed. The virus pellet was thus separated and resuspened in culture medium with Target Cells.

In this study two groups of tubes containing 3-4 logs/ml of virus were tested. One group was exposed to from 5% to 50% of ether by volume and incubated at room temperature for 5 minutes, the second group was exposed to the same graduated amounts of ether and incubated for 10 minutes.

After 4 weeks, the positive culture tubes (virus unexposed to ether) showed growth of virus but all test tubes containing plasma spiked with HIV-I and exposed to ether showed negative growth indicating that no infectious virus could be recovered after incubation of HIV-I spiked plasma with as little as 5% diethyl ether (V/V) for 5 minutes at room temperature (22° +/- 2° C) .

We repeated the study at a larger scale. This time we spiked the plasma with HIV-I to contain 7 log₁₀ TCID_5Q/ml HIV-I. The results went beyond those of the first study and showed that up to 7 logs of virus could be inactivated by exposure to 5% ether for 5 minutes.

III. Having discovered that H-9 lymphocytes could be killed by low concentrations of ether, I decided to determine the lowest concentration of ether and exposure-time needed to kill the infected H-9 lymphocytes. We then conducted a third study exposing HIV-I/infected H-9 lymphocytes to graduated amounts of ether (5%, 10%, 15% and 20%) with exposure time of 5 minutes or 10 minutes. Incubation of HIV-I/H-9 infected cells with 10% or greater (V/V) diethyl ether for 5 or 10 minutes at 22° +/- 2° C inactivated all infectious virus. The HIV-I/H-9 infected cells treated with diethyl ether were not able to infect additional target cells when co-cultivated with equal numbers of H-9 cells.

In summary my studies demonstrated the following: a) No infectious virus could be recovered after 4 weeks from infected plasma containing up to 7 logs of HIV-I exposed to 5% ether at room temperature for 5 minutes. b) No infectious virus could be recovered after 4 weeks from HIV-I/infected H-9 lymphocytes at a concentration of 0.5 x 10 /ml exposed to 10% ether for 5 minutes at room temperature. c) Living H-9 lymphocytes could be killed by exposing them to low concentrations of ether, 10% for 5 minutes, at room temperature. d) Ether in concentrations up to 50% will not damage or destroy the antibodies, complement, blood factors, enzymes, etc. present in the plasma. e) Ether will kill the living blood cells, in the concentrations necessary to kill the virus.

From these studies it becomes clear that there would be a prohibitive problem in the use of ether if it were to be injected directly in the blood (intravenously) to effect the proposed treatment. It should be pointed out that ether was used for decades, and it may still be used in some parts of the world, in small amounts for general anethesia and it has always been recognized as being the safest anesthetic agent. However, if based on the kilograms of body weight or based on the blood volume, sufficient amounts of ether are administered to produce the therapeutic concentration of 10% by volume in the plasma in order to kill both the cell-free virus and the infected cells containing replicating virus, the same concentration would also kill all the healthy cellular elements which are vital to the patient (red cells, granulocytes, platelets) . This of course is not compatible with life; because total loss of all red cells will cause immediate cessation of oxygenation; and total loss of platelets will dangerously jeopordize the clotting mechanism. Another problem would be that the volume of ether administered at one time to attain such concentration would also be incompatible with life because of its effects on the central nervous system.

The only way to accomplish the goal of using organic solvents, such as diethyl ether for treatment of HIV infections, is to treat the infected blood in an extracorporeal apparatus. In accordance with the apparatus and methods of the present invention, the healthy cellular elements (red cells, platelets, and also granulocytes, if need be) are first removed from the infected blood and returned to the patient. Then the infected plasma containing cell-free virus and replicating virus inside the infected cells is mixed and treated with sufficient amounts of organic solvent, namely diethyl ether, (about 10% or more) . The mixture is agitated at room temperature in order to prevent the ether from getting separated from plasma because of its volatility. After the treatment is completed, the ether is removed and the treated plasma is returned to the patient. Diethyl ether is highly volatile and it has a boiling point of only 34.431° C. Removal of ether from treated plasma is accomplished by distillation below 56° C under vacuum or by centrifugal forces under vacuum. To make certain that no residual ether is remaining in the treated plasma, a combination of the two techniques may be used.

The treatment apparatus (described hereinafter) is operated either continuously or in cycles. Therefore, the flow of infected blood from the patient (or from a container) to the apparatus and the flow of treated blood from the apparatus back to the patient (or the container) may be continuous or intermittent. In any case the treatment is continued until the entire blood is made substantially free from viable cell-free virus and from viable infected white cells containing replicating virus. When the initial treatment or treatments have reached a point when the viable infected white cells have been substantially eliminated from the blood circulations as indicated by blood tests, from then on it would not be necessary to include the white cells in the plasma to be treated by organic solvents. At that point the infected plasma is separated from all the blood cells and the infected plasma containing cell-free virus is treated by organic solvents, preferably ether. This modification of technique is advantageous because after the initial treatments when the infected white cells in the blood have been killed and the fresh and healthy T-4 lymphocytes and monocyte-macrophages have entered the blood circulation from the hematopoietic system, it is desirable to preserve these healthy cells and not subject them to the effects of ether any more. The T-4 helper lymphocytes play an important role in maintaining the patient's immune system. One of the main reasons why the immune system of the AIDS patient becomes weak and the patient becomes prone to opportunistic infections such as pneumocystic carinii pneumonia, etc., is that the patient keeps losing his healthy T-4 helper lymphocytes.

Method for Treating Blood Compositions Containing Viable Cells

The present invention is also aimed at preventing the virus contaminated red blood cells and platelets from getting transfused to recipients. Thus the invention will reduce the risk of spreading HIV infections or hepatitis and other diseases caused by enveloped viruses via transfusion. Since the main bulk of viral load of an infected unit of blood is in the plasma which is separated from the red cells and platelets, there is only a small number of virus remaining with the red cells or platelets. This small number of viruses can be inactivated by low concentrations of organic agents which will not be detrimental to the cells. If necessary, the low viral load of the cells can further be reduced to a minimum by washing off the virus which will then make it possible to inactivate them by very low concentrations of organic agents without damaging the red blood cells or platelets. Any infected white cells which may be mpanying the red cells or platelets may be removed by filtration systems.

After separation of plasma from the cells, it is estimated that the remaining number of virus with the red cells and platelets is about 1 log /ml or less when these cells are resuspended in a physiologically compatible fluid such as normal saline. Based on my study results, 1 log.. -/ml of virus can be inactivated by a concentration of as little as 0.75% ether (v/v) .

The potential harmful effects of organic agents on the red cells and platelets can be further reduced by the following process. After the plasma is separated from the red cells or platelets and before contacting them with organic agents, the cells are resuspended in a physiologically compatible liquid such as normal saline. The suspension is agitated on a shaker for a few minutes to wash off the viruses from the cells. The cells are separated from the liquid which will contain most of the viral load of the cells. The separation is accomplished by conventional means such as filtration or centrifugation. The process is repeated as necessary, e.g., 2-3 times or more, until a point is reached that after resuspension of the cells in physiologically compatible liquid, the viral load of the liquid is only a fraction of 1 log /ml of virus. At that point, it is contemplated that the concentration of the organic agent needed to inactivate a fraction of 1 log /ml of virus would be about 0.5% to 0.75% or less.

Thus in accordance with the present method, the concentrations of organic agents e.g. diethyl ether needed to inactivate the viral load of the red blood cells and platelets are reduced to the levels that are not harmful to the cells. Based on my studies, a concentration of 10% ether is needed to kill the cells. The organic agents are then removed by conventional means such as centrifugation or distillation under vacuum. The vapors of the ether are chilled, condensed to liquid, and collected in a reservoir. The treated cells or platelets are collected in suitable sterile containers for transfusion. After the completion of removal of the organic agent, samples are taken from the treated cells and tested by sensitive devices, such as a gas chromatograph to make certain that the residues are within an acceptable range for transfusion.

According to the present method, the organic agents e.g. diethyl ether, are used in the concentration range of from about 0.01% to about 25%, and preferably in the range of from about 0.1% to about 2.5% in a physiologically compatible liquid such as normal saline. The organic agents will dissolve the lipid material in the envelope of the virus. The dissolution of the lipid material will cause the glycoprotein spikes of the envelope with its antigen system to collapse. Thus the virus will lose its infectivity.

Apparatus For Treating Blood From A Patient

There is shown in Fig. 1 a schematic drawing of an apparatus 2 operating in accordance with the present invention. The bulk of the components are commercially availiable, therefore, the details of their construction will not be given here.

It is demonstrated in FIG. 1 that infected blood is drawn from the patient 4 via the intravenous tubing 6. Typically the withdrawal of blood is done via a needle or catheter inserted in the right antecubital vein. In the preferred embodiment, the flow of blood at withdrawal from the patient 4 into the apparatus and the return of the treated blood from the apparatus back to the patient is continuous. The return of the treated blood is also accomplished by the use of a needle or catheter inserted into the left antecubital vein. The rate of blood flow may be effected by pump 12. Several kinds of such pumps are commercially available. The preferred embodiment of the apparatus comprises an enclosed, air tight, operating chamber 13 (represented in dashed lines) where the actual treatment of the infected plasma and infected cells is carried out. The operating chamber 13 and other related equipment may be disposed in a housing (not shown) .

The treatment with ether and with other inflammable organic agents is conducted in the enclosed, air tight operating chamber 13, in complete absence of oxygen. Before each treatment session, a pump 15 connected with the chamber 13 evacuates all the air from within the chamber. The chamber 13 is then filled with a non-flammable, non- combustible gas 17, such as nitrogen. A gauge and control mechanism 19 maintain the pressure of nitrogen within the chamber 13 considerably higher than the atmospheric pressure (e.g., at 2 or 3 atmospheric pressure) to prevent any potential air leak from outside into the chamber. In this manner, even if there is a spark created by one of the mechanical or electrical components of the apparatus, the spark could not ignite the ether because of the total lack of oxygen.

Anticoagulants are preferably injected into the extracorporeal blood stream at 8 near the point where blood is withdrawn from the patient. The most common anticoagulants are: Trisodium citrate, Sequestrene (EDTA) , and heparine.

An occluded vein sensor 10 is preferably used in the path of the blood flow to prevent the formation or continued existence of bubbles in the blood stream. Pump 12 propels the infected blood into centrifuge 14 which separates the red cells and white cells from plasma. The centrifuges 16, 18 and filtration system 20 separate the healthy cellular elements (granulocytes and platelets) from the infected cells (lymphocytes and monocyte-macrophages) . Intravenous fluids are added at 22 to these healthy cells (red cells, granulocytes, and platelets) , and pump 24 returns them to the patient via the tubing 26. The infected plasma containing cell-free virus and the infected cells containing replicating virus are propelled by pump 28 into mixer 30 where they are mixed with organic solvent preferably ether coming from container 32. The agitator 34 mixes the ether with plasma and infected cells and will not allow ether to get separated from plasma because of its volatility. After treatment is effected, the mixture of treated plasma, lymphocytes, monocyte-macrophages and ether is propelled by pump 36 into the distillation station 38 where the mixture is heated inside water bath 44 to temperatures of 50°-52° C by electric coils 46. The suction machine 42 aspirates the ether and collects it in container 40. The mixture of plasma and cells still containing some residual ether is propelled by pump 48 into centrifuge 50 where suction machine 54 removes the residual ether separated by centrifuge 50 from plasma and collects it in container 52. The treated plasma is collected in collector 56.

A gas chromatograph 58 equipped with automatic sampling mechanism, takes repeated samples at regular intervals, from the contents of the collector 56 after treatment has been effected, and determines the residues of ether or any other organic agent used, in parts per million or parts per billion depending on the agent used. This is to ensure that the residues of agents used are within the acceptable range, and that the treated plasma or treated cells are safe to be returned to the patient. Pump 60 returns the treated plasma with killed cell-free virus and killed infected cells, to the patient at 62.

After treatment is effected, in the process of removal of ether by centrifuge-vacuum systems and/or distillation systems, cooling mechanisms or compressors 23 are used to compress the vapors of ether, which is then collected in bottles.

It should be pointed out that although this preferred embodiment has been described as a continuous operation and continuous blood flow, the treatment of blood infections caused by enveloped viruses according to the instant invention may also be accomplished by batched technique as described below. The batched technique is particularly useful in blood banks to treat the units of blood, plasma or blood products before releasing them for transfusion. The technique will prevent the transmission of diseases caused by enveloped viruses from blood donors to transfusion recipients, e.g. hepatitis type B.

As it was pointed out above, during the course of treatment when a point is reached when all the infected lymphocytes and monocyte-macrophages have been eliminated from the blood circulation, as indicated by blood tests, from then on it would not be necessary to include the white cells in the plasma to be treated by organic solvents. At that point after centrifuge 14 has separated the blood cells from plasma, the blood cells will all be returned to the patient with the aid of intravenous fluids 22 and pump 24 via line 26; and the centrifuges 16, 18 and filtration system 20 will be bypassed.

Apparatus For Treating Blood Compositions Containing Viable Cells

Referring now to Fig. 2, the preferred embodiment of the apparatus 2A for treating the blood compositions con¬ taining viable cells such as red cells and platelets has basically the same design as the system described above with some modifications. The modifications include filtration mechanisms for separating the red cells and platelets from any infected white cells, mechanisms for washing off the virus from the red cells and platelets, mechanisms for resuspending the cells in physiological fluids, and various other components such as tubing, pumps, etc., necessary for carrying out the method for treating the red cells and platelets. In the following description, the components previously described with reference to Fig. 1 are given the same numerals of reference. The bulk of the components of the apparatus are commercially available. Therefore the details of their construction will not be given here. In this embodiment, the actual treatment of the infected red cells and platelets is also carried out in an enclosed air tight operating chamber 13 (represented by dotted line) . The operating chamber 13 and other related equipment may be disposed in a housing (not shown) .

This embodiment also conducts the treatment with ether and with other inflammable organic agents in the enclosed air tight operating chamber 13, in total absence of oxygen. The chamber is preferably double walled and has a door with a sealing mechanism (not shown) . Before each treatment session, pump 15 connected with chamber 13 evacuates all the air from within the chamber. The chamber 13 is then filled with a non-flammable, non-combustible gas 17, such as nitrogen. A gauge and control mechanism 19 maintain the pressure of nitrogen in the chamber 13 considerably higher than the atmospheric pressure (e.g. at 2 or 3 atmospheric pressure) to prevent any potential air leak from outside into the chamber. In this manner, even if there is a spark created by one of the mechanical or electrical components of the apparatus, the spark could not ignite the organic agent (ether) because of the total lack of oxygen.

It is demonstrated in Fig. 2 that the red cells or platelets having viruses on them are drawn from a container 3 via tubing 6 and pumped by pump 12 into a washing container 5. The rate of the cell flow is effected by pump 12. Several kinds of such pumps are commercially available.

Anticoagulants may be added to the cells at point 8 near where the cells are withdrawn from container 3. The most common anticoagulants used are sodium citrate, heparin, and ethylene-diamine-tetra-acetic- acid (EDTA) .

A physiogically compatible liquid 7 such as normal saline is added via line 9 to the withdrawn red cells or platelets in the washing container 5. An agitator 16 connected with the washing container 5 agitates the cells, together with the added physiologically compatible liquid 7, and washes off substantial numbers of the virus from the cells, significantly reducing the viral load. Pump 21 propels the washed cells and the physiologically compatible liquid to a centrifuge 23 where the cells are separated from the physiologically compatible liquid. Fresh physiologically compatible liquid is added via line 25 to the washed and separated cells, and the cells are resuspended in the fresh physiologically compatible liquid inside centrifuge 23. Pump 27 propels the resuspended cells to mixer 30 where ether is added to them from reservoir 32.

Agitator 34 connected with the mixer 30 agitates the mixture of ether, cells, and physiologically compatible liquid to maximize the effects of the ether on the remaining viruses, and prevents the ether from getting separated from the physiologically compatible liquid because of its volatility. After the completion of treatment, the mixture of treated cells, physiologically compatible liquid, the ether, and the inactivated viruses, is propelled by pump 36 into the distillation station 38 where the mixture is heated, e.g. in a water bath 44 to temperatures of from 37-52 degrees C by electric coils 46. The suction machine 42 aspirates the ether and by the aid of a condenser (not shown) collects it in container 40.

The mixture still containing some residual ether is propelled by pump 48 into centrifuge 50 where suction machine 54 removes the residual ether separated by centrifuge 50 from the cells, and collects it by the aid of condenser 23 in reservoir 52. The treated cells or platelets, now free of viable enveloped viruses and free of residual ether are collected in collector 56.

A gas chromatograph 58 equipped with an automatic sampling mechanism, takes repeated samples from the contents of the collector 56 after treatment has been completed, and determines the residues of ether or any other organic agent used, in parts per million or parts per billion depending on the agent used. This is to ensure that the residues of agents used are within the acceptable range, and that the treated cells or platelets are safe to be used for transfusion. Pump 60 propels the treated cells or platelets which are now free from viable virus, via tubing 62 into a fresh and sterile container 64 which is then sealed.

The design of the apparatus has been described as related to the treatment of red blood cells and platelets contaminated with enveloped viruses. The same basic design with some modifications can be used, according to the methods of this invention, for treatment of whole blood infected with or carrying enveloped viruses.

Such modifications comprise mechanisms for separating the whole blood into a red cell component including platelets, and a plasma component including white cells. The red cells and platelets are treated together or separately as has been described above. The plasma component is treated with organic agents such as diethyl ether in an amount and over a period of time sufficient to inactivate both forms of the virus, e.g., cell-free virus of HIV and the replicating form of the virus which is inside the infected lymphocytes and macrophages. After the two components of blood have been treated, they may be separately packaged or may be combined and packaged together to reconstitute whole blood.

General Considerations

Suitable organic agents for use in the present invention include: ethers, halogenated ethers, halogenated hydrocarbons, chlorinated hydrocarbons, chloroform, acetone, and alcohols. Ethers, which in small concentrations have effectiveness similar to ethyl ether may also be used, including ethyl vinyl, methyl, propyl, methyl isopropyl, methyl butyl, ethyl propyl, and methyl propyl ethers.

Halogenated ethers suitable for use include methoxyflurane (2,2-dichloro-l,1-difluoroethyl methyl ether, Penthrane) ; enflurane (2-chloro-l,1,2-trifluoroethyl difluoromethyl ether, Ethrane) ; isoflurane (l-chloro-2,2,2- trifluoroethyl difluoromethyl ether, Forane) ; and fluroxene (2,2,2-triffluoroethyl vinyl ether). Halogenated hydrocarbons suitable for use include halothane (2-bromo-2chloro-l,1,1-trifluoroethane, Fluothane) ; trichloroethylene; dichloromethane (methylene chloride) ; ethyl chloride; ethylene; 1,1,1-trichloroethane; and xylene.

Alcohols suitable for use in the present invention include ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol, and isopentanol.

A variety of physiologically acceptable or compatible liquids may be used for washing off the viruses from the cells and the platelets, such as normal saline (isotonic saline solution, 0.9% sodium chloride in water), 5% dextrose in water, lactated Ringer's solution, low molecular weight dextran, a solution of dextran and isotonic saline, human serum, purified animal serum, and others.

As to what kind of equipment to use to accomplish component separation and treatment will make no difference, as long as the equipment used and the procedure followed are in accordance with the present invention. What is currently used in the field of blood component separation, are a variety of centrifugal separators, or as they are called in the industry, cell separators. Filtration systems are also used in conjunction with cell separators to filter cells from platelets, or to filter the types of white cells from each other.

The number and location of the cell separators, filters, and the types and locations of other pieces of equipment shown in the drawings, and discussed herein illustrate the concept that: (a) necessary pieces of equipment are used to separate the infected components of blood; (b) these components are then treated with certain amounts of organic agents over given periods of time, then appropriate equipment are utilized to remove the organic agent from the treated components, and after removal; (c) the treated components are tested to determine their safety, by measuring their residues of organic agents; and thereafter, (d) if the residues are acceptable, the treated components are returned to the patient, containerized for transfusion, or they may be combined and packaged together to reconstitute whole blood.

It should be understood that those who are experienced in the design and construction of this type of apparatus, will select the most suitable materials of construction, and most efficient pieces of equipment available. It should also be understood that additions, modifications, improvements, simplifications, and changes may be made; including changes in the placement of various pieces of equipment in the operating chamber and in the housing, giving consideration to the safety, efficiency, and easiness of operating the apparatus. For example, electronic equipment, computers, circuit boards, microprocessors, controls, relays, special wiring, switches, etc. may be added. Other additions such as disposable units, containers, reservoirs, etc. may be made; or exclusion of certain things which may not be safe to use, or which may not be efficient, or may be too bulky or cumbersome, etc. , may be made.

Claims

CLAIMS :

1. An apparatus for treatment of viral infections of blood compositions containing viable cells caused by infective enveloped viruses having a lipid envelope and spikes covered by glycoproteins, said apparatus comprising the following elements operatively connected with each other: a first reservoir containing a physiologically compatible fluid suitable for washing off enveloped viruses from said viable cells and suspending said cells; a washing mechanism connected with said first reservoir and adapted to receive blood compositions containing viable cells from a source for mixing said compositions with said physiologically compatible fluid and washing off a substantial number of said enveloped viruses from said viable cells and thereby reducing the viral load of said viable cells; a first component separator connected with said washing mechanism and with said first reservoir for separating said washed viable cells from physiologically compatible fluid containing the washed off viruses and introducing fresh physiologically compatible fluid to said washed and separated viable cells to suspend said washed and separated viable cells in said fresh physiologically compatible fluid; a second reservoir connected with said first component separator containing organic agents in liquid form selected from the group consisting of ethers, halogenated ethers, halogenated hydrocarbons, chlorinated hydrocarbons, chloroform, acetone, and alcohols: a mixing mechanism connected with said first component separator and with said second reservoir for mixing and treating said supension of washed viable cells and fresh physiologically compatible fluid with said organic agents; said mixing mechanism mixing and treating said suspension of washed viable cells and fresh physiologically compatible fluid with said organic agents in an amount and over a period of time sufficient to dissolve the envelope and destroy the glycoprotein covered spikes of enveloped viruses remaining on said washed viable cells (cell-free virus) thereby depriving said viruses of means for penetrating and infecting healthy cells, without damaging the viability of said cells; a removing mechanism connected with said mixing mechanism for substantially removing said organic agents from the treated cells after the completion of said treatment; a second component separator connected with said removing mechanism for separating and withdrawing any residual organic agents from said treated cells; a measuring mechanism connected with said removing mechanism for measuring the residues of said organic agents in said treated cells, after removing said organic agents, to determine whether said residues are within the acceptable range and said treated components are safe for reuse; and mechanisms for propelling said compositions and fluids, as needed at various points, during the course of treatment.

2. An apparatus according to claim 1 wherein said washing mechanism comprises an agitator mechanism for mixing said compositions in said physiological compatible fluid and agitating the mixture sufficient to wash off substantial numbers of said viruses from said viable cells.

3. An apparatus for treatment of viral infections of blood caused by infective enveloped viruses having a lipid envelope and spikes covered by glycoproteins, said apparatus comprising the following elements operatively connected with each other: component separators adapted to receive infected blood from a source and for separating said infected blood substantially into infected components including plasma and white cells, and substantially healthy or uninfected components including red cells and platelets; at least one reservoir containing organic agents in liquid form selected from the group consisting of ethers, halogenated ethers, halogenated hydrocarbons, chlorinated hydrocarbons, chloroform, acetone, and alcohols; a mixing mechanism connected with said component separators and with said at least one reservoir for mixing and treating said infected components with said organic agents; said mixing mechanism mixing and treating said infected components with said organic agents in an amount and over a period of time sufficient to dissolve the envelope and destroy the glycoprotein covered spikes of said viruses (cell-free virus) thereby depriving said viruses of means for penetrating and infecting healthy cells, thus inactivating said enveloped viruses, or sufficient to kill the infected white cells containing virus (replicating virus) thereby stopping the maturation of the virus within said infected white cells without damaging the biochemical constituents of plasma including antibodies, complement, clotting factors, emzymes, and blood factors; a removing mechanism connected with said mixing mechanism for substantially removing said organic agents after the completion of said treatment; a measuring mechanism connected with said removing mechanism for measuring the residues of said organic agents in said treated components, after removing said organic agents, to determine whether said residues are within the acceptable range and said treated components are safe for reuse; mechanisms for introducing suitable fluids into said components, as needed at various points, throughout the cycle of treatment; and mechanisms for propelling said components, as needed at various points, during the course of treatment.

4. An apparatus according to claim 3 including an enclosed operating chamber enclosing at least said mixing mechanism in which said treatment is conducted.

5. An apparatus according to claim 4 in which said operating chamber is air tight and is void of oxygen.

6. An apparatus according to claim 5 in which said operating chamber is filled with a non- combustible, non-flammable gas.

7. An apparatus according to claim 5 in which said non-combustible, non-flammable gas has a pressure at least equal to the atmospheric pressure.

8. An apparatus according to claim 5 in which said non-combustible, non-flammable gas is nitrogen.

9. An apparatus according to claim 3 including shakers and agitators connected with said mixing mechanism for shaking said mixer mechanism and agitating the contents thereof to facilitate efficient mixing and treating.

10. An apparatus according to claim 3 including return mechanisms connected with said component separators for returning said red cells and platelets to said source or to a receptacle, and with said measuring mechanism for returning said treated plasma and white cells, separately or in combination, to said source or to a receptacle.

11. An apparatus according to claim 3 including a mechanism for introducing anticoagulants and intravenous fluids to said infected blood or said components, as needed, during the course of treatment.

12. An apparatus according to claim 3 including temperature control mechanisms connected with said mixing mechanism, said removing mechanism, and said measuring mechanism whereby the process of mixing and treating said infected blood components with said organic agents; the process of removing said organic agents from the treated components; and the process of measuring said residues of said organic agents after the treatment is completed, are conducted and maintained at temperatures below 56° C.

13. An apparatus according to claim 3 including vapor-recovering and condensing mechanisms connected with said removing mechanism for recovering and condensing removed vapors of said organic agents, after the completion of said treatment.

14. An apparatus according to claim 3 including a mechanism connected with said component separators for substantially separating granulocytes from said infected white cell components; and a mechanism for returning said granulocytes to said source or to a receptacle.

15. A method for treatment of blood compositions containing viable cells to render said compositions free of infective enveloped viruses having a lipid envelope and spikes covered by glycoproteins, said method comprising the steps of:

(a) suspending said blood compositions in a physiologically compatible fluid suitable for washing off enveloped viruses from said viable cells and suspending said cells;

(b) contacting said suspended blood compositions with organic agents in liquid form selected from the group consisting of ethers, halogenated ethers, halogenated hydrocarbons, chlorinated hydrocarbons, chloroform, acetone, and alcohols, in an amount and over a period of time sufficient to dissolve the envelope and destroy the glycoprotein covered spikes of said viruses (cell-free virus) thereby depriving said viruses of means for penetrating and infecting healthy cells, thus inactivating said enveloped viruses without damaging the viability of said cells;

(c) separating and substantially removing said organic agents from the treated blood compositions to render said treated blood compositions suitable for reuse.

16. The method according to claim 15 including the step of prior to contacting said blood compositions with said organic agents; substantially reducing the viral load of said blood compositions.

17. The method according to claim 16 wherein said step of substantially reducing the viral load of said blood compositions comprises

(i) washing off substantial numbers of said viruses from said viable blood cells with said physiologically compatible fluid,

(ii) separating and removing said physiologically compatible fluid containing said washed off viruses from said viable blood cells;

(iii) repeating steps (i) and (ii) as necessary to further reduce the viral load of said washed viable blood cells;

(iv) resuspending said washed viable cells in a fresh amount of said physiologically compatible fluid; performing steps (b) and (c) as recited in claim 15 on the suspension of said washed viable blood cells in said fresh physiologically compatible fluid.

18. The method of treatment according to claim 15 in which said ethers comprise substantially diethyl ether.

19. The method according to claim 15 wherein said steps of suspending said compositions in a physiologically compatible fluid and contacting them with said organic agents, and said step of separating and substantially removing said organic agents from said compositions are carried out at temperatures below approximately 56° C.

20. A method for treatment of whole blood to render said whole blood free of infective enveloped viruses having a lipid envelope and spikes covered by glycoproteins, said method comprising the steps of:

(a) separating said whole blood substantially into a plasma component including white cells, and a red cell component including platelets;

(b) mixing and treating said plasma component with organic agents in liquid form selected from the group consisting of ethers, halogenated ethers, halogenated hydrocarbons, chlorinated hydrocarbons, chloroform, acetone, and alcohols, in an amount and over a period of time sufficient to dissolve the lipid envelope and destroy the glycoprotein covered spikes of said viruses (cell-free virus) thereby depriving said viruses of means for penetrating and infecting healthy cells, thus inactivating said enveloped viruses, or in an amount and over a period of time sufficient to kill the infected white cells containing virus (replicating virus) thereby stopping the maturation of the virus within said infected white cells without damaging the biochemical constituents of plasma including antibodies, complement, clotting factors, emzymes, and blood factors;

(c) suspending said red cell component in a physiologically compatible fluid suitable for washing off enveloped viruses from said viable cells and suspending said cells;

(d) contacting said suspended red cell component and physiologically compatible fluid with said organic agents in an amount and over a period of time sufficient to dissolve the lipid envelope and destroy the glycoprotein covered spikes of said viruses (cell-free virus) thereby depriving said viruses of means for penetrating and infecting healthy cells, thus inactivating said enveloped viruses.

(e) separating and substantially removing said organic agents from said plasma component and said red cell component; and (f) combining said treated plasma component and said treated red cell component to form reconstituted whole blood suitable for transfusion.

21. The method according to claim 20 including the step of prior to contacting said plasma component and said red cell component with said organic agents; substantially reducing the viral load of said red cell component.

22. The method according to claim 21 wherein said step of substantially reducing the viral load of said red cell component comprises

(i) washing off substantial numbers of said viruses from said red cell component with said physiologically compatible fluid,

(ii) separating and removing said physiologically compatible fluid containing said washed off viruses from said red cell component;

(iii) repeating steps (i) and (ii) as necessary to further reduce the viral load of said washed red cell component;

(iv) resuspending said washed red cell component in a fresh amount of said physiologically compatible fluid; performing steps (c) and (f) as recited in claim 20 on the suspension of said washed red cell component in said fresh physiologically compatible fluid.