Interferon and other antiviral agents, with special reference to influenza: a Memorandum*

Memorandaare state- Les Mimorandums ments concerning the exposent les conclu- Ml Vlemoranda conclusions or recom- sions et recommen- mendations of certain dations de certaines WHO scientific meet- reunions scientifiquesMerIl/l lnorandu1ms */ / ings; they are signed de l'OMS; ils sontby the participants in signes par les partici- the meeting. 'pants d ces reunions. Bulletin of the World Health Organization, 56 (2): 229-240 (1978) Interferon and other antiviral agents, with special reference to influenza: a Memorandum* Recent progress in the production of interferons and in knowledge of their mode of action has opened new possibilities for their prophylactic or therapeutic use, for example, in the treatment of influenza. Such use still needs further evaluation as do the presently available chemical antiviral drugs. The activity of interferon against other viral infections is also promising and preliminary trials on their effect on certain tumours have begun at a number of centres. There is, however, a needfor quality control of interferon preparations and recommendations for the production and use ofinterferon as well asforfurther research are included in this Memorandum. The discovery of interferon by Isaacs & Linden- mann in 1957 brought hope of combating viral infec- tions with a substance naturally produced by cells in response to viral aggression. Unexpectedly, the first applications revealed difficulties in obtaining con- sistent results because there were many unknown factors in the chemical nature of interferons, their mode of production by cells, and their mode of action on virus maturation. However, progress has recently been made which again opens up possibili- ties for the use of interferon. The purpose of this Memorandum is to review the present state of re- search on interferons and to identify facts on which a consensus can be reached. Because one of the first promises of the use of interferon was the prevention * This Memorandum was drafted by the signatories listed on page 240 on the occasion of a consultation held at the World Health Organization, Geneva in October 1977. Re- quests for reprints should be addressed to Virus Diseases, Division of Communicable Diseases, World Health Organiz- ation, 1211 Geneva 27, Switzerland. of influenza, particular attention is paid to this field as well as to the identification of those chemical drugs that could be used at present to prevent or treat this disease in the perspective of an epidemic or pandemic situation. As the efficacy of interferon on respiratory tract infections still needs further evaluation, other pro- mising applications have been reviewed as well. One of the difficulties in studies of interferon has been for some time the lack of reference preparations and the need for guidance on quality control. In order to help towards further progress in these fields, this Memo- randum summarizes the present position regarding the properties and mode of action of interferon, the source of interferon and its preparation, the inducers of interferon, the potential use of interferon in epi- demic situations with particular reference to influ- enza, and the quality control of interferon prepara- tions and other antiviral agents used for the treat- ment of influenza. The Memorandum also includes recommendations for future research. 3686 -229- MEMORANDUM PROPERTIES AND MODE OF ACTION OF INTERFERON Interferons are small proteins that are able to inhibit the replication of animal viruses; they are released after an appropriate induction in cells of animal species ranging from fish to mammals. The genetic information for the production of interferons as well as for the proteins necessary for their action is located in the cellular genome. CHEMICAL AND PHYSICAL PROPERTIES All interferons studied at present are glycoproteins with apparent molecular weights ranging from 12 000 to 160 000, depending on the host and the cell type used for their synthesis. Interferons are not antigenic in the homologous animal species but spe- cific antibodies can be obtained in a heterologous host after immunization. Early difficulties experi- enced in the production of such immune sera were probably due to the small amount of interferon protein present in the preparations. The molecules are resistant to a considerable range ofpH but at low pH the stability of the molecules is increased. All interferon preparations can be kept frozen at -20°C for long periods of time without significant loss of antiviral properties but with most interferons the activity is lost rapidly by heating to 56°C. Although their chemical structure is unknown, all interferons contain polysaccharide residues. In the case of leucocyte interferon, it has been shown that polysaccharides may constitute 15-20 %Y of the mole- cule, but the polysaccharides do not play a detectable role in the antiviral action. Two different interferons have been described from human cells: one produced in leucocytes and the other in fibroblasts. These interferons are distin- guishable by differences in their physicochemical properties and antigenic structure as well as in their stabilities and host specificities. In the case of fibro- blast interferon, it has been shown that the genes responsible for its production are located in both chromosome 2 and chromosome 5. The specific ac- tivity of pure interferons is very high, ranging from about 109 to 1010 units/mg of protein. BIOLOGICAL EFFECTS The antiviral effect of interferons is normally re- stricted to cells of the same animal species as the producer cells. In some cases, however, heterologous activity has been observed. Interferons inhibit the replication of a wide range of both DNA and RNA animal viruses, although the sensitivity of individual viruses to the action of interferon varies. In addition to their antiviral effect, interferon preparations can (a) decrease cell replication and inhibit tumour growth, as shown in vitro by colony formation and in vivo by studies with transplanted or virus induced tumours; (b) inhibit the replication of certain intracellular organisms such as rickettsias and protozoa; (c) change the sensivity of cells to toxins and hormones; and (d) inhibit circulating antibody formation and cell mediated immunity as well as enhance phagocytosis and cytotoxicity of lympho- cytes. Moreover, interferon seems to play a role in the regulation of the immune system. Some of these effects, however, could be due to biologically active impurities. The primary site of action appears to be on the cell membrane where the interferon molecule interacts with the cell by means of an interferon-specific recep- tor system. This system consists of a binding site made of gangliosides and an activator site probably consisting of glycoproteins. For induction of the antiviral effect, the integrity of the cell membrane is necessary. An interferon-specific modification of the cell membrane components is required as a primary step for antiviral activity and could probably explain many of the other biological effects. The cells also produce new products such as a protein/kinase and a nuclease, as well as other less precisely defined pro- teins. Inhibition of newly synthesized virus-specific pro- teins is probably due to alteration of the methylated cap of an RNA molecule and inhibition of synthesis of the polypeptide at the ribosomal level. Modifica- tion of RNA and transfer-RNA may occur in interferon-treated cells and, with large amounts of interferon, overall cellular RNA and protein synthesis may also be depressed. SOURCES OF INTERFERON AND ITS PREPARATION In general, cells make little interferon and for only antigenicity and species specificity of interferons a short period of time. Very large numbers of cells demands that a preparation intended for human use are therefore needed for large-scale production. The must be produced in human cells. At present, three 230 INTERFERON AND OTHER ANTIVIRAL AGENTS main sources of human interferon are available: leucocytes, diploid fibroblasts, and lymphoblastoid cell lines. Other potential sources of human inter- feron are transformed human epithelial or fibroblast- oid cell lines. Some such cell lines are relatively good producers of interferon and production may be fur- ther increased by genetic manipulation. HUMAN LEUCOCYTE INTERFERON At present, leucocytes are the main source of human interferon and most of the current knowledge on the pharmacokinetics, toxicity, and clinical use- fulness of interferon is based on studies with this material. As the trend in transfusion practice is towards the use of component therapy rather than whole blood, leucocytes are available in large num- bers for the production of interferon. It has been estimated that a modern blood transfusion centre serving a population of one million inhabitants could yield about 1014 leucocytes per year for the produc- tion of interferon and with present technology over 1011 units of interferon could be derived from this number of cells. The leucocytes are purified by lysing the few re- maining red cells with ammonium chloride or by low speed centrifugation. Although the lymphocytes are the main producers of interferon, they are not rou- tinely separated from the polymorphonuclear cells. The leucocytes are incubated in a suspension culture and " primed " with a small dose of interferon. The production of interferon thereafter is induced with a large dose of Sendai or Newcastle disease viruses. Different strains of Sendai virus differ considerably in their capacity to induce interferon production and compounds such as polyinosinic-polycytidylic acid (poly(I)-poly(C)) are almost inactive in this system. The crude interferon harvested after overnight incubation contains about 40 000 units/mi. Although fresh blood is used routinely, the yield of interferon is not adversely affected by storage of the blood for 1-2 days at 4°C. A method has been developed by which human leucocyte interferon, which is stable, can be concen- trated 5000-fold and purified 100-fold on a large scale with over 50% recovery. The interferon is dissolved in acid ethanol and the impurities are selectively precipitated by increasing the pH. The final product contains up to 108 units of inter- feron/ml and the specific activity is over 106 units/mg of protein. The partially purified preparations of human leu- cocyte interferon are very stable and can be stored for at least one year at 4°C without significant loss of activity. No stabilizing additives are required. HUMAN DIPLOID FIBROBLAST INTERFERON Human diploid fibroblast cell strains have several advantages over other cell substrates. They are nor- mal cells by karyotype analysis and tumorigenicity is unlikely. The cells can be propagated by serial pas- sage, thus providing a large quantity of uniform cells and they can be exhaustively examined for the ab- sence of adventitious agents as well as tumorigenicity before use by means of a " cell seed " system. Limita- tions include the finite lifetime of normal diploid fibroblasts and the fact that these cells will not grow unless anchored to a substrate. Large-scale culture systems for such cells have been developed and these human diploid fibroblast cell strains are already being used for the production of several established virus vaccines. Induction of interferon with viruses or synthetic polynucleotides such as poly(I)-poly(C) gives rise to only poor yields offibroblast interferon. Combined induction with poly(I)-poly(C) and sequential inhibition of protein and RNA synthesis (so-called " superinduction ") leads to satisfactory yields of interferon in the range of 105_106 units per 107 cells when certain cell strains are used. Cycloheximide and actinomycin D usually serve as metabolic inhib- itors in the superinduction process. A limitation of this method is that, since actinomycin D is an irre- versible inhibitor, each cell culture can be superin- duced only once. Recent work, however, suggests that the use of a reversible inhibitor at this stage may permit induction of the same cells for up to three times. A potential induction of type-C RNA viruses by inhibitors of protein synthesis used in the super- induction schedule should be considered in the char- acterization of the cell strain used for interferon production. After removal of the metabolic inhibit- ors and during interferon synthesis, serum or a plasma protein faction must be added to the culture medium. Heterologous serum, however, is not ac- ceptable for the preparation of fibroblast interferon for human use. High producer cells have been devel- oped from neonatal foreskin (FS4), embryonic skin (VG2S), and fetal lung tissue (Searle 17/1) and a search for other suitable human diploid fibroblast cell strains is in progress. Cell strains WI-38 and MRC-5, which have been used extensively for the manufacture of vaccines, produce unsatisfactory 231 MEMORANDUM yields of interferon. Based on the experience with leucocyte interferon, it may be concluded that one human dose of fibroblast interferon should contain about 106_107 units and for clinical application, therefore, crude fibroblast interferon will have to be concentrated about 100-fold. In order to obtain such large quantities of crude fibroblast interferon under workable conditions, mass cell culture technology such as multisurface propagators and microcarrier techniques are currently being studied. Several methods of concentration and partial puri- fication of crude fibroblast interferon have been described, including ultrafiltration, fractionation with ammonium sulfate, and affinity chromato- graphy. The fact that fibroblast interferon is less stable than leucocyte interferon must be taken into consideration during this process. The clinical value of fibroblast interferon, including the optimal dose to be used, has yet to be established. HUMAN LYMPHOBLASTOID INTERFERON Transformed human lymphoblastoid cells are pro- duced either from lymphomas or from normal leuco- cytes transformed by Epstein-Barr virus. Such cells often show karyotype abnormality and can be shown to contain copies of the EB virus genome. In addition, many lymphoblastoid cell lines behaves as transplant- able tumours when inoculated into immunosup- pressed hosts. At present, therefore, the use of lymphoblastoid interferon should be considered with great caution. Lymphoblastoid cell lines have the great advan- tage for interferon production that the cultures are continuous. They may also be grown on a very large scale in stirred suspension cultures, using established fermentor technology. It is likely that large-scale availability of the cells will favourably affect the cost of interferon. At present only one cell line, designated Namalva, has been well characterized and routinely produces high levels of interferon. In chemical and immuno- logical properties, Namalva interferon closely resem- bles leucocyte interferon. Namalva cells have been induced on a pilot scale in fermentors using para- myxovirus inducers and yields have been achieved that are comparable to those obtained with fresh leucocytes. INDUCERS OF INTERFERON A major problem which, until now, has prevented the use of interferon on a large scale is its very limited availability as well as the high cost of the little interferon that has been made available. Chem- ical compounds have been shown to be effective in inducing interferon but a number of compounds that have been found to be effective in rodents, particu- larly when given orally, have been shown to be inactive in primates. Some compounds, such as a copolymer of divinyl ether and maleic acid anhy- dride, or polyacrylic acid, are moderately active inducers but cannot be degraded and may be carci- nogenic. Poly(I)-poly(C) is a potent inducer of inter- feron in mice and is effective both prophylactically and therapeutically against a variety of virus infec- tions. Furthermore it has an inhibitory effect on a number of tumours. Unfortunately, it is a very poor interferon inducer in man, and in monkeys and chimpanzees it does not induce any detectable inter- feron. This ineffectiveness in primates may be related to the presence in primate serum of an enzyme complex which hydrolyses and inactivates poly(I)- poly(C). A complex of poly(I)-poly(C) with poly I-lysine and carboxymethyl cellulose (poly ICLC) is partially resistant to such hydrolysis and when it is adminis- tered to man or nonhuman primates good levels of serum interferon are produced. Up to 15 000 units of interferon per ml of serum have been produced in cynomolgus monkeys and 6000 units/ml in rhesus monkeys. The compound has been tested in nonhuman pri- mates against a number of serious viral diseases. It has a prophylactic effect in simian haemorrhagic fever. When given to monkeys 6 hours after inocula- tion of a large quantity of yellow fever virus, about 75% of the treated animals survived, compared with a 100% death rate in the untreated monkeys, and they developed good antibody titres. Similarly, one dose of the drug plus one dose of rabies vaccine is highly effective in postexposure prophylaxis of severe rabies infection in monkeys. In chimpanzee carriers of hepatitis B virus, the virus disappeared during the course of the development of serum interferon, but evidence of hepatitis B virus replication reappeared when the drug was withdrawn. Poly ICLC is also a potent immune adjuvant when used in conjunction with several vaccines. A preliminary study was car- 232 INTERFERON AND OTHER ANTIVIRAL AGENTS ried out in man to determine how high a dose could be tolerated and what levels of interferon would be produced. Up to 15 000 units of interferon per ml of serum have been found but these levels are associated with toxic reactions that can be attributed either directly to the drug or to the large amount of interferon. Lower doses of the drug can induce high levels of interferon, giving up to a few thousand units of interferon per ml of serum. Fever was the major side-effect with occasional leucopenia. At the very highest doses of the compound, thrombocytopenia has been observed. Treatment with this interferon inducer differs from treatment with interferon in several respects. The cost of treatment with the drug is at present only a fraction of the cost of treatment with interferon. Higher levels of serum interferon are obtained with the drug than with exogenous interferon. By and large, poly ICLC activates the immune system,. whereas interferon may be inhibitory; on the other hand, the toxicity of the inducer may be greater than that of interferon, although this aspect requires fur- ther study. The antitumour action of interferon has been demonstrated in murine models with a small number of tumour cells. Tumour immunity may be necessary for the action of interferons. Thus individ- uals who previously have been treated by other means and who have retained their immunity in whole or in part are desirable subjects for these studies. Large-scale controlled tests should be orga- nized to pursue the encouraging results suggested by individual observations. POTENTIAL USES OF INTERFERON IN EPIDEMIC SITUATIONS As interferon becomes more readily available, par- ticularly from buffy coat leucocytes, it is important to assess its possible role in the prophylaxis or modification of influenza. Success in prophylaxis as well as in the treatment of influenza with interferon has been reported from the USSR. However, in prophylactic trials in volunteers in the United King- dom more than one million units per patient were required as well as frequent intranasal sprays before signifiant effects were observed. Further controlled field trials are required, therefore, to define an opti- mal dose, as well as a better delivery system. The inability to determine when any one patient is ex- posed during the influenza season makes such pro- phylaxis difficult. Considering the large number of persons at risk during epidemic influenza, consider- able quantities of interferon would be required even for limited field application, but such quantities of this material are not available. Before interferon can be considered as an alternative to immunization or antiviral chemotherapy, therefore, its potential should be further explored. For example, little or no data are available on the local toxicity or long-term effects of interferon in the respiratory tract. Studies are under way to determine in vitro resistance to viral infection of cells obtained from the nasal epithelium of individuals given different dosages of interferon intranasally. In addition, inhibitors or inactivators of interferon have been found in nasal secretions, which together with the nasal clearance mechanisms explain the need for large doses of interferon for the prophy- laxis of influenza. Locally applied interferon appears to be as active in herpes simplex keratitis as the antiviral com- pounds currently in use. In addition, some encourag- ing preliminary results have been obtained in de- creasing the frequency of recurrent herpes simplex keratitis. Because of their prolonged presence in the tissues during infection, the herpesviruses have long pre- sented appealing targets for systemic antiviral che- motherapy and the use of interferons in immunosup- pressed patients is worth studying. Current studies have concentrated on infections with herpes zoster, herpes simplex, and cytomegalovirus in cancer pa- tients or those who have received organ transplants. A recent randomized double-blind placebo-control- led study demonstrated that cancer patients treated with large doses of interferon (35 x 106 units/day) have had fewer visceral complications with herpes zoster as well as less postherpetic neuralgia than untreated controls. Furthermore, they had a shorter course ofnew vesicle formation in the primary dermatome as well as distal cutaneous dissemination. As vesicle formation takes 1 or 2 days to be manifested in the patient, therapy must be started early in the course of the infection. Further studies are required to determine the minimum dose and duration of treatment for the maximum antiviral effects. Con- trolled trials are in progress to determine whether late prophylaxis or early treatment of varicella in children with leukaemia is possible. Prophylaxis of herpes simplex infection is being attempted in transplant recipients using fibroblast as 233 MEMORANDUM well as leucocyte interferon. Interferon is also being given to patients requiring surgery involving the trigeminal ganglion. Cytomegalovirus excretion in infants has been reduced with large doses of inter- feron but virus excretion returned to former levels after treatment ceased. Despite the transient nature of this effect, prophylaxis with either fibroblast or leucocyte interferon is being attempted at two centres in transplant patients who are at high risk of cyto- megalovirus infection. Preliminary results from one centre suggest that cytomegalovirus infection is not modified. Therapy of chronic hepatitis B infection is attract- ing much interest. Both leucocyte and fibroblast interferons are being used at several centres, and the infectious hepatitis B viral particles are the most affected by treatment. This effect is prolonged in some patients while others appear to be resistant. More extensive studies are required to evaluate the application of interferon in persistent hepatitis B infection. The most important long-term aims for such therapy would be (a) elimination of infectivity and (b) improvement of liver function. Diseases caused by Marburg, Ebola, and Lassa viruses are serious because of their severity and ability to spread in the hospital setting. A patient who became infected with Ebola virus and who subsequently recovered was treated with interferon soon after the onset of symptoms; however, since he was also given plasma containing convalescent antibody, no definite conclusions as to the efficacy of interferon could be drawn in this case. Several patients with symptomatic rabies have been treated systemically and intrathecally with leucocyte inter- feron but all have died and whether or not life was prolonged is not clear. Studies with interferon are in progress in relation to a number of chronic viral infections such as progressive multifocal leucoencephalopathy and sub- acute sclerosing panencephalitis. Consideration is also being given to the use of interferon in the treatment of certain acute arbovirus infections. In addition to the studies in Sweden, employing inter- feron to prevent relapse in osteosarcoma, prelimi- nary trials are under way in a number of centres with other tumours. Fever, fatigue, lethargy, bone marrow depression- and a transient reduction in the number of lympho, cytes have been observed in patients treated with leucocyte and fibroblast interferon. Higher levels of serum transaminase have also been consistently noted at the commencement of treatment. An im- mediate hypersensitivity type of reaction has been observed in the skin at the site of injections. In the case of some of these reactions, a rough dose- response relationship has been found but as more purified preparations of interferon have become available some side-effects appear to be less severe. The possibility that on rare occasions severe reac- tions may occur should be borne in mind. QUALITY CONTROL OF INTERFERON PREPARATIONS The following notes are included as a guide to those national control authorities who are formulat- ing requirements for the production and control of interferons. No details of tests are included because it is considered appropriate for these to be specified by the national control authority. CONTROL OF SOURCE MATERIALS Control of cell substrates The cell substrate used shall be approved by the national control authority. Freedom from contaminating viruses. The methods used shall be approved by the national control au- thority. Control of virus or other inducing agent The viruses or inducers used shall be approved by the national control authority. The appropriate tests include: 1. Viruses (a) identity (b) purity 2. Inducers (a) chemical purity CONTROL OF PRODUCTION METHODS The method used for the production shall be approved by the national control authority. All pro- cedures shall be carried out under aseptic conditions. 234 INTERFERON AND OTHER ANTIVIRAL AGENTS The time of sampling as well as the volume of each sample taken during the production shall be deter- mined by the national control authority. PURIFICATION AND CONCENTRATION OF THE INTERFERONS The methods of separation of the interferons as well as their purification and concentration from the supernatant fluids shall be approved by the national control authority. They shall be shown to remove or inactivate the virus inducer used and to remove substances that may give rise to allergic reactions in man. FILLING AND FREEZE DRYING The blending and filling of the concentrated and purified interferon shall be done under aseptic condi- tions. Any stabilizer added to the product shall be approved by the national control authority. When the contents of the ampoules are freeze-dried the limits of the moisture content of the dried prepara- tion in the final ampoules shall be approved by the national control authority. TESTS ON THE PRODUCT IN THE FINAL CONTAINERS The product in the final containers shall be sub- jected to the following tests: 1. Sterility-freedom from viruses, bacteria, myco- plasmas, and fungi. Tests for freedom from viruses present difficulties. Tests in heterologous systems should be considered and these should be specified by the national control authority. 2. Freedom from abnormal toxicity. 3. Freedom from endotoxins. The limits of the test shall be determined by the national control author- ity. 4. Potency. Many methods are available for the measurement of biological activity and the method used shall be approved by the national control au- thority. The potency shall be declared in reference units. 5. Purity. The number of reference units per mg of total protein shall be measured. The requirement for purity shall be approved by the national authority. The tests shall also include tests for substances used during the production and the upper limits allowable for such substances shall be specified by the national control authority. 6. Moisture content. The upper limit of moisture content shall be approved by the national control authority. LABELLING The label on the container shall state: (a) the international name; (b) the source of the interferon; (c) the potency in reference units; (d) the name of the manufacturer; (e) the number of the batch; and (f) the licence number of the manufacturer. In addition, the label on the package shall state: (a) the stabilizing agent (if any) and its concentra- tion; (b) the human dose; and (c) the total number of units in the ampoule. In addition, the leaflet shall state: (a) a brief description of the preparation of the product; (b) any contraindications to the use of the pro- duct; and (c) the conditions for which the product is indi- cated and the recommended human dose. STANDARDS FOR INTERFERON Comparison of results from different laboratories working with interferon is difficult. This is due pri- marily to reliance on bioassay for the quantification of interferon and the variety and inconsistency of the assay systems used. It is desirable, therefore, to develop international standards to permit the com- parison of results both within and between labora- tories. This is particularly important for clinical studies in order that dosages can be compared. There are currently four research reference reagents that are widely distributed. These were established in 1969 and are made available by the National Insti- tute for Biological Standards and Control in the United Kingdom and by the National Institute of Allergy and Infectious Diseases in the USA; they include mouse, rabbit, chicken, and human leucocyte interferon. In addition, there is a reference prepara- tion for leucocyte interferon in the Tarasevic Insti- tute in the USSR. Since 1969, these reagents have been widely used and considerable information has been obtained on the stability and application of these interferons. However, although they have con- 235 MEMORANDUM tributed significantly to the comparison of results from different laboratories, several limitations must be considered. When the same interferon sample is assayed re- peatedly in the same laboratory considerable varia- tion has been observed. Greater variations have been observed when the same interferon has been titrated in different laboratories. When using a reference preparation, the adjustment is made as a two-step process: 1. the relative potency of a laboratory reference preparation against the reference standard. 2. the assignment of a relative potency for an unknown test sample, determined in a single bio- assay determination, simultaneously with the labora- tory reference. The results of the unknown test samples are then expressed in terms of the two-step calculation with the assumption that the two comparisons move in the same direction. In general this may be true, but they may move in opposite directions. There are other complicating factors in the standardization including different assay systems, different dose- response slopes, and the use of different cells. When laboratory reference preparations are used, they should be compared simultaneously as many times as possible with research reference reagents. Preferably, a uniform assay system should be recom- mended. The results of these comparative titrations should be stated, together with the standard devia- tion and/or range of values, and the number of titrations that were carried out should be made known. It would be helpful if the uncorrected titres were reported, together with the comparative titres of the standard with the reference preparation. There is a clear need for a reference preparation for human fibroblast interferon, and there is also a need for specific antisera to human leucocyte, fibro- blast, and lymphoblastoid interferons for the pur- poses of identification. OTHER ANTIVIRAL AGENTS FOR INFLUENZA Most substances with antiviral activity have been identified by empirical testing of a large number of chemical compounds in an attempt to inhibit selec- tively the synthesis of viral components at concentra- tions that will not destroy the normal metabolic activities of the host cell. There are, however, several biological and biochemical processes that are essen- tial for viral replication and which are not essential for the survival of the cell. These processes may be more appropriate targets for selective attack by anti- viral drugs and may be listed as follows: 1. Attachment of the virion to specific cell recep- tors. 2. Transcription of early viral messenger RNA by the transcriptase of the virion. 3. Translation of early proteins from viral mes- senger RNA. 4. Replication of viral nucleic acid by virus-coded polymerases. 5. Post-translational cleavage of proteins and as- sembly of virions. 6. Regulation of viral gene expression. The following is a brief summary of information relevant to the prospects of antiviral chemotherapy of influenza. Review of the literature reveals a re- markable variation in the interpretation of results of experimental and field studies, thus emphasizing the need for further research. AMANTADINE Amantadine is a synthetic amine which is very stable. It is rapidly absorbed after oral administra- tion and in man the majority is excreted unaltered in the urine. Amantadine has been shown to have both in vitro and in vivo antiviral activity against strains of influenza A virus. It has also been shown to have in vitro activity against parainfluenza and rubella vi- ruses, but it lacks activity against influenza B, mea- sles, and other RNA viruses and DNA viruses. There are some reports which indicate that the mode of action of amantadine is inhibitory, prevent- ing or delaying entry of the virus into the cell. There also seems to be some action on the processes of uncoating and release of viral RNA and, on the basis of the lymphocytic choriomeningitis (LCM) virus model, amantadine may act at a second stage in the growth cycle. The yield of LCM virus was reduced even when the addition of amantadine was delayed for as long as 20 hours after infection. There are reports from the USSR suggesting that the antiviral effect is on the RNA-dependent RNA polymerase of 236 INTERFERON AND OTHER ANTIVIRAL AGENTS influenza virus. In tests with radiolabelled com- pounds and fowl plague virus, it was shown that amantadine or rimantadine had no effect on attach- ment and had a minimal effect on uncoating. Radio- labelled virus could be detected in the nuclei of host cells but no transcription occurred. It was concluded, therefore, that the major mechanism of action is on the polymerase. Amantadine has a dopaminergic effect in the cen- tral nervous system. It does not depress the immune response. Metabolism. Amantadine is rapidly absorbed after oral administration, reaching maximum blood levels in man after about 2-4 h, with a half-life of 10-24 h. About 90% of the drug is excreted unchanged in the urine after oral administration. In animals, when blood levels of the drug are low, relatively high concentrations have been found in the tissues, especi- ally the lungs. Normal dose in man. 200 mg daily as a single dose or in two divided doses. Toxicity. Very high doses are required in animals before toxic effects are observed and when these occur they are primarily related to the central ner- vous system. The drug is embryotoxic and terato- genic in rats when given at 12 times the dose for man (50 mg/kg/day), but this effect is not observed in rabbits at up to 25 times the recommended dose. Side-effects in man. About 10% of healthy volun- teers have mild reactions to an initial single dose of 100 mg, and 10-15% to a single dose of 200 mg. Although the incidence of reactions in healthy volun- teers decreased with continuous doses at the same level, side-effects occurred on average in 3-7% of healthy adults. Data are also available from a large number of elderly patients taking the drug continu- ously but for an unspecified period of time for the treatment of Parkinson's disease. Side-effects occur- red in 2-5% of those treated and included livedo reticularis and ankle oedema, nervous excitement, difficulty in concentration, dizziness or lightheaded- ness, orthostatic hypotension, urinary retention, slur- red speech, ataxia, and depression. Insomnia and lethargy may also occur. Nausea, anorexia, vomiting, dry mouth, tremors, and skin rash have occasionally been reported. More serious side-effects include con- gestive cardiac failure, psychosis, and leucopenia. Hallucinations and feelings of detachment have oc- curred. These effects appear to be dose-related and disappear on withdrawal of the drug. Doses of four times the recommended dose have caused convul- sions. Prophylaxis ofinfluenza A. In double-blind studies, amantadine was effective in preventing experimental and natural infection with influenza A. In most trials, there was at least a 50% lower incidence of illness and/or symptoms, fewer virus isolations and a 50-70% lower incidence of serological evidence of infection. Large field studies indicate that amanta- dine provides about 50% protection against infection and more than 60% protection against clinical ill- ness. Protection ceases when drug treatment is stopped, indicating that it should be continued for the entire period of exposure or, at least, for a period long enough to cover the development of antibody if immunization is employed during the epidemic. It is important to note that there are reports of the development of resistant strains in treated tissue cultures as well as in animals, but such strains have not been detected in man. In studies in which resis- tant strains were reinoculated into mice together with a wild strain, the resistant strain disappeared, sug- gesting a reduced ability to replicate. Because of the concern about side-effects, aman- tadine should be used with care for prophylaxis. It should not be used throughout the influenza season, and its best use might be for prophylaxis at the outbreak of influenza in a given community, treat- ment continuing for perhaps 2 weeks. Treatment of influenza A. Amantadine has been shown to have a clear therapeutic effect, although less impressive than the prophylactic effect, in the early treatment of acute influenza, resulting in a significant reduction in the duration of fever. In studies that included measurement of pulmonary function, it was observed that in naturally acquired influenza A infection, amantadine reduced the dura- tion of increased peripheral airway resistance after the acute phase of illness. This effect may be impor- tant in the more rapid return of patients to full activity. Treatment has a minimal or no measurable effect on the shedding of virus and there is no significant impairment of antibody formation. Precautions. Amantadine should not be given to patients with a history of epilepsy or of gastric or duodenal ulceration. Care should be exercised in elderly patients with cerebral atherosclerosis and in patients receiving treatment with stimulants of the central nervous system. The drug should also be used with caution in patients with cardiovascular, hepatic, 237 MEMORANDUM or renal disorders and in patients with recurrent eczema or psychosis. Amantadine may enhance the effects of trihexyphenidyl hydrochloride, benzatro- pine, and orphenadrine and doses of these drugs should be reduced if given concurrently with amanta- dine. ANALOGUES OF AMANTADINE Rimantadine Rimantadine is a synthetic analogue closely related to amantadine. It appears to be somewhat more active in influenza A both in vitro and in vivo and it is better tolerated. The mode of action and metabolism of rimantadine are similar to those of amantadine. Dose in man. 150-200 mg twice daily. There are some reports that the dose can be reduced to 100 mg daily. Toxic effects include vomiting, nightmares, and anxiety, but these are less frequent with the reduced dose. Other derivatives Numerous analogues of amantadine and rimanta- dine have been tested, and two compounds have recently been studied in the USSR. A monoalkyl derivative of rimantadine appears to be equally effec- tive against influenza B as against influenza A. The second compound is a rimantadine polymer with a plasma expander that delays excretion, presumably by delaying absorption; the dose can therefore be reduced so that instead of daily treatment patients can be treated every third day. RIBAVIRIN Ribavirin is a synthetic nucleoside analogue of guanosine. This drug has been shown to be active in vitro against a wide range of both DNA and RNA viruses. It is most active against the viruses of influ- enza A and B and parainfluenza. Ribavirin appears to be superior to amantadine both in vivo and in vitro against experimental influenza and parainfluenza in animals. Mode of action. It is phosphorylated in the liver and other tissues to ribavirin 5'-monophosphate, an inhibitor of inosine monophosphate dehydrogenase, thus blocking a critical step in the synthesis of guanosine nucleotides. Ribavirin is not virucidal, has no effect on viral adsorption, penetration, or uncoat- ing, and does not induce interferon. It acts intracel- lularly to inhibit viral nucleic acid synthesis, proba- bly in the guanine synthetic pathway. Its metabolites appear rapidly in the urine. Acute toxicity. The LD50 in rhesus monkeys is more than 10 g/kg. Subacute and chronic toxicity. A dose of 90-200 mg/kg/day for 30 days administered to albino rats resulted in a reduction in weight gain. Daily oral doses of 200 mg/kg for 30 days given to immature rhesus monkeys led to a reduction in weight gain and some haematological changes including depression of haemoglobin, haematocrit, and erythrocyte count. The anaemia was progressive but reversible when the drug was discontinued. In chronic toxicity studies, immature rhesus monkeys received daily doses of 30, 60, and 120 mg/kg orally for 6 months; significant decrease in the white blood cell count was observed with a dose of 120 mg/kg/day. A teratogenic effect was observed in rats treated orally with 10 mg/kg/day during days 6-15 of gesta- tion. A dose of 10 mg/day was embryotoxic in the rabbit. There was no suppression of the humoral antibody response in animals given up to 100 mg/kg. Weak depression of cellular immunity was noted with doses approaching toxic levels of the drug, i.e., 100-200 mg/kg/day. Side effects in healthy volunteers included mild frontal headaches, abdominal cramps, and fatigue when given 600 mg/day for 28 days. A dose of 1200 mg/day for 13 days caused anaemia, which was reversible when drug administration ended. Therapeutic trials. The results of clinical trials with influenza A (and with viral hepatitis types A and B) are conflicting and its therapeutic efficacy remains to be established. ISOPRINOSINE In animal experiments, antiviral activity was found against influenza A and some other viruses. How- ever, 2.5 g twice daily by mouth for 10 days was no more effective than a placebo in a double-blind trial with 30 volunteers infected, 48 hours after starting treatment, by intranasal inoculation of influenza A/ Hong Kong/l/68 virus. 238 INTERFERON AND OTHER ANTIVIRAL AGENTS RECOMMENDATIONS 1. Recent clinical studies of exogenous interferons of human cellular origin have shown promise for the treatment of certain acute and chronic viral infec- tions, as well as certain neoplasia. These findings should encourage and promote continued research on the development, production, and application of human interferon. 2. Exotic virus diseases with high mortality consti- tute an opportunity for clinical studies of interferon. 3. A centre should be established for the collation of data on interferon preparations and findings in clinical tests. 4. Standards for interferons, particularly standard assay techniques, should be developed and the neces- sary substrates should be made available. 5. The data concerning the currently avail- able research reference (human leucocyte, rabbit, mouse) reagents should be reviewed to determine whether they are indeed ready to be established as international standards and how they should be used. 6. A research reference reagent for human fibro- blast interferon has been prepared and should be made widely available. Specific antisera to human leucocyte, fibroblast, and lymphoblastoid interferon should be produced and made available. 7. Research on methods of production and testing of interferons should be encouraged. An example would be the research and development of DNA recombinants leading to the production of inter- ferons in prokaryotic cells. 8. Attention needs to be given to the comparative evaluation of alternative approaches to the control of epidemic influenza, including vaccines as well as antiviral drugs. The combined use of vaccines and antiviral agents should also be investigated in field trials under well controlled conditions. 9. Antiviral drugs such as amantadine and riman- tadine appear to be effective in the prophylaxis and therapy of influenza but there is a need for further research on these drugs. Comparative studies should be conducted to define more precisely their efficacy, toxicity, and effects on the central nervous system and behaviour patterns. FUTURE RESEARCH In spite of the advances that have been made in the preparation and experimental use of interferon in man, little is known either of its precise chemical composition or its mode of action. These areas require further investigation and attention should be devoted to the discovery and practical utilization of the actual cellular control factors that are induced in cells on exposure to interferon. Of very great impor- tance is the production of batches of very high purity for assays of reactogenicity and efficacy in man since some of the purported toxicities and actions of interferon may in fact be due to biologically active impurities, such as lymphokines, in the interferon preparations in current use. Much attention needs to be given to improving the production of human interferons of acceptable purity at the lowest possible cost. Although there is now emphasis on production from human leucocytes and human diploid fibroblasts, alternative methods of production involving tank culture of cells on micro- carriers, as well as the possible use of human lym- phoblastoid or even cells of neoplastic origin, should be considered. Methods should be developed for the production of interferon without the need for serum in the culture medium. Efforts should be made to- wards the chemical synthesis of interferon after more is known about the purification and sequencing. The synthesis of interferon by prokaryotic cells into which the human genetic material has been intro- duced by genetic recombination should be investi- gated. The different interferons should be characterized and the production and study of those that are particularly useful should be encouraged. Methods for safety assessment should be developed that will assure freedom of the product from contaminating viruses, such as those of hepatitis, and other harmful contaminating substances. Clinical studies should be carried out to establish optimal regimens for treating acute and chronic viral infections and neoplastic diseases from the stand- point of both safety and efficacy, realizing that even 239 .6 MEMORANDUM the most pure interferon may carry inherent adverse effects. The possible use of interferons for the preven- tion or curtailment of disease in limited outbreaks is worthy of investigation. A search should be conducted for safe and effec- tive inducers of interferons that may be equal to or better than those currently available for the preven- tion and treatment of diseases in man. * * * F. Assaad, Division of Communicable Diseases, World Health Organization, Geneva, Switzerland. P. Bres, Division of Communicable Diseases, World Health Organization, Geneva, Switzerland. K. Cantell, State Serum Institute, Helsinki, Finland. T. Cartwright, G. D. Searle & Co., England. C. Chany, Hopital Saint-Vincent-de-Paul, Paris, France. S. G. Dzagurov, State Institute for Standardization and Control of Medical-Biological Preparations (Tarasevic Institute), Moscow, USSR. G. J. Galasso, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. L. J. Higy-Mandic, Division of Prophylactic, Diag- nostic and Therapeutic Substances, World Health Organization, Geneva, Switzerland. M. R. Hilleman, Merck Institute for Therapeutic Research, West Point, PA, USA. G. N. Hlabic, Department of Vaccines and Other Biological Products, Ministry of Health, Moscow, USSR. H. B. Levy, National Institute of Allergy and Infec- tious Diseases, National Institutes of Health, Be- thesda, MD, USA. M. Majer, Behringwerke AG, Marburg (Lahn), Federal Republic of Germany. T. C. Merigan, Stanford University Medical Center, Division of Infectious Diseases, Stanford, CA, USA. F. Perkins, Division of Prophylactic, Diagnostic and Therapeutic Substances, World Health Organiza- tion, Geneva, Switzerland. P. de Somer, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium. A. J. Zuckerman, London School of Hygiene and Tropical Medicine, London, England. 240