DK174934B1 - 5-Amino-(1-beta-d-rebofuranosyl) imidazole and triazole - Google Patents

Abstract

Allergy, autoimmune and neural diseases and diseases associated with restricted blood flow are treated by administration of 5-amino-(1-beta-D-ribofaranosyl) imidazol carboxamide (I) or 1-beta-D-ribofuranosyl 1,2,4-triazole-3 carboxamide (II) opt. with reagents which enhance the effects of (I) and (II). Enhancing agents include erythro-9-(2-hydroxy 3-nonyl) adenine. HCl, coformycin, 2'-dioxycoformycin and dipyridamole.

Description

in DK 174934 B1

The invention relates to a method for screening purine nucleoside compounds or analogs for their ability to enhance cellular synthesis and release of adenosine.

5 Adenosine belongs to the J class of biochemicals called purine nucleosides and is an important biochemical cell regulatory molecule, as described by Fox and Kelly in Annual Reviews of Biochemistry, Vol. 47, page 635, 1978, which affects a wide variety of cell types and I® is responsible for myriad biological effects. Adenosine is e.g. a potent vasodilator, immune cell function inhibitor, mast cell degranulation activator, granulocyte activation inhibitor, and a putative inhibitory neurotransmitter. Given the broad biological activity spectrum of adenosine, considerable forces have been, and still are, directed to the establishment of practical therapeutic uses of the molecule and its analogues. -.----------------------------

Since adenosine or similar purine nucleosides are believed to act at the level of the cell plasma membrane by binding to receptors attached to the membrane, previous work has focused on increasing the extracellular concentrations of the molecule.

Unfortunately, at the concentrations to be administered to a patient to maintain an effective extracellular therapeutic concentration, adenosine or its analgesic toxic, and therefore, the administration of adenosine alone has little therapeutic use. As a result, other ways to maintain locally high extracellular adenosine concentrations have been sought. The 3 most commonly used are: a) interfering with adenosine uptake with reagents specifically blocking adenosine transport as described by L · - - - - - - - 2 DK 174934 B1

Paterson et al., Annals of the New York Academy of Sciences, Vol. 255, page 402, 1975; b) inhibition of adenosine metabolism, which in turn makes cellular adenosine available for extracellular transport and out-of-cell use 5 as described by Carson and Seegmiller in The Journal of Clinical Investigation, vol. 57, page 274, 1976; of adenosine analogs made to turn to adenosine cell plasma membrane receptors with lower dissociation constants than adenosine.

There are a wide variety of chemicals that inhibit the cellular uptake of adenosine. Some do so specifically and are essentially competitive inhibitors of adenosine uptake and others non-specifically inhibit. Theophylline acts as a competitive inhibitor, while dipyridamole, and a host of other chemicals, including colchicine, phenethyl alcohol and papaverine, inhibit uptake non-specifically.

Methods for increasing the extracellular concentrations of adenosine by altering adenosine metabolism are mainly based on the use of chemicals which inhibit enzymatic degradation of adenosine. Most of this work has been focused on identifying adenosine deaminase inhibitors that convert adenosine to inosine. Adenosine deaminase is inhibited by coformycin, 2, -deoxycoformycin or erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride.

Some adenosine analogs have been prepared which have structural modifications to the purine ring, alterations in 30 substituent groups attached to the purine ring, and modi

fixations at, or changes in, the binding site of the carbohydrate moiety. Halogenated adenosine derivatives have been found to be extremely promising and have, as described by - 3 DK 174934 B1

Wolff et al., 1 the Journal of Biological Chemistry,

Vol. 252, p. 681, 1977, biological effects similar to the effects that adenosine induces.

Although all three of the above techniques have advantages 5 over the use of adenosine alone, they have several drawbacks, the most important being that they are dependent on chemicals that have adverse therapeutic side effects, mainly because they have to be administered at doses that are toxic. , and because they affect 10 most cell types non-selectively. As described in Purine Metabolism in Man, (eds. De Baryn, Simmonds and Muller), Plenum Press, New York, 1984, most cells in the body carry receptors for adenosine. As a result, the use of methods that generally increase adeno-15 concentrations in the body causes dramatic changes in normal cellular physiology in addition to controlling diseases that benefit from adenosine therapy.

__________________ The above discussion is to be understood to mean that a ---- -------- method that will increase extracellular concentrations of adenosine or adenosine analogs without complex side effects and which will allow to increase adenosine concentrations is corrected. selectively against the cells that will benefit most from this will have significant therapeutic use. Therefore, it would be advantageous to develop a method for screening various purine nucleoside compounds for their ability to increase cellular synthesis and release of adenosine.

A novel method of curing diseases based on increasing the extracellular concentration of adenosine, or adenosine nanalogs, using purine nucleosides, in particular the nontoxic purine nucleosides, 5-amino-1-D-ribofu, is disclosed. -ranosyl) imidazole-4-carboxamide (AICA riboside), and Ι-β-D-4 ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and allopurinol riboside (or allopurinol base which is converted to the riboside). The latter reagents are administered to a patient and taken up and converted into 5 mono- and sometimes triphosphate cells. Adenosine or inosine is generated from adenosine triphosphate during rapid cellular energy utilization, such as during seizures or under reduced blood flow, by a series of intracellular biochemical reactions. Inosine production is 10 much greater than adenosine production (about 100 to 1). The compounds then diffuse out of the cells and are consequently present in the nearest extracellular environment at high concentrations. Purine nucleosides such as AICA riboside increase adenosine production.

The increase in adenosine release is due in part to a reduction in 5 'nucleotidase activity. It can be seen from the following diagram that with reduced 5'-nucleotidase activity, IMP is not converted as quickly to inosine and more nucleotide is converted to AMP, which accumulates and is converted to adenosine. The KM for AMP (1 mM) is approx. 10 times higher than that of IMP (1 mM). Since adenosine concentrations are not significantly altered in the patient and changes occur only in areas of rapid AMP use, the treatment method does not generally cause adenosine release.

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Diagram 1. Adenosine metabolism.

This diagram shows the pathway by which adenosine ------- _d is different — or broken down — —a f — cell x, _, and adenosine can also be transported into cells or released from cells.

The 2'-deoxyadenosine metabolism may utilize some of these pathways: 1, S-adenosylmethionine methyltransferases; 2, S-adenosyl homocysteine hydrolase; 3, adenoside deaminase; 4, purinnucleoside phophorylase; 5 & 6, xanthine oxidase; 7, transport mechanisms; 8, adenosine phosphorylase (not established); 9, adenosine kinase; 10, 5'Nucleotidase and non-specific phosphatase; 11, adenylate kinase; 12, nu-cleoside diphosphokinase; 11, adenylate cyclase.

Therefore, it is clear that purine nucleosides, AI-CA riboside, ribavirin and allopurinol are a medically beneficial agent for establishing a high local extracellular concentration of adenosine without the toxicity or non-specific uptake problems associated with other methods. which regulates extracellular 1 - 6 DK 174934 B1 adenosine concentrations.

In the drawing, the figures show the following.

FIG. 1. In vitro effect of 5-amino-1- (β-D-ribo-furanosyl) imidazole-4-carboxamide on adenosine excretion 5 by lymphocytes.

FIG. 2. β-hexosaminidase release from control and rebavirin-treated mast cells. Mouse bone marrow mast cells grown for 13-7 days in media alone (Γ 1) or in 10 M ribavirin) were subjected to calcium ion exchange. Percent β-hexosaminidase release from quiescent and stimulated cells is shown as mean values +/- standard deviations of two values from 7 trials. x = significantly different from control cells (p <0.05). Similar results were obtained with DNP-BSA antigen stimulation of anti-DNP IgE-sensitized mast cells.

FIG. 3. Dose-reaction effects of ribavirin on mast cell O-hexosaminidase release. Mast cells were grown in media alone (control) or il, 10 or 20 μΜ ribavirin for 6 days, washed, and subjected to 20 A23187 (| 1) and net β-hexosaminidase release determined. Ribavirin-treated cells released significantly less β-hexosaminidase at all concentrations tested when exposed to A23187. Median content and spontaneous release did not differ in 25 control and ribavirin-exposed cells. The mean values +/- standard deviations of double determinations from 3 trials are plotted graphically.

The present invention relates to a method for screening purine nucleoside compounds 30 or analog thereof for their ability to enhance cellular synthesis and release of adenosine, characterized by administering a first to cultured cells a first mixture DK 174934 B1 7! (b) to said cultured cells administer a calcium ionophore or 2-deoxyglucose, to activate a net-C i .. __1 ____ 1__1.1__i__Λ__1 J --- j uw oucuuoiuti WQLUirxxstiiit? , c) determine the concentration or amount of adenosine released by the cultured cells, and d) compare the concentration or amount to the concentration or amount of adenosine released from a 10 or more set of control cells.

The effect of purine nucleosides, AICA riboside or ribavirin, on adenosine transport can be shown either in vitro or in vivo, with the effective concentration of each molecule being 10 μΜ-ΐ00μΜ. When applied to the cells in vitro, AICA riboside or ribavirin can be dissolved in an aqueous solution and added directly to the culture media. It is expected that the administration of dis-molecules by patients will be nitrated, since the purine nucleosides are not readily degraded by enzymes in the body or by exposure to the low pH found in the stomach. There is no a priori reason to believe that the drug cannot also be administered intravenously or by direct intramuscular injection, topically or by inhalation.

Upon contact with activated energy-demanding target cells, the purine nucleosides, AICA riboside or ribavirin, are transported intracellularly by a plasma membrane-bound diffusion transport system where they can be phosphorylated by adenosine kinase to obtain 30 purine nucleotide monophosphate. The latter chemical can be converted to the triphosphate, an intermediate in the novel purine nucleoside synthesis.

By controlling the amount of adenosine, and thus the efficacy of the AICA riboside or ribavirin treatment, the degree of increased extracellular transport is recorded by isolating the aqueous fluid surrounding the cells and determining the amount present in the media by chromatographic techniques. as described by Matsumoto et al., The Journal of Biological Chemistry, Vol. 254, page 8956, 1979. In the tissue culture experiments, 1 mmolar deoxycoformycline was added to the aqueous fluid to prevent adenosine destruction by adenosine deaminase. In addition, the residual 10 amount of adenosine in the cells was analyzed by high pressure liquid column chromatography after isolation from the cells as described by Matsumoto et al., The Journal of Biological Chemistry, Vol. 254, page 8956, 1979.

Since the amount of extracellular adenosine can be regulated by increasing or decreasing the concentration of AICA riboside or ribavirin found in the cellular fluid environment, the amount of extracellular adenosine can be increased by increasing the amount of administered AICA riboside or ribavirin if the adenosine concentration is too low. to be medically beneficial. Similarly, if excessive, the amount of adenosine can be reduced by reducing the amount of the purine nucleoside.

It is believed that AICA riboside or ribavirin treatment will benefit patients suffering from a wide range of diseases. Eg. For example, patients suffering from allergies, especially asthma, hay fever, chronic urticaria, urticaria pigmentosa and eczema, can expect to benefit from pu rinnucleoside therapy. as discussed in B. Benacerra and A.

Unanue, Textbook of Immunology (Williams & Williams, Λ 30 Baltimore / London, 1979), is the key to suppressing allergic reactions to preventing the release of pharmacologically active substances from mast cells. Mast cells are large basophilic stained cells with extensive granules containing substances such as histamines which are released by the mast cell during the allergic reaction and which are necessary to support the allergic reaction. The release of these pharmacologically active substances, which is found in 1 cell cell, is called "urea granulation". Chemicals that prevent degranulation thus have a beneficial effect on reducing the rate of the allergic reaction. Patients suffering from allergies can be successfully treated with AICA ribosid or ribavirin, as these molecules prevent mast cell degranulation. Mast cell activation causes the release of prostaglandins and leukotrienes (non-preform mediators), such as slow-acting drug in anaphylaxis.

In addition, patients suffering from autoimmune disorders may be relieved if treated with one or the other purine nucleoside. Autoimmune diseases occur naturally and involve an im - mouth reaction to the individual 's own tissue. To initiate an autoimmune reaction, it is necessary that different immune cells interact to support the reaction.

Chemicals that interfere with the necessary cell-cell interactions can thus be expected to inhibit the course of the disease. An immune cell type required for the formation of an autoimmune reaction is the T lymphocyte. Since it is well known that adenosine is toxic to T lymphocytes, it should be presumed that administration of AICA riboside or ribavirin will eradicate or inhibit this immunity of immune cells and thus be of considerable therapeutic benefit to individuals suffering from autoimmune disorders. In addition, adenosine inhibits granulocyte production of oxygen free radicals.

In addition to allergies and autoimmune diseases, it should be possible to use AICA riboside or ribavirin in the treatment of diseases originating or exacerbated by insufficient blood flow through a given organ. Eg. For example, angina pectoris, transient ischemic attacks or migraine headaches can be treated by administering one or the other purine nucleoside.

This is to be expected as the adenosine is known as a potent vasodilating agent which acts by reducing vascular smooth muscle contraction.

In addition to acting as a vasodilating agent, AICA increases riboside or ribavirin collateral blood flow through another mechanism. Granulocytes often adhere to microvasculature in areas of restricted blood flow. Both drugs can cause granulocytes to loosen, thereby helping to restore normal blood flow. Thus, the uptake of AICA riboside or ribavirin by smooth muscle cells followed by subsequent release of the adenosine should cause vasodilation and / or removal of granulocytes.

Another disease associated with restricted blood flow is myocardial arrhythmia. Although limited blood flow initiates arrhythmia, the exact cause is unknown. However, lipid peroxidation of oxygen groups is known to be arrhythmogenic. Since the latter is secreted by granulocytes, AICA riboside or ribavirin inhibition of granulocyte activation can be expected to control arrhythmia. Granulocytes are also found in higher concentrations in areas of arteriosclerosis. Suppression of their activation Λ may reduce the release of other mediators for 30 arrhythmias.

The invention is further illustrated by the following non-limiting examples.

EXAMPLE 1

Regulation of mast cell degranulation with the purinucleotide, 5-amino-1- (β-D-rlbofuranosyl) imidazole-4-carboxy-5β-1β

The degranulation of mast cells plays an important role in allergic diseases such as asthma. Thus, a means of preventing degranulation provides a means by which the disease can be controlled.

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To detect increased excretion of adenosine from 15 mast cells, cells were first isolated from 250 g1 rats by flushing the pleural and peritoneal wells with Ca ++ and Mg ++ free Tyrode buffer containing 0.1 g gel _____letlne and 5 mg heparin in 100 ml. After washing the cells once by centrifugation at 100 x g in the above buffer, they were resuspended in Tyrode's buffer containing 0.1 g of gelatin and 1 mg of DNase per ml. 100 ml of buffer, filtered through nylon screen cloth and suspended at a concentration of 3-7 x 107 cells with cell nuclei per cell. ml. Next, the mast cells were purified on metrizamide gradients by suspending 2 ml of the 2 ml cells! cushion structures consisting of 22.5% (w / v) metrizamide in Tyrode's buffer with gelatin and DNase, and centrifuged at 500 x g for 15 minutes at room temperature.

The obtained cell pellets were washed twice and resuspended with 5-10 x 10 6 cells with cell nuclei per cell. ml and 4-5 ml were applied to 30 ml of a 3-9% (w / v) continuous metrizamide gradient. Centrifugation at 35 x g for 12 minutes at room temperature separated the red blood cells from the mast cells. Mast cells obtained by this technique usually make up more than 90% of the pelleted cells.

Effect of AICA ribosol on degranulation 5

Inhibition of degranulation by AICA riboside was demonstrated by showing that AICA riboside inhibits degranulation induced by the calcium ionophore A23187, which is expressed by the release of the acid exoglycosidase, B-hexosaminidase. A23187, 1 µg / ml, with or without AICA riboside is added to 2-5 x 10 6 mast cells at 37 ° C in Tyrode's buffer and the amount of released mast cell B-hexosaminidase is measured. In the presence of 100 μ molar AICA riboside, only 17.6% of B-hexosaminidase is released, whereas 28.8% is released when AICA riboside is not present. Thus, AICA riboside inhibits mast cell granulation. The percent release of B-hexosaminidase and the method of measuring the enzyme was carried out as described by Schwartz et al., Journal 20 of Immunology, Vol. 123, October 1979, page 1445.

EXAMPLE 2 AICA ribosil enhancement of adenosine release from lymphoblasts

Adenosine is a known inhibitor of certain sides of the immune response. Thus, an agent for regulating adeno-zine concentrations enables favorable treatment of 30 patients suffering from autoimmune diseases as they usually occur as a result of overactive immune cell reactions.

_i 13 DK 174934 B1

Isolation of lymphoblasts

The human spleen lymphoblast cell line WI-L2 was used to detect the effect of AICA riboside ζ vs X Æ J - lj -Λ- J _ ____ ~ QU9uvoxuj.ii ^ ivcj.oc · ^ cxiciiiiicus luatuLie uy tsy compounds described by Hershfield et al., Science, Vol.

197, page 1284, 1977. The cell line was stored in RPMI

1640 cell culture media supplemented with 20% fetal calf serum and 2 nM glutamine and grown in an atmosphere of 10 5% carbon dioxide in air. Since fetal calf serum contains purine metabolizing enzymes to produce the effect of AICA riboside, WI-L2 cells were incubated in RPMI 1640 medium supplemented with 20% heat-inactivate, dialyzed fetal bovine serum, 2 mM glutamine and 10 mM 15 4- (2-hydroxyethyl) - 18-piperazine ethanesulfonic acid buffer, pH 7.4 (Calbiochem).

[_____ _____ yirknlnq_of_AICAribos.ld_ Påadenos.infrXqjLv.else ___ _____________ 20 FIG. 1 shows that AICA riboside, ranging from 100-500 ymolar, increases adenosine release from lymphoblasts.

Ca. 1.4 nanomoles of adenosine / 10 7 WI-2 cells are spontaneously excreted and this number is increased to ca. 2.3 nano-25 moles at 500 ymolar ACIA riboside.

The amount of adenosine released from the cells to the supernatant, or the amount of nucleosides remaining in the cells, is determined by mixing 30 µl of cooled 4.4 N perchloric acid to the supernatant or 300 µl of 30 cooled 0.4N perchloric acid to the pelleted cells and centrifuging at 500 xg for 10 minutes at 4 ° C.

Each supernatant obtained is neutralized with 660 µl of a solution containing 2.4 g of tri-n-octylamine (Alamin in General Mills) in 12.5 ml of 1,1,2-trichloro-trifluoroethone ( Freon-113) solvent mixture as described by Khym, Clinical Chemistry, Vol. 21, page 1245, 1975. After centrifugation at 1500 x g for 3 minutes 5 at 4 ° C, the aqueous phase is removed and frozen to -20 ° C until assayed for adenosine or for nucleosides. Adenosine is determined isocratically on a C-18 microBondapak reverse phase column equilibrated with 4 mmolar potassium phosphate, pH = 3.4: acetonitrile 60% (95: 5 10 v / v) buffer. Adenosine eluted after 8-10 minutes and its identity was confirmed by its sensitivity to adenosine deaminase and by solution with adenosine standards. In addition, the extracted samples from the pelleted cells can be analyzed for nucleosides by high pressure hydrogen chromatography on a Whatman Partisil-10 (SAX) column equilibrated with 10 mmolar potassium phosphate, pH = 3.78, and eluted with a linear gradient to a 0.25 molar potassium phosphate, 0.5 molar KCl, pH = 3.45. Continuous measurement was performed by absorbance at 254 and 280 nm. Peaks are determined quantitatively by comparison with high pressure liquid chromatography analyzes of suitable standards.

EXAMPLE 3 Effect of AICA Ribosil on Adenosine Release in Neuroblastoma Cells According to the Invention

Neuroblastoma cell lines were grown in media and under conditions as described in Example 2. The media was supplemented with 0.005 mM - 0.5 M AICA riboside and micro-molar amounts of the calcium ionophore A23187. Under these conditions, the cells secreted at least 2 times more adenosine than the control cells.

EXAMPLE 4

Effect of ribavirin on mast cell granulation - Bone marrow obtained from Balb / C mouse females was grown in a 1: 1 mixture of Razin media and conditioned media prepared from co-cultured splenocytes from C57B1 / 6J and C3H mice in the presence of concanavalin A as described by Razin ., the Proc. Natl. Acad. Sci.

10 VSA 281 2559-2561, 1981. After weekly transfer and at least 15 days in tissue culture, the cells obtained were 90% pure mast cells and 95% viable as measured by Trypan blue exclusion. Cells exposed to ribavirin during culture were washed 3 times before use in experiments. Parallel cell cultures cultured in media alone were used as controls for pharmacologically engineered mast cells. Cell fluid was measured by cell counting at specific times and comparing the instantaneous number of ribavirin treated cells with the number of cells grown in media alone.

β-hexosaminidase was chosen as a representative granule-associated preformed mast cell mediator because it is easy to quantify and its release is almost exactly the release of parallel histamine. Mouse bone marrow mast cells were centrifuged at 200 xg for 5 minutes, washed 3 times in Tyrode's buffer containing no divalent cations, sensitized for 30 minutes at 37 ° C with anti-DNP (dinitrophynyl phosphate) igE (1 µg / 10® cells), and was exposed to either DNP-BSA antigen. ^ (175 ng / 3 x 10 5 cells) or A23187 (10 µg / ml / 3 x 10 5 cells) in 400 μΐ complete Tyrodes buffer for 10 minutes at 37 ° C. Reaction mixtures were centrifuged at 200 x g for 10 minutes and supernatant and pellet β-hexosaminidase concentrations were tested by hydrolysis of p-nitrophenyl-β-D-glucosamide as described by Schwartz et al. in J. Immunol 123, 1445 (1979). Spontaneous β-hexosaminidase release was determined in unexposed cells. The net% of β-hexosaminidase released is defined as follows:

Net% β-hex release. = super, [β-hex] _ -spontaneous 10 super, [β-hex] + pellet released [β-hex] wherein [β-hex] is β-hexosaminidase and super. is supernatant. When exogenous adenosine was present in the reaction mixture, it was added simultaneously with the secretion stimulant.

Mouse bone marrow mast cells exposed to A23187 or DNP-BSA antigen released 8-15% of the total cell β-hexosaminidase, a preformed, granule-associated mediator. Ribavirin (10 μΜ) added concomitantly with mast cell stimulation does not affect β-hexosaminidase release. However, mast cells incubated for 3-7 days in 10 μΜ ribarvirin washed and exposed to A23187 showed a marked impairment of β-hexosaminidase release compared to corresponding cells grown in media alone (Fig. 4). Exposure to ribavirin did not alter the mast cell mediator content (i.e., total cell β-hexosaminidase concentration) nor cell viability, and spontaneous release of β-30 hexosaminidase was similar in the two cell groups. The dose-response relationship between ribavirin exposure and pre-formed mediator release is shown in FIG. 5. Although 1 μΜ ribavirin for 6 days significantly inhibits mediator release, maximal inhibition is clearly between ΙΟμΜ and 20 μΜ.

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Claims (2)

A method for screening purine nucleoside compounds or analogs thereof for their ability to enhance cellular synthesis and release of adenosine, characterized in that a (a) to cultured cells is administered a first mixture containing the purine nucleoside compound or analogs to be screened , (b) administering to said cultured cells a calcium ionophore or 2-deoxyglucose to activate a net adenosine triphosphate catabolism; (c) determining the concentration or amount of adenosine released by said cultured cells; and (d) comparing said concentration or amount to said concentration or amount. adenosine released from one or more sets of control cells. The method of claim 1, characterized in that the cultured cells comprise a neuroblastoma cell line. The method according to claim 1, characterized in that In addition, the cultured cells comprise WI-L2 in the human splenic lymphoblast cell line. «4 _i DK 174934 B1 * 2.4-. «JS td S 22 u vo C 20 ·. srw I.8- · / Di / ^ j - 'ω / 05 / x I.6- · J ω yzy' M / w / o / z I.4 · / ω t - D <I.2- Lol- 1-1 | 1 1- I00 200 300 400 500. AICA-R (FINAL CONCENTRATION MEDIA J), Vi) FIG. I o Η ω 40Γ w 30Γ 5 WII ri CONTROL W ω u_i w Ο ^ RIBAVIRIN W «5 30- tu Μ ω 20- £ (Λ <ω S 2 £ <Μ 2 S ζ 20 - ο II '0- ο 33 —. —3Ε ~ ι g 5- * * »i É 5- -ti ..1-¾¾ - UL_ SPONTAN + A23IB7 x 2 Z 2 FIG. 2 Eh -v- O RIBAVIR FIG. C