【病毒外文文獻(xiàn)】1990 Intranasal Treatment of Picornavirus and Coronavirus Respiratory Infections in Rodents Using 7-Thia-8-Oxoguanosine

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Antiviral Chemistry and ii an intranasal encephalomyocarditis EMC virus infection in adult mice Concentrations of 0 3 and 1 7 thia 8 oxoguanosine delivered intra nasally to rats 24 and 18 hours before virus inoculation were highly protective against the otherwise lethal coronavirus infection A 1 concentration of drug administered 4 and 8 hours after virus challenge increased mean survival times of rats but did not increase numbers of survivors Intranasal treatment of an EMC infection produced moderate improvements in mean survival times and survival Titrations of EMC virus indicated 300 fold reductions in nasal titres in drug treated relative to placebo control animals on days 2 4 following virus challenge The distribution of r 4 C 7 thia 8 oxoguanosine was determined shortly after intranasal delivery to mice and rats Approxi mately 50 of total doses were deposited in the inner noses and mouths of both species Most of the rest was found on in the outer noses stomachs tracheas oesophagi and lungs By analogy the infecting viruses were deposited on in the same organs and tissues of each species The results suggest that containment of the viruses primarily occurred in the nasopharyngeal area prior to their spread to the lungs rat coronavirus or the brain EMC where fatal pathologies were manifest Intranasal application of an interferon inducing nucleoside analogue rep resents a new approach for the study of treatment of the common cold Received 26 September 1989 revised 23 November 1989 Present address and for correspondence Department of Animal Dairy and VeterinarySciences Utah State University Logan Utah 84322 5600 USA Introduction Rhinoviruses and coronaviruses account for 50 and 20 of common colds in humans respectively Tyrrell 1988 The development of antiviral agents has primarily focused on rhinoviruses as targets Certain types of compounds have been identified which inhibit the uncoat ing of rhinovirus particles AI Nakib and Tyrrell 1987 Diana et al 1989 Kelleyet aI 1988 thus preventing virus replication One of these substances was recently shown in a clinical study to suppress COmmon colds when given prior to virus challenge AI Nakib et al 1989 Interferon has been given prophylactically to persons in settings in which other family members were afflicted with colds Douglas et al 1986 Hayden et sl 1986 Monto et el 1989 but side effects from such treatments are common and efficacy is questionable Monto et al 1989 We recently reported that the novel immune modulator 5 amino 3 I3 D ribofuranosylthiazolo 4 5 d pyrimidine 2 7 3H 6H dione 7 thia 8 oxoguanosine Fig 1 is a broad spectrum antiviral agent Smeeet aI 1989 1990a inhibiting many DNA and RNA viruses in vivo Depending upon the infection model efficacy of the compound can be achieved between 10 and 200mgkg 1when admini stered prophylactically The nucleoside induces interferon and activates natural killer cells in vivo Smee et al 1990b but does not inhibit the growth of cultured cells at 500fLM In mice the 50 lethal dose of7 thia 8 oxoguanosine is 320mgkg 1when given as a single intraperitoneal i p injection Smee al 989 Divided daily doses of 20Cfmgkg 1or lessdo not appear to be deleterious to mice even when administered for 10 or more consecutive days By l p administration 7 thia 8 oxoguanosine was active against coronavirus infections in suckling rats initiated by intranasal virus inoculation and against encephalomyocarditis EMC virus infections in mice when the virus challenge was i p For the present studies the intranasal route of virus challenge was used with rat coronaviruses and EMC viruses as animal models for the common cold The rat coronavirus infection is probably the animal model closest to coronavirus induced common colds Parker et al 1970 Human rhionoviruses have not been adapted to rodents except for one report in which unusual and tedious procedures were necessary to document virus replication in mice Yinand Lomax 1986 The EMC virus 48 D F Smee H A Alaghamandan M L Bartlett and R K Robins Fig 1 Structure of 7 thla B oxoquanoslne which is a picornavirus related to human rhinoviruses has been used by others to evaluate the efficacy of interferon preparations Sim and Cerruti 1987 presumably as an indicator of antirhinovirus activity Since mice receiving EMC virus die of encephalitis this is an imperfect model for rhinovlrus induced common colds However we have observed that EMC when administered intranasally rep licates in the nose and lungs of infected mice Suppression of EMC virus replication in the nose would therefore be akin to inhibition of rhinovirus replication in the upper respiratory tract In this communication 7 thia 8 oxogua nosine is shown to have antiviral activity in the intranasal EMC and rat coronavirus models when administered intranasally Results For rat coronavirus infection suckling rats were treated intranasally with 7 thia 8 oxoguanosine Table 1 Prophy lactic treatments of 0 3 and 1 solutions were signifi cantly effective at increasing the numbers of survivors relative to the placebo control A more moderate effect was achieved using the 0 1 solution and 0 03 was inactive Some increases in mean survival times were evident in treated groups of rats that died A second study was conducted to determine whether intranasal treat ments given after virus challenge would still be protective to rats Table 2 Prophylactic treatment with a 1 solution of compound starting at 24 hours produced a substan tial cure rate from the infection but treatments starting four hours after virus challenge did not However this therapy caused significant increases in mean survival times relative to the respective placebo control There were differences in the severity of infection in the two placebo groups 39 survival for rats pretreated with placebo compared to 0 survival for animals receiving virus first which may have affected the results For intranasal EMC virus infection intranasal treatment with 7 thia 8 oxoguanosine provided moderate protec tion from mortality and some increase in mean survival Table 1 Dose response intranasal treatment of coronavirus infected rats with 7 thia 8 oxoguanosine Solution Dose Survivorsl Mean survival given mM mg kg Total time days Placebo 0 0 15 OJ 9 0 1 5 c 0 03 1 2 2 0 5 OJ 10 4 0 9 0 1 3 6 6 5 13 38 d 10 8 1 4 e 0 3 10 22 11 13 85 d 11 0 2 8 1 30 66 9 13 69 d 11 3 1 5 a Concentration of 7 thia 8 oxoguanosine in bicarbonate buffer 201 L1 of which was administered intranasally to each rat 24 and 18 hours before intranasal virus challenge The dose corresponding to each amount is given in the adjacent column b Mean survival time days of rats that died Survivors lived through 21 days c Standard deviation d Statistically significant p 0 05 determined by the two tailed Fisher exact test e Statistically significant p 300 fold reductions in virus titres in the noses of treated mice on days two to four indicating a marked antiviral effect in the upper respiratory tract Spleen and brain virus concentra tions were also suppressed in mice receiving the nucleo side By day six the virus broke through to produce high titres in the brain which resulted in mortality thereafter Surprisingly 7 thia 8 oxoguanosine did not produce a strong suppressive effect on lung virus titres Table 2 Effects of timing of 7 thia 8 oxoguanosine treatment on a rat coronavirus infection in suckling rats Dose Times of Survivorsl Mean survival mg kg treatment Total time days Placebo 24 18 11 28 39 10 5 1 4 66 24 18 26 28 93 d 12 0 1 4 Placebo 4 8 0 28 OJ 6 7 1 3 66 4 8 0 27 OJ 9 3 1 4 a Administered intranasally at the times indicared relative to virus challenge b Mean survival time days of rats that died Survivors lived through 21 days c Standard deviation d Statistically significant p 0 001 determined by the two tailed Fisher exact test e Statistically significant p 0 001 determined by the two tailed r test Intranasal treatment ofpicornavirus and coronavirus respiratory infections 49 Table 3 Effects of intranasal and intraperitoneal treatments of 7 thia 8 oxoguanosine on an intranasal EMC virus infection in mice Virus LD so 10 32 Dose Treatment Survivors Mean survival mgkg l route Total time days Placebo Ln 6 16 38 5 4 1 5 d 20 Ln 12 16 75 8 0 1 4 e Placebo i p 2 16 13 4 9 1 1 50 l p 7 16 44 8 6 4 0 e 100 i p 12 16 75 9 0 2 4 e Placebo Ln 1 16 6 3 7 o r 20 l n 4 16 25 5 7 1 6 e Placebo l p 1 16 6 4 9 1 2 50 l p 5 16 31 7 1 2 3 e 100 l p 12 16 75 8 0 1 6 e a Half daily doses were administered 24 and 18 hours before virus inoculation b Ln intranasal 20 I of a 1 solution of 7 thia 8 oxoguanosine i p intraperitoneal c Mean survival time days of mice that died Survivors lived through 21 days d Standard deviation e Statistically significant p 0 005 determined by the two tailed r test f Statistically significant p 0 002 determined by the two tailed Fisher exact test The distribution of 14C 7 thia 8 oxoguanosine on in various organs and tissues following intranasal admini stration was determined Table 4 In both mice and rats the inner noses and mouths received about 50 of the total dose Mice which were anaesthetized for drug administration received 9 of the dose in the lungs and 11 in the stomach In contrast unanaesthetized rats got 20 of the dose in their stomachs and only 1 in lungs The deposition of virus containing media in mice and rats following intranasal dosing would be similar to the dis tribution of 7 thia 8 oxoguanosine since similar inocu lation techniques were used Discussion These results demonstrate that a topically applied inter feron inducing immune modulator provided protection to mice and rats infected with viruses related to those causing common colds in humans From the rat studies we showed that prophylactic or at best very early treatments were necessary to provide a benefit This suggests that prophylactic application such as in house hold settings may be appropriate to achieve clinical efficacy This is the strategy researchers are using to evaluate interferon preparations Douglas et aI 1986 Hayden et et 1986 Monto et al 1989 for example The schedule of treating in divided daily doses was worked out in many Viral systems Smee et al 1989 1990a and appears to be optimal Neither more frequent treatments nor divided doses given for several days in succession increases efficacy There may be some merit to intermittent treatments divided daily doses given every Days after virus challenge o 2 3 4 5 6 0 2 3 4 5 6 Fig 2 EMC virus titres in organs or tissues of mice treated with 7 thia 8 oxoguanosine Intranasal treatments were the same as in Table 3 Each data point represents the arithmetic mean titre from five mice e nucleoside at 20 mgkg l all mice in the placebo group were dead by day six 50 D F Smee H A Alaghamandan M L Bartlett and R K Robins Mouse 5 3 2 9 b Rat 12 10 3 27 Table 4 Distribution of 4C 7 thia 8 oxoguanosine after intranasal treatment of adult mice and suckling rats Species Outer nose Percent of Total Administered Dose Standard Deviation Inner Tracheal nose Mouth Oesophagus Lungs Stomach 30 9 19 6 3 1 9 7 11 6 15 44 10 27 2 4 4 24 4 13 30 12 21 7 4 2 1 1 22 13 19 44 13 30 2 7 0 3 10 41 a Animals were sacrificed 5 minutes after administration of compound and then autopsied Values are averages for six mice or five rats Mice were anaesthetized for intranasal treatment whereas rats were not b Range three or four days however but more studies are necessary to confirm this For these studies virus challenges of 10 times the LO so were used which generally created severe infections It took a larger dose than this to kill mice with EMC virus using the intranasal route of virus challenge coupled with intranasal drug treatment fable 3 In the design of these studies we wanted to get high percentages of mortality in the placebo control since it is more difficult to achieve statistical significance with fewer deaths in this group In so doing the effects of the antiviral agent may also be reduced in these severe infections This is illustrated by comparing the activity of intranasal 7 thia 8 oxoguano sine at 19 and at 32 LO so fable 3 The overall number of survivors was increased in treated mice receiving 10 LO so of virus It should also be noted that the virus dose did not seem to make a difference to the intraperitoneal activity of 7 thia 8 oxoguanosine in the same study From the EMC virus studies it was evident that systemic l p administration of the compound was superior to the intranasal route for overall efficacy especially at the high input of 32 LO so of EMC per mouse This also applies to studies we reported earlier with the rat coronavirus model Smee et al 1989 For i p delivery higher doses of compound could be given and more extensive drug distribution achieved which probably leads to greater immune activation and interferon induction In addition 7 thia 8 oxoguanosine is very poorly absorbed orally at least in rodents larger animals have not been examined yet The nucleoside would clear from the nose and mouth via the stomach and intestinal tract where it would leave the body without having any systemic effect All of these factors may account for superior efficacy of l p relative to intranasal administration From the standpoint of safety and practicality however it makes sense to treat the common cold topically Locally induced interferon coupled with activation of immune cells may be all that is required to achieve efficacy whereas whole body induc tion of interferon and immunopotentiation could have significant side effects Whether or not EMC virus is a relevant model for the common cold is debatable since the mice die from encephalitis The results presented here show that sub stantial decreases in nasal virus titres were achieved by treatments with 7 thia 8 oxoguanosine which is akin to inhibition of common cold viruses in the respiratory tract What ultimately kept the mice alive longer than placebo controls was the suppression of virus replication in the brain From unpublished studies we have conducted with Semliki Forest virus the effect of a single day s treatment with the nucleoside causing some reduction in mortality lasts no morethan five days These datacorrelate well with those shown in Fig 2 which illustrate the suppression of EMC virus in the brain for about five days after which time virus breakthrough occurred leading to mortality In the experiment reported in Table 2 there was a marked difference in survival between placebo groups 39 survival in one group versus 0 in the other In the first group having 39 survival the placebo was giver on day three and the virus on day four The second group received virus on day three followed by placebo treatment four hours later The increased survival of the first group may have resulted from a placebo effect or from increased resistance of the host toward the pathogen since susceptibility to infection is age dependent Parker et aI 1970 or from a combination of these factors It is also possible that administration of intranasal fluid after virus challenge may have increased the severity of the infection We did not anticipate these results when the experiment was designed Results of radioactivity distribution studies of 14C 7 thia 8 oxoguanosine showed that 50 of the recovered compound went into the nose and mouth Thus the nucleoside was well targeted to exert its antiviral effect We used 20 gram rats for these experiments instead of 6 gram rats for infections to facilitate autopsy The dis tribution of compound in 6 versus 20 gram animals is probably similar exceptthat less compound probably Can be administered internally to the smaller rats i e more compound may be deposited on the outer nose or Intranasal treatment ofpicornavirus and coronavirus respiratory infections 51 sneezed away In mice only about 10 of available drug went into the lungs which may be part of the reason why EMC virus tit res were only moderately suppressed in lung tissue The fact that more compound went to mouse lungs than rat lungs can be attributed to the use of anaesthesia in mice which made it easier for them to breathe nucleoside into their lungs Virus titrations of rat coronavirus infected lungs or nasal areas were not conducted because of the unavailability of a suitable cell culture system We have had very poor success producing the primary rat kidney cells mentioned by Parker et al 1970 that are only susceptible to virus infections when used within one week after initial prepar ation of the cells from rat kidneys The LBC cell line that supports the replication of rat coronavirus could not be obtained from those who developed it Hirano et al 1985 It is assumed that the in vivo antiviral activity against rat coronavirus is correlated with decreases in virus titres as was shown for EMC virus The concept of using intranasally applied interferon inducers to treat virus infections is not a new one but goes back over two decades De Clercq and Merigan 1969 reported beneficial effects of topically applied poly I poly C against intranasal vesicular stomatitis virus infections in mice Aerosol administration of poly I poly C was found to be active against mouse influenza infections Gerone et al 1971 Against respiratory diseases in humans intranasal poly I poly C moderately suppressed common cold symptoms caused by rhinovirus 13 and type A2 influenza virus Hill et aI 1972 A double stranded RNA of fungal origin referred to by some investigators as Stato Ion was effective intranasally against influenza virus in mice Kleinschmidt and Streightoff 1971 but was later shown only to delay the onset of symptoms and to cause slight reductions in virus titre against experimental rhino virus type 4 infections in humans Aoki et aI 1978 More recently the interferon inducer bropirimine significantly reduced symptoms of experimental infectious bovine rhinotracheitis virus disease in cattle when given as a prophylactic intranasal medication Wierenga 1985 To date no interferon inducer has been approved for the treatment of any viral infections in humans or animals however The present results using the water soluble nucleoside 7 thia 8 oxoguanosine suggest that this agent deserves further consideration with a view to treatment of the respiratory infections caused by rhino viruses and coronaviruses Materials and Experimental procedures Compound 7 thia 8 oxoguanosine was synthesized using the published procedure Nagahara et al 1990 It was soluble in 2 sodium bicarbonate pH 8 6 8 8 up to 10mg rnr Smee et al 1989 The placebo used for animal studies was a solution of 2 sodium bicarbonate 14Cl 7 thia 8 oxoguanosine was custom synthe sized by Moravek Biochemicals Brea CAl Viruses and cells Rat coronavirus 8190 strain and encephalomyocarditis virus EMC strain were purchased from the American Type Culture Collection ATCC Rockville MD L929 cells were also obtained from ATCC EMC virus was propagated in L929 cells Smeeet al 1989 whereas rat coronavirus was passaged in three day old suckling rats Parker et al 1970 and the virus was harvested from rat lungs at six days Animal experiments Swiss Webster female mice and pregnant Fischer rats were purchased from Charles River Labs Wilmington MA Viruses were pre titrated in these animals to identify doses Whichwere 10 times the 50 lethal dose LD so Each experiment was COn ducted with 10 LD so except where indicated Twenty gram mice were treated with Ketamine Park Davis Co Morris Plains NJ at 1OOmg kg 1 prior to intranasal inoculation of 20 fl 1 of EMC virus Three to four day old about six grams Fischer rats were not anaesthetized for intranasal instillation Parkeret al 1970 of 20 ul of virus Suckling rats appear to be more sensitive than adult mice to fluid which may get into their lungs when anaesthetized and many of them die from such treatment In these models the mice die from EMC encephalitis Sim and Cerruti 1987 andthe rats die from coronavirus pneumonia Parker et aI 1970 For drug efficacy studies mice received