各种药物的干扰素诱导和抗病毒性能的比较
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Several synthetic substances have now been demonstrated to be active interferon inducers
in various animal species (4, 8, 10, 11, 14). Two of these, polyriboinosinid [poly(I:C)] and bis-diethylaminoethylfluorenone (tilorone HCI), have been the object of considerable research and speculation concerning eventual use as antiviral agents (1, 7, 10, 17). Therefore, both represent compounds against which any new interferon inducer might be compared in terms of interferon-inducing and antiviral properties. In the studies described in this report, a new low-molecular-weight interferon inducer (2amino-5-bromo-6-methyl-4-pyrimidinol, [U25,166]) first reported in 1976 by Nichol et al. (13) was compared with poly(I:C) and tilorone HCl. These studies were undertaken to determine the spectrum of antiviral activity and evaluate parameters that might effect eventual clinical utility, including the state of hyporeactivity that develops secondary to virus infection or as a consequence of repeated doses of inducer.
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MATERIALS AND METHODS Mice. Female ICR strain mice (Upj:tuc-ICR) were provided by Upjohn Rearing and Procurement and were housed under conditions of constant temperature and a 12-h light cycle, with food and water provided ad libitum. After being received from the breeder, mice were housed as described for at least 1 week to allow them to adjust to the new environment before being used. Cells. Murine interferon assays were carried out in a cloned continuous line of mouse L-cell fibroblasts (L2,9) originally obtained from the American Type Culture Collection (ATCC) Cell Repository. Media. Eagle minimum essential medium (MEM; Microbiological Associates) containing 10% fetal calf serum (Reheis Chemical Co., Phoenix, Ariz.), 100 U of penicillin per ml, and 50 ,ug of streptomycin per ml was used to propagate and maintain all tissue cultures. Viruses. Vesicular stomatitis virus (VSV), Indiana strain, was obtained from the ATCC (Rockville, Md.). Pools of VSV were propagated in primary chicken embryo cell monolayers. The stock preparation of VSV used in these studies had a titer of 2 x 107 plaque-forming units (PFU)/ml when assayed in L, mouse cells.
VOL. 11, 1977
INTERFERON-INDUCING PROPERTIES OF U-25,166
985
The strain of encephalomyocarditis (EMC) virus used in these studies was obtained from the ATCC. Young adult mice (16 ± 2 g) were infected by the intranasal route. Brains were removed after onset of paralysis and were homogenized as a 10% suspension in MEM. The pool had a titer of 9.5 x 10f PFU/ ml on ',929 cells. A2 influenza virus (A2/AA/2160, H2N2), obtained from H. E. Renis (Upjohn), had previously been mouse adapted, and the pool used in these studies was prepared by intranasal inoculation of the virus. Lungs were harvested 48 h after infection, homogenized as a 10% suspension in MEM, and stored at -70°C. The HWC strain of Herpesvirus hominis type 1 (HVH-1) was propagated in primary rabbit kidney cells and titered 107 PFU/ml on these cells. Semliki Forest virus (SFV) was obtained originally from S. Baron (National Institutes of Health [NIH]) and was propagated on primary chicken embryo cells. The pool used in these studies titered 2 x 108 PFU/ml on primary chicken embryo cells. West Nile virus was obtained through the NIH (ATCC VR82, catalog no. V554-001-552). Suckling ICR mice were injected intracerebrally, and 48 h later mice were sacrificed and brains were removed and homogenized as a 10% suspension in MEM. The pool titered 5 x 108 PFU/ml on primary chicken embryo cells. Friend leukemia virus (FLV) originally obtained from the ATCC (ATCC 245) was injected intraperitoneally (i.p.) into 25- to 30-g ICR mice. Spleens were removed 21 days later and homogenized as a 10% suspension in MEM. After clarification (2,000 x g for 15 min), supernatant fluid was removed and stored at -70°C. Inducers. Tilorone HCl (bis-diethylaminoethylfluorenone) was generously furnished by Richardson-Merrell (Cincinnati, Ohio) and was dissolved in phosphate-buffered saline at a concentration permitting an inoculum of 250 mg/kg orally (p.o.). Poly(I:C), prepared by P. L. Laboratories (Milwaukee, Wis), was dissolved in phosphate-buffered saline and was administered i.p. at 100 Ag/mouse. U-25,166, purchased from Aldrich Chemical Co. (Milwaukee, Wis.), was suspended in aqueous 1% carboxymethylcellulose and unless otherwise noted was adminstered at 1,000 mg/kg p.o. X-irradiation. Mice received 650 R of whole-body irradiation from a Van de Graaff accelerator. WBC counts. Total leukocyte (WBC) counts were determined by collecting 0.02 ml of blood from the orbital sinus with heparinized capillary tubes. Blood was diluted in a Unipette (Becton-Dickinson) containing 1.1% ammonium oxalate, and cells were counted microscopically in a Neubauer hemacytometer. Thin-layer blood smears were also prepared for differential WBC counts after staining with Wright stain. Interferon assay. Confluent monolayers of L,2, cells grown in 35-mm plastic petri dishes (Falcon Plastics) were treated with 1 ml of an appropriate interferon dilution overnight at 37°C. The plaque reduction assay used VSV as the challenge virus
in various animal species (4, 8, 10, 11, 14). Two of these, polyriboinosinid [poly(I:C)] and bis-diethylaminoethylfluorenone (tilorone HCI), have been the object of considerable research and speculation concerning eventual use as antiviral agents (1, 7, 10, 17). Therefore, both represent compounds against which any new interferon inducer might be compared in terms of interferon-inducing and antiviral properties. In the studies described in this report, a new low-molecular-weight interferon inducer (2amino-5-bromo-6-methyl-4-pyrimidinol, [U25,166]) first reported in 1976 by Nichol et al. (13) was compared with poly(I:C) and tilorone HCl. These studies were undertaken to determine the spectrum of antiviral activity and evaluate parameters that might effect eventual clinical utility, including the state of hyporeactivity that develops secondary to virus infection or as a consequence of repeated doses of inducer.
984
MATERIALS AND METHODS Mice. Female ICR strain mice (Upj:tuc-ICR) were provided by Upjohn Rearing and Procurement and were housed under conditions of constant temperature and a 12-h light cycle, with food and water provided ad libitum. After being received from the breeder, mice were housed as described for at least 1 week to allow them to adjust to the new environment before being used. Cells. Murine interferon assays were carried out in a cloned continuous line of mouse L-cell fibroblasts (L2,9) originally obtained from the American Type Culture Collection (ATCC) Cell Repository. Media. Eagle minimum essential medium (MEM; Microbiological Associates) containing 10% fetal calf serum (Reheis Chemical Co., Phoenix, Ariz.), 100 U of penicillin per ml, and 50 ,ug of streptomycin per ml was used to propagate and maintain all tissue cultures. Viruses. Vesicular stomatitis virus (VSV), Indiana strain, was obtained from the ATCC (Rockville, Md.). Pools of VSV were propagated in primary chicken embryo cell monolayers. The stock preparation of VSV used in these studies had a titer of 2 x 107 plaque-forming units (PFU)/ml when assayed in L, mouse cells.
VOL. 11, 1977
INTERFERON-INDUCING PROPERTIES OF U-25,166
985
The strain of encephalomyocarditis (EMC) virus used in these studies was obtained from the ATCC. Young adult mice (16 ± 2 g) were infected by the intranasal route. Brains were removed after onset of paralysis and were homogenized as a 10% suspension in MEM. The pool had a titer of 9.5 x 10f PFU/ ml on ',929 cells. A2 influenza virus (A2/AA/2160, H2N2), obtained from H. E. Renis (Upjohn), had previously been mouse adapted, and the pool used in these studies was prepared by intranasal inoculation of the virus. Lungs were harvested 48 h after infection, homogenized as a 10% suspension in MEM, and stored at -70°C. The HWC strain of Herpesvirus hominis type 1 (HVH-1) was propagated in primary rabbit kidney cells and titered 107 PFU/ml on these cells. Semliki Forest virus (SFV) was obtained originally from S. Baron (National Institutes of Health [NIH]) and was propagated on primary chicken embryo cells. The pool used in these studies titered 2 x 108 PFU/ml on primary chicken embryo cells. West Nile virus was obtained through the NIH (ATCC VR82, catalog no. V554-001-552). Suckling ICR mice were injected intracerebrally, and 48 h later mice were sacrificed and brains were removed and homogenized as a 10% suspension in MEM. The pool titered 5 x 108 PFU/ml on primary chicken embryo cells. Friend leukemia virus (FLV) originally obtained from the ATCC (ATCC 245) was injected intraperitoneally (i.p.) into 25- to 30-g ICR mice. Spleens were removed 21 days later and homogenized as a 10% suspension in MEM. After clarification (2,000 x g for 15 min), supernatant fluid was removed and stored at -70°C. Inducers. Tilorone HCl (bis-diethylaminoethylfluorenone) was generously furnished by Richardson-Merrell (Cincinnati, Ohio) and was dissolved in phosphate-buffered saline at a concentration permitting an inoculum of 250 mg/kg orally (p.o.). Poly(I:C), prepared by P. L. Laboratories (Milwaukee, Wis), was dissolved in phosphate-buffered saline and was administered i.p. at 100 Ag/mouse. U-25,166, purchased from Aldrich Chemical Co. (Milwaukee, Wis.), was suspended in aqueous 1% carboxymethylcellulose and unless otherwise noted was adminstered at 1,000 mg/kg p.o. X-irradiation. Mice received 650 R of whole-body irradiation from a Van de Graaff accelerator. WBC counts. Total leukocyte (WBC) counts were determined by collecting 0.02 ml of blood from the orbital sinus with heparinized capillary tubes. Blood was diluted in a Unipette (Becton-Dickinson) containing 1.1% ammonium oxalate, and cells were counted microscopically in a Neubauer hemacytometer. Thin-layer blood smears were also prepared for differential WBC counts after staining with Wright stain. Interferon assay. Confluent monolayers of L,2, cells grown in 35-mm plastic petri dishes (Falcon Plastics) were treated with 1 ml of an appropriate interferon dilution overnight at 37°C. The plaque reduction assay used VSV as the challenge virus
