Antitumor activity and pharmacology of 1-beta-D-arabinofuranosylcytosine-5'-stearylphosphate: an orally active derivative of 1-beta-D-arabinofuranosylcytosine.

The antitumor activity of 1-beta-D-arabinofuranosylcytosine-5'-alkylphosphates (CnPCAs) against L1210 leukemia in mice after oral administration was demonstrated. The optimum length of the alkyl group on the phosphate moiety of CnPCA for exhibiting a high antitumor activity was found to be between tetradecyl (C14) and tricosyl (C23). The most active alkyl derivative in this system was found to be 1-beta-D-arabinofuranosylcytosine-5'-stearylphosphate (C18PCA). The optimum and minimum effective doses of C18PCA were 100 and 6.25 mg/kg/day (q1d, day 1 to day 5), respectively. The maximum T/C% of C18PCA was approximately 220. The antitumor activity of C18PCA was not greatly dependent on the treatment schedule and route. Plasma concentration of 1-beta-D-arabinofuranosylcytosine (ara-C) remained in the range of 0.4 to 0.75 nmol/ml [corrected] for 24 h after oral administration of 100 mg/kg (170 mumol/kg) of C18PCA. These results indicate that C18PCA administered per orally is absorbed intact through the gastrointestinal tract and area-C is released of long period of time. C18PCA is regarded as an orally active depot form of ara-C.

Determination of base composition of DNA by high performance liquid chromatography of its nuclease P1 hydrolysate.

The base composition of DNA was directly determined by high performance liquid chromatography (HPLC) of its nuclease P1 hydrolysate. This method can satisfactorily be employed even if the sample of DNA contains RNA or the amount of the sample is very small.

Sensitive radioimmunoassay for cytarabine and uracil arabinoside in plasma.

A sensitive and specific radioimmunoassay for cytarabine (ara-C) or uracil arabinoside (ara-U) was established by using [3H]ara-C and anti-ara-C antiserum or [3H]ara-U and anti-ara-U antiserum. The antiserum was prepared by immunizing rabbits with ara-C or ara-U hapten conjugated to human albumin. In the present assay, as low as 2.5 ng/ml (0.01 microM) of ara-C or 5 ng/ml (0.02 microM) of ara-U in blood plasma samples could be detected. The cross-reaction of ara-C or ara-U structurally related compounds with each antiserum was so small that the nucleosides could be determined without any purification procedure. The plasma concentration of ara-C and ara-U in mice following oral administration of an antileukemic agent, cytosine arabinoside-5'-stearylphosphate (C18PCA) (100 mg/kg), or ara-C (48 mg/kg) was determined by using this method. In the case of C18PCA administration, ara-C was detectable in blood for at least 24 hours longer than in the case of ara-C administration. This method is suitable for the analysis of ara-C and ara-U, the metabolites of depot drugs of ara-C such as C18PCA.

Plasma cyclic nucleotides in spontaneously hypertensive rats: hyperresponse to acute hot stress.

An acute hot stress caused a sharp increase in plasma cyclic AMP and cyclic GMP in both spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). This hot stress-induced increase in plasma cyclic AMP was observed even after chemical sympathectomy elicited by 6-hydroxydopamine or depleting the catecholamine stores in adrenergic neurons by tyramine or reserpinization, but was no longer observable after beta-adrenergic blockade by propranolol, blockade of autonomic ganglia by hexamethonium, adrenodemedullation or anesthesia by pentobarbital. These results indicate that the initial stimulation of the central nervous system evoked the release of catecholamines from the adrenal medulla which could activate adenylate cyclase via the stimulation of beta-adrenoceptors on the cell surface. The increment of plasma cyclic GMP was not influenced by prior blockade of the peripheral autonomic nervous system, but was totally abolished by pentobarbital, indicating that cyclic GMP generated within the central nervous system in response to the hot stress would be directly related to its increase in the peripheral blood stream. The plasma cyclic AMP and cyclic GMP responses were greater in adult SHR than in young SHR and young and matured WKY. The predominant response of plasma cyclic AMP might be due to a greater release of catecholamine from the adrenal medulla in matured SHR. The hyperresponse of plasma cyclic GMP in adult SHR remains to be fully elucidated. The increased cyclic nucleotide responses in SHR might be an important factor in the maintenance of hypertension.

Comparison of the in vitro and in vivo anti-herpes activities of 1-beta-D-arabinofuranosylthymine and its 5'-monophosphate.

In vitro and in vivo anti-herpes activities of 1-beta-D-arabinofuranosylthymine 5'-monophosphate (ara-TMP) were compared with those of 1-beta-D-arabinofuranosylthymine (ara-T). On a molar basis ara-TMP was almost as active as ara-T against six strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) as monitored by a cytopathogenicity-inhibition and a plaque reduction assay in human embryonic lung fibroblast cells. When tested against experimental encephalitis in mice inoculated intracerebrally with HSV-1, intraperitoneal or intravenous treatment with 150 mg/kg/day of ara-TMP or 100 mg/kg/day of ara-T, for 5 days was effective in increasing in the mean survival time of mice. For a single dose of ara-TMP, intravenous administration was more effective than intraperitoneal or oral administration. However, oral administration of ara-T was the most effective of the treatment regimens used. Substantial plasma levels of ara-T were detected for a longer time after oral administration of ara-T than after intravenous administration of ara-TMP or ara-T, suggesting that the efficacy of oral administration of ara-T may be correlated with the maintenance of the substantial blood drug levels.

Inhibitory effects of antiherpesviral thymidine analogs against varicella-zoster virus.

Thymidine analogs highly active against herpes simplex virus were compared in their inhibitory action against seven strains of varicella-zoster virus by a plaque reduction assay. E-5-Bromovinyl-arabinosyluracil (BV-ara-U) was most active, followed by E-5 chlorovinyl-arabinosyluracil, E-5-bromovinyl-2'-deoxyuridine (BV-dUrd), 2'-fluoro-5-methyl-arabinosyluracil, 2'-fluoro-5-iodo-arabinosylcytosine, arabinosylthymine, 5-vinyl-arabinosyluracil, acycloguanosine, and 5-iodo-2'-deoxyuridine, in order to decreasing activity. BV-ara-U was more than 10 times as active as BV-dUrd and almost completely inhibited plaque development of five strains of varicella-zoster virus at a concentration as low as 1 ng/ml.

Antiherpesviral and anticellular effects of 1-beta-D-arabinofuranosyl-E-5-(2-halogenovinyl) uracils.

1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil (BV-ara-U) and 1-beta-D-arabinofuranosyl-E-5-(2-chlorovinyl)uracil (CV-ara-U) were tested for their anti-herpesviral activity in virus rating method, a plaque reduction method, and a virus yield reduction method, using human embryonic lung fibroblast (HEL-F) cells, At a concentration as low as 0.1 microgram/ml, both drugs exerted a marked inhibitory effect on the development of cytopathogenic effect induced by herpes simplex virus type 1 (HSV-1) infection and on the multiplication and plaque formation of HSV-1. Neither BV-ara-U nor CV-ara-U was significantly active against HSV type 2 (HSV-2). They scarcely inhibited growth of HEL-F cells, mouse L, and murine leukemia cells. Compared with 1-beta-D-arabinofuranosylthymine and 5-iodo-deoxyuridine, BV-ara-U and CV-ara-U were more than 10 times as active against HSV-1 and much less active against HSV-2. BV-ara-U was as active as E-5-(2-bromovinyl)-2'-deoxyuridine against HSV-1 and less inhibitory to growth of HEL-F cells. Cellular deoxyribonucleic acid synthesis was not significantly influenced by the new derivatives of arabinosyluracil, even at a concentration as high as 300 microgram/ml. The derivatives showed extremely marked inhibition of deoxyribonucleic acid synthesis in HSV-1-infected cells, whereas their inhibitory effect on deoxyribonucleic acid synthesis in HSV-2-infected cells was much lower than that in HSV-1-infected cells. These findings indicate that BV-ara-U and CV-ara-U are selectively inhibitory to HSV-1 multiplication.

Urinary excretion of cyclic nucleotides and principal electrolytes in healthy humans of different ages.

Urinary excretion of cyclic AMP, cyclic GMP, inorganic phosphorus, calcium, sodium, potassium and creatinine was measured in 138 healthy male and 104 healthy female humans from 2 to 68 years old. The range of cyclic nucleotide excretion was as follows: cyclic AMP (mumol/day), 1.01-10.89; cyclic GMP (mumol/day), 0.13-2.00; cyclic AMP (mumol/g creatinine), 1.52-8.93; cyclic GMP (mumol/g creatinine), 0.11-1.87. The 242 volunteers were grouped into seven classes according to age: A, 2-9 years old; B, 10-19; C, 20-29; D, 30-39; E, 40-49; F, 50-59 and G, 60-68. Average excretion (mumol/day) of cyclic AMP in class A (2.62 +/- 0.29 for males and 2.30 +/- 0.18 for females) was significantly smaller than that in other classes (4.59 +/- 0.12 for males and 3.90 +/- 0.13 for females) (p less than 0.01). Such a significant difference was not observed in cyclic GMP excretion. In terms of mumol/g creatinine, however, average excretion of both cyclic AMP and cyclic GMP in class A was greater than that in other classes. The amounts of urinary cyclic AMP and cyclic GMP (mumol/day) were correlated with age in the subjects from 2 to 16 years. A reverse correlation between the amounts of both nucleotides (mumol/g creatinine) and age was found in the young subjects. No correlation between the excretion of either urinary cyclic nucleotide and age was found in adults. A significantly positive correlation between cyclic AMP (mumol/day) and inorganic phosphorus (g/day) was found (r = 0.50 for males and 0.56 for females) (p less than 0.01). This correlation suggests that urinary cyclic AMP might reflect the activity of parathyroid hormone in normal humans. There was no significant correlation between cyclic GMP and electrolytes tested. The above results are considered to provide basic data for clinical evaluation of relevant disorders.

Changes in the concentration of cyclic nucleotides dependent on their antibodies in plasma.

In the process of immunization of rabbits with cyclic AMP or cyclic GMP, the plasma concentration of cyclic AMP or cyclic GMP was markedly elevated as the titer of the antiserum increased. The i.v. injection of anti-cyclic AMP or anti-cyclic GMP antiserum into rats or mice also caused a marked increase in the respective cyclic nucleotide in plasma. On the other hand, an injection with the antiserum did not cause any significant increase in urinary cyclic nucleotides. Therefore, the effect of the antiserum seems to be different from that of glucagon or carbamylcholine which increases cyclic AMP or cyclic GMP respectively in both plasma and urine. The injection of the antiserum, but not nephrectomy, caused a significant increase in plasma cyclic nucleotides in adrenalectomized rats. This made it unlikely that the antiserum-induced increase was due to the inhibition of urinary excretion. The plasma cyclic nucleotides, increased by an i.v. injection with the antiserum, servived enzymatic hydrolysis by phosphodiesterase in vitro; most of them proved to be bound to the antibody. It was concluded that the increases in plasma cyclic nucleotides in the process of cyclic nucleotide-immunization were mainly due to decreased metabolic clearance of plasma cyclic nucleotides.

Antiherpesviral activity and inhibitory action on cell growth of 5-alkenyl derivatives of 1-beta D-arabinofuranosyluracil.

Antiherpesviral activity of 5-vinyl-1-beta-D-arabinofuranosyluracil was as high as that of 1-beta-D-arabinofuranosylthymine, whereas the former was less inhibitory to cell growth than the latter. 5-Propenyl- and 5-butenyl-1-beta-D-arabinofuranosyluracil were less active than 5-vinyl-1-beta-D-arabinofuranosyluracil.

A new antitumor enzyme, L-lysine alpha-oxidase from Trichoderma viride. Purification and enzymological properties.

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 has been purified to homogeneity. The enzyme shows absorption maxima at 277, 388, and 466 nm and a shoulder around 490 nm and contains 2 mol of FAD/mol of enzyme. The enzyme has a molecular weight of approximately 116,000 and consists of two subunits identical in molecular weight (about 56,000). In addition to L-lysine, L-ornithine, L-phenylalanine, L-tyrosine, L-arginine, and L-histidine are oxidized by the enzyme to a lesser extent. Several lysine analogs such as delta-hydroxylysine are oxidized efficiently. Balance studies showed that 1 mol of L-lysine is converted to an equimolar amount of alpha-keto-epsilon-aminocaproate, ammonia, and hydrogen peroxide with the consumption of 1 mol of oxygen. alpha-Keto-epsilon-aminocaproate spontaneously is dehydrated intramolecularly into delta 1-piperideine-2-carboxylate in the presence of catalase, and is oxidatively decarboxylated into delta-aminovalerate in the absence of catalase. The Michaelis constants are as follows: 0.04 mM for L-lysine, 0.44 mM for L-ornithine, 14 mM for L-phenylalanine, and 1.6 mM for oxygen with L-lysine.

Effect of treatment with 1-beta-D-arabinofuranosylthymine of experimental encephalitis induced by herpes simplex virus in mice.

1-beta-D-Arabinofuranosylthymine (ara-T) was examined for its therapeutic efficacy against encephalitis in mice inoculated intracerebrally with herpes simplex virus. Intraperitoneal treatment with 100 mg of ara-T per kg twice daily for 4.5 days was as effective as treatment with 50 mg of arabinosyladenine 5'-monophosphate per kg. Under the same conditions, doses of 5-iododeoxyuridine or arabinosylcytosine (50 mg/kg each) were not effective. Even when the virus inoculum was as high as 320 or 3,200 50% lethal doses, ara-T increased the life span significantly. Oral treatment with 27 mg of ara-T per kg produced a modest increase in the mean survival time, equal to that of 50 mg of ara-T per kg administered intraperitoneally or subcutaneously. A single dose of ara-T, 800 mg/kg intraperitoneally or 400 mg/kg orally, was effective. The 50% lethal dose of ara-T administered intraperitoneally and that administered orally were more than 10 and 15 g/kg, respectively. The therapeutic indexes (maximal tolerated dose divided by minimal effective dose) in multiple intraperitoneal treatments and in multiple oral treatments were estimated to be more than 25 and 100, respectively.

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method.

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).

Interferon production in human diploid cell strains derived from embryonic lungs.

Twenty-three strains of human diploid cells derived from embryonic lungs were tested for production of interferon by "superinduction." Strain HAIN-55 produced a relatively high level of interferon. The optimal concentration of cycloheximide for superinduction was essentially equal to that reported with foreskin fibroblasts. On the other hand, actinomycin D at a concentration of 4 to 16 microgram/ml enhanced the production of interferon more strikingly than at a concentration of 1 microgram/ml, which was usually employed for superinduction in the foreskin fibroblasts. Inhibition of interferon production was observed when fetal bovine serum was added to the medium during treatment with metabolic inhibitors for superinduction. Minimal essential medium was superior to Eagle's basal medium as growth medium for interferon production, and serum added after removal of metabolic inhibitors could be replaced by bovine serum albumin. The yield of interferon produced under the best conditions in this study, with strain HAIN-55, was more than 10,000 reference units/ml.

In vitro antiherpesviral activity of 5-alkyl derivatives of 1-beta-D-arabinofuranosyluracil.

Several 5-alkyl derivatives of 1-beta-d-arabinofuranosyluracil (araU) were tested for antiherpesviral activity and inhibitory action on cell growth in human embryonic lung fibroblasts. 1-beta-d-Arabinofuranosylcytosine, 9-beta-d-arabinofuranosyladenine, and 5-iododeoxyuridine (IUdR) were included as reference materials. Among the 5-alkyl derivatives of araU, arabinosylthymine was the most active, followed by 5-ethyl- and 5-propyl-araU. 5-Ethyl-araU was as active as IUdR and more active than 9-beta-d-arabinofuranosyladenine against herpes simplex virus (HSV) type 1 and did not inhibit cell growth at a concentration as high as 1,000 mug/ml. 5-Butyl- and 5-methoxymethyl-araU, as well as araU, exhibited relatively low activity. The araU derivatives tested were as active against HSV WT-34, an isolate from a patient with keratitis, as against HSV type 1. Against an IUdR-resistant isolate, HSV WT-20, arabinosylthymine was less inhibitory than IUdR. Deoxyribonucleic acid synthesis in HSV type 1-infected cells was markedly inhibited by arabinosylthymine, IUdR, and 5-ethyl-araU, whereas cellular deoxyribonucleic acid synthesis in uninfected cells was significantly inhibited by IUdR but not by arabinosylthymine or 5-ethyl-araU.