Stimulation of natural killer cell activity and inhibition of proliferation of various leukemic cells by purified human leukocyte interferon subtypes.

One of several human leukocyte interferon subtypes A (LeIF-A), obtained in purified form from a gene cloned in Escherichia coli, stimulated human peripheral blood natural killer cell activity, whereas another human leukocyte interferon subtype D (LeIF-D) had no effect with the use of K562 as target cells. With Daudi as target cells, both LeIF-A and LeIF-D stimulated natural killer cell activity. A hybrid human leukocyte interferon, NH2-terminal 61 amino acids and COOH-terminal 104 residues of LeIF-A and LeIF-D, respectively (LeIF-AD) showed greater stimulation than did LeIF-A, but the stimulation did not exceed that of natural buffy coat interferon. A mixture of equal antiviral units of LeIF-A and LeIF-D was no more effective than was LeIF-A alone. The cloned interferon subtypes showed differential effects on the proliferation of three human leukemic cell lines: Daudi (B-cell lymphoblastoid leukemia); BALL 1 (B-cell acute lymphoblastic leukemia); CCRF-HSB-2 (T-cell acute lymphoblastoid leukemia). Growth of Daudi cells was generally most sensitive to all the interferons tested, LeIF-A, -D, -AD, and a buffy coat preparation; no viable cells remained after 120-hr exposure to 1000-unit/ml doses of the interferons. BALL 1 was relatively resistant to the interferon subtypes tested including LeIF-AD, but this cell lines was very sensitive to a preparation of natural buffy coat interferon. CCRF-HSB-2 showed some sensitivity to all the interferons with greatest sensitivity to LeIF-A (10% of the viable cells were detected after 1000 units/ml exposure for 120 hr). In contrast to the leukemic cell lines tested, human amnion cells (WISH) and the human erythroid leukemia, K562, were resistant to the antiproliferative activity of the interferons.

Importance of treatment regimen of interferon as an antitumor agent.

A highly purified hybrid human leukocyte interferon, IFN-alpha AD, produced in Escherichia coli, has been used to define optimum treatment conditions for L1210 leukemia in mice. Treatments prior to tumor inoculation were ineffective. Treatments from the third day post-tumor inoculation were most effective, and treatments every third day were more effective than were regimens involving more frequent treatments. IFN-alpha AD was effective in vivo against tumors formed from a line of L1210 cells resistant to IFN-alpha AD in cell cultures. These and other results indicate the importance and nature of indirect mechanisms of action for efficacy of interferons against tumors.

Effects of cloned human leukocyte interferons in the human tumor stem cell assay.

Clonogenic tumor cells from fresh biopsies of human cancers were cultivated in vitro and tested for sensitivity by continuous exposure to pharmacologically achievable concentrations of either of two highly purified human leukocyte interferon subtypes (IFN-alpha A and IFN-alpha D) prepared by recombinant DNA methods. The interferons were compared on a weight basis at concentrations of 0.4 and 4.0 ng/ml (equivalent to 80 and 800 units of interferon activity for IFN-alpha A and 2.0 and 20 units for IFN-alpha D). Inhibition of tumor colony-forming units (50% of control or less) was observed in 38.1% of the 273 tumors tested against IFN-alpha A, and in 16% of the 71 tumors tested against IFN-alpha D. Of the tumor types with at least ten samples tested against IFN-alpha A, the percentage of cases exhibiting inhibition was as follows: melanoma (51.7%), lung cancer (50%), myeloma (33.4%), ovarian cancer (33.9%), sarcoma (33.3%), adenocarcinoma of unknown primary (30.4%), breast cancer (28%), acute leukemia (30.8%), and renal cancer (23%). More marked inhibition (30% of control or less) was observed in 18.7% of all tumors tested against IFN-alpha A. Of 60 melanomas tested, 18 (30%) exhibited marked in vitro inhibition of growth with IFN-alpha A. Although a smaller number of tumors (71) were tested against IFN-alpha D on a weight basis, it appeared, in general, to be slightly less active than IFN-alpha A (p less than 0.01), and only 8% of tumors tested exhibited marked inhibition over the same dosage range of interferon. Comparison of the dose-response curves for the 68 tumors tested simultaneously against both interferons did not reveal marked interpatient differences in the inhibition curves, although IFN-alpha D was slightly less active overall. Tumors exhibiting at least 50% inhibition of tumor colony formation also proved to be sensitive to a significantly larger number of cytotoxic drugs (tested simultaneously) than the tumors not inhibited with interferon (p less than 0.0001 for IFN-alpha A). We conclude that the in vitro clonogenic assay may aid in targeting tumor types most likely to exhibit interferon sensitivity and assist in case selection for entry into clinical trials with cloned interferons.

Human alpha and gamma interferon analogs in rabbits with herpetic keratitis.

Complete gene synthesis methods have been used to construct analogs of human interferons (IFNs): these include a consensus of the known human IFN-alpha S, designated IFN-alpha Con1 and a variant of human IFN-gamma, designated IFN-gamma 4A. These interferons, in purified form, were used topically against herpes simplex virus type 1 (HSV-1) induced ocular keratitis in rabbits. Eyes pretreated with IFN-alpha Con1 had decreased signs of infection and a lower incidence of HSV-1 positive trigeminal ganglia (3 of 14 positive) compared to the placebo treated (10 of 14 positive). IFN-alpha Con1 was as effective as natural IFN-alpha subtypes on a units basis, despite the very high specific activity of this analog. IFN-gamma 4A used under similar conditions do not result in beneficial effects with treatments beginning 24 or 48 hr before or 4 hr after virus inoculation. Rabbits with confirmed latent HSV infection were treated topically with IFN-alpha Con1 (10(6) units per eye each day) either before or before and after attempts to intentionally reactivate the infection by bilateral iontophoresis of 6-hydroxydopamine plus topical epinephrine treatment of the corneas. These IFN-alpha Con1 treatment regimens along with intentional reactivation during latency did not: (1) lessen the frequency of inducible ocular shedding episodes; (2) alter the mean time of 3-5 days between attempts to reactive latent infection and the appearance of HSV in tears; or (3) significantly change the incidence of HSV-positive trigeminal ganglia (83-100% HSV positive).

Relationship between the antiviral effects of interferons and their abilities to depress cytochrome P-450.

Several hybrid human interferons have now been constructed by recombinant DNA techniques. Two of these hybrid interferons, IFN-alpha AD(Bgl) and IFN-alpha AD(Pvu) differ by only three amino acids, but IFN-alpha AD(Bgl) was fifteen times more potent than IFN-alpha AD(Pvu) in antiviral activity towards infection of mouse L-929 cells by vesicular stomatitis virus. Only the hybrid with the greater antiviral activity in the mouse depressed cytochrome P-450, aminopyrine N-demethylase and benzo[a]pyrene hydroxylase in the liver. These experiments demonstrate that minor changes in amino acid structure not only have a major effect on the antiviral properties of interferon but also influence the ability of interferon to depress cytochrome P-450 in the liver.

Induction and depression of cytochrome P-450-dependent mixed-function oxidase by a cloned consensus alpha-interferon (IFN-alpha CON1) in the hamster.

A novel analogue of human alpha-interferon (IFN-alpha CON1) was tested for its ability to modify the hepatic cytochrome P-450-dependent mixed-function oxidase system in the hamster. This cloned interferon was derived by selecting the most frequently observed amino acid sequences at each position in the known human alpha-interferon subtypes. IFN-alpha CON1 had a biphasic effect on cytochrome P-450 and related drug biotransformation in the hamster causing an initial increase followed by a significant depression. IFN-alpha CON1 also had a biphasic effect on cytochrome P-450 in the lung, adrenal and spleen but only a depressant effect in the kidney. This effect was not due to morphological damage and followed the species specificity for this type of interferon. Both the increase and the decrease in cytochrome P-450 could be prevented by the administration of the protein synthesis inhibitor puromycin. Various isozymes of cytochrome P-450 induced by phenobarbital, beta-napthaflavone and clofibrate were also depressed by this interferon. The results presented in this report suggest that IFN-alpha CON1 interferon will likely depress drug biotransformation in humans because the antiviral effects and the "anti-cytochrome P-450" effect of interferons cannot be separated, and this interferon has antiviral properties in both hamster and human cells. Clinically relevant drug interactions may be common during the concomitant use of this interferon and other drugs that are metabolized by cytochrome P-450.

Modulation by a human interferon of antitumor effects of cyclophosphamide against a lymphosarcoma in hamsters.

Administration to hamsters of a highly purified human leukocyte interferon subtype, IFN-alpha A, obtained by recombinant DNA methods, abolished the efficacy of high doses of cyclophosphamide (2.5 mg/hamster) against the TBD 932 lymphosarcoma. The effect was more pronounced with concomitant than with sequential treatments and did not occur with melphalan. Antagonistic effects occurred at high interferon doses (10(5) I.U./treatment), but an additive or synergistic positive effect occurred at lower interferon doses (10(3) I.U./treatment) and at lower, non-protective, doses of cyclophosphamide. These effects may be due to immunomodulatory responses induced by the drugs involved.

Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets.

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) . (polycytidylic acid) even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.

Antiviral and side effects of interferons produced by recombinant DNA techniques as tested in rhesus monkeys.

Human interferon type alpha 2 (HuIFN-alpha 2) produced by Escherichia coli was found to be as active as natural leukocyte interferon in protecting rhesus monkeys against intradermal vaccinia virus infection. HuIFN-beta 1 produced in E. coli had similar but less pronounced activity. HuIFN-alpha 2 induced fever but not leukopenia, while HuIFN-beta 1 had opposite effects. Concurrent treatment with acetosalicylic acid and prednisolone/azothioprine combinations did not interfere with the efficacy of the human interferons.

Natural and cloned human leukocyte interferon in herpesvirus infections of rabbit eyes.

Human buffy-coat interferon and a leukocyte subtype (LeIF-A) obtained by gene cloning in Escherichia coli were equally effective in ameliorating the course of herpetic keratitis in rabbits when applied topically at a daily dose of 0.5 X 10(6) units per eye for six days commencing one day before infection. When given intramuscularly at the same dose, there was no alteration in the course of the disease. Topical treatments with LeIF-A were equally effective in suppressing epithelial damage seven days after infection, regardless of whether treatment commenced one day before infection or two days after infection. However, treatment from the day before infection also caused a significant decrease in epithelial defect two days after infection.

The synthetic 32-46 fragment of human growth hormone increases insulin and glucagon levels in the conscious dog.

Human growth hormone (hGH, 22 K) has an acute insulinlike effect not observed with the 20 K variant form of hGH that lacks amino acids 32 to 46 (deletion peptide, hGH32-46). The possibility that hGH32-46 increases insulin secretion was examined by infusing hGH32-46 (1.6 nmol/kg/min) or saline in a crossover design study into each of four conscious 16-hour-fasted dogs for three hours (0 to 180 minutes) following a 40-minute control period. At 90 minutes, plasma glucose was raised to and maintained at 170 mg/dL by glucose infusion for three hours (until 270 minutes). After a lag period of 30 minutes hGH32-46 infusion caused glucagon to increase (P less than 0.05) by 67 +/- 20 pg/mL and insulin tended to rise by 8 +/- 3 microU/mL. Saline tended to cause glucagon and insulin to decline slightly (by 17 +/- 8 pg/mL and 6 +/- 2 microU/mL); hGH32-46 increased (P less than 0.05) tracer determined (3H-3-glucose) glucose production by 1.13 +/- 0.66 mg/kg/min while saline had no effect. Neither treatment changed plasma glucose (100 +/- 4 to 105 +/- 3 mg/dL with hGH32-46; 99 +/- 4 to 99 +/- 4 mg/dL with saline). Induction of hyperglycemia (168 +/- 2 mg/dL) caused glucagon concentrations to fall similarly to about 50 pg/mL with and without hGH32-46. Insulin rose in both protocols but to a greater extent (P less than 0.05) with hGH32-46 (+67 +/- 18 v +35 +/- 13 microU/mL at 180 minutes).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparisons of dose-response data for various standard and recombinant DNA-derived human interferons.

Dose responses of the NIH standard IFN-alpha and IFN-beta preparations were compared with recombinant DNA-derived IFN-beta (IFN-beta 1) and various IFN-alpha subtypes, molecular hybrids and mixtures. Cytopathic effect assays were employed using vesicular stomatitis virus on human HeLa and bovine MDBK cells. A natural peripheral blood lymphocyte and recombinant DNA-derived IFN-gamma were also included in the comparisons. Two-tailed t-tests between slopes showed no significant differences in any pair-wise comparison using crude or highly purified preparations.

The inadequacy of rectal temperature measurements for assessing the effects of antiviral drugs on influenza virus infection of ferrets.

From 6 experiments in which 99 ferrets were infected with influenza virus A/Finland/74 and treated with various agents which suppress virus shedding and other parameters of infection, we assessed whether rectal temperature correlated with nasal virus shedding. A number of temperature and virus-shedding related parameters were determined for each experiment but statistical analysis showed little correlation between them, although an elevated temperature occurred at some time after infection. The pooled data also suggested that temperature and virus shedding parameters are not clearly related. The analysis indicates that intermittent rectal temperature measurements are unsatisfactory for determining the efficacy of anti-influenza agents in ferrets.

Reevaluation of human alpha interferons against herpesvirus infection of the rabbit eye.

Because of reported differences in potency, recombinant DNA-derived human alpha interferons (IFNs) were reevaluated for use against acute Herpes Simplex Virus type 1 (HSV-1) infection of the rabbit eye. The IFNs used topically were IFN-alpha 2, (IFN-alpha 2) and a consensus of known human IFN-alpha s, designated IFN-alpha Con1. Prophylactic treatment with IFN-alpha Con1 at 1 or 15 X 10(6) U/eye/day beginning 48 hours before HSV-1 inoculation and therapeutic treatment with 5 or 15 X 10(6) U/eye/day beginning 4 hours after inoculation with either IFN-alpha Con1 or IFN-alpha 2 appeared to prevent or significantly reduce the development of corneal epithelial involvement. The effects were dose dependent with no evidence for decreased activity at the higher dose. The duration of HSV-1 shedding into tear film was not significantly reduced.

Antiviral effects of highly purified bacteria-derived human leukocyte interferons (subtypes A and D) and a human fibroblast interferon against herpes virus infection of the rabbit eye.

Highly purified preparations of two recombinant-DNA derived human leukocyte interferon subtypes (LeIF-A and LeIF-D) and a similarly derived fibroblast interferon were compared for efficacy against herpes simplex virus, type 1, infection of the rabbit cornea. LeIF-D appeared to be more effective than LeIF-A especially when compared on the basis of interferon units. The fibroblast interferon showed no significant effects. The greater activity of LeIF-D compared with LeIF-A could be due to greater direct antiviral effects, as observed in rabbit cell cultures.