Antiherpes activity of the immunomodulator OK-432, a streptococcal preparation, in immunosuppressed mice

The antiviral activity of OK-432, an antitumor agent originating from Streptococcal preparations, against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). Intraperitoneal administration of OK-432 to mice 1 day after treatment with 200 mg CY/kg prevented death due to HSV-2 encephalitis in a dose-dependent manner. When the immunosuppressed mice were given OK-432 prior to HSV-2 infection, both by the intraperitoneal route, virus growth in the peritoneal cavity was significantly suppressed. Following with OK-432, the number of macrophages in immunosuppressed mice was increased to a significantly greater extent than the numbers of lymphocytes and polymorphonuclear leukocytes. The intrinsic antiviral activity of macrophages against HSV-2 as well as the natural killer (NK) activity against YAC-1 target cells was significantly enhanced by OK-432 in immunosuppressed mice.     

Effects of oral administration of streptococcal preparation OK-432 on thoracic duct lymphocytes

One KE of streptococcal preparation OK-432 (PIC) was given orally to 6-week old male Wistar-Imamichi rats twice weekly, and control animals received comparable volumes of saline alone. Samples of thoracic duct lymph and posterior vena caval blood were collected prior to and 4 and 8 weeks following PIC. The T cell count of thoracic duct lymphocytes was about three times greater than that of venous blood specimens, and increased significantly in animals following PIC. Controls showed only a slight increase followed by a decrease, so that by 8 weeks of treatment with PIC, the thoracic duct T cell count revealed a significantly greater number in the PIC group than in the controls. In addition, after 8 weeks the per cent T cell subpopulation in thoracic duct lymph increased significantly in the treated group, but it declined in the control group. Concerning T cell subsets in thoracic duct lymphocytes, the percentage of suppressor/cytotoxic T cells showed a significant elevation in the control group after 4 weeks compared with the treated group. No other blood or lymph variables reached statistical significance in this respect.     

Induction of Th1-type cytokines by lipoteichoic acid-related preparation isolated from OK-432, a penicillin-killed streptococcal agent

We have isolated the lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by an affinity chromatography on CNBr-activated Sepharose 4B-bound TS-2 monoclonal antibody (mAb) that neutralizes interferon (IFN)-gamma-inducing activity of OK-432. In in vitro experiments using human peripheral blood mononuclear cells (PBMC), OK-PSA induced IFN-gamma, interleukin (IL)-2, IL-12, IL-18, tumor necrosis factor (TNF)-alpha and TNF-beta that are generally called "Th1-type cytokines" both in protein and in mRNA levels. Furthermore, the neutralizing test using cytokine-specific antibodies demonstrated that IL-18 plays a most significant role for IFN-gamma- and killer cell-inducing ability of OK-PSA among the other cytokines tested. These findings clearly indicated that OK-PSA, an LTA-related molecule, is a main effective component of OK-432, and is a potent inducer of Th1-type cytokines by T cell and natural killer (NK) cell activation mediated by monocytes-derived IL-18, and that it may be a useful immunotherapeutic agent for the patients with malignancies better than original OK-432.     

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation,by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.     

Antitumor activity of trienomycin A on murine tumors

The antitumor activity of a novel ansamycin antibiotic, trienomycin A, against various murine tumors was studied with two treatment schedules. The intraperitoneal injection of the antibiotic showed remarkable antitumor activity on sarcoma 180 and P388 leukemia at doses of 160 or 320 mg/kg, showing 151% and 100% increase in life span, respectively. Trienomycin A inhibited the growth of Ehrlich and Meth A cells in vitro at doses of 0.1-0.4 microgram/ml when the cells were exposed to the antibiotic for 72 hours. The incorporation of [3H]thymidine into acid precipitable material in HeLa cells was slightly more marked than that of [3H]uridine and [3H]leucine when the cells were exposed to 0.04 or 0.08 microgram/ml of trienomycin A for 4 hours. It appeared that trienomycin A showed antitumor activity by direct cytotoxic action.     

Release of tumor necrosis factor (TNF) into mouse peritoneal fluids by OK-432, a streptococcal preparation

A cytotoxic factor was induced in peritoneal fluid by injection of OK-432 in mice which had been primed with OK-432. Two-step stimulation (priming and eliciting) was always necessary to induce the cytotoxic factor. OK-432-primed mice did not produce soluble cytotoxic factor spontaneously and no cytotoxic activity was detected in the mice treated by a single injection of OK-432 as an eliciting agent. This observation was also confirmed by in vitro experiments. Only when activated macrophages were incubated with OK-432 (or with lipopolysaccharide) was cytotoxin released into medium supernatant. High doses of OK-432 were required to prime mice for the production of cytotoxic factor, whereas a small amount was enough to elicit. The peritoneal cytotoxic factor obtained by OK-432 injection appears to be identical to tumor necrosis factor in the serum for the following reasons: The two factors are similar in mode of cytotoxic action. Both are produced from macrophages. They are similar in physicochemical characteristics. The cytotoxicity of the peritoneal cytotoxic factor was totally abolished by anti-TNF serum.     

Effect of cimetidine on the absorption of orally administered tetracycline.

1 Six healthy female subjects received orally two 250 mg tetracycline tablets either with 400 mg cimetidine or placebo. In a separate experiment six healthy female volunteers received 500 mg tetracycline as a suspension on the fifth day of a 6 day regime of 400 mg cimetidine or placebo, 8 hourly and at bedtime. 500 mg tetracycline as tablets was also given with cimetidine only. 2 Following a single dose of cimetidine the mean peak tetracycline plasma concentration was significantly reduced by 1.2 microgram/ml and the area under the plasma concentration, time curve was decreased by 40%. The 72 h urinary tetracycline excretion was diminished by approximately 30%. 3 Tetracycline had no effect on the plasma concentration, time profile of cimetidine. 4 Administration of 400 mg cimetidine 8 hourly and at night for 6 days and dosing with 500 mg tetracycline as tablets or suspension on the fifth day produced no alteration in tetracycline kinetics. 5 It was concluded that under clinical circumstances it is unlikely that cimetidine produces a clinically significant alteration in tetracycline absorption or disposition. 6 The bioavailability of tetracycline from tablets and suspension was found to be similar (31.2% and 36.9% of the dose excreted in the urine over 72 h). 7 The mean renal clearance of tetracycline was 83.8 ml/min (95% confidence interval +/- 9.2 ml/min) and was unaltered by cimetidine treatment.     

Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes by the streptococcal preparation OK432

In vitro overnight exposure to the streptococcal preparation OK432 of blood and tumor-associated lymphocytes enhanced their lytic activity against autologous, freshly isolated tumor cells from malignant pleural effusions of cancer patients. This enhancement was mediated through factors that were distinct from interferon (IFN) and interleukin 2 (IL 2). Treatment with IFN-alpha, -beta, or -gamma, or IL 2 failed to augment autologous tumor killing (ATK) activity, although the treatment enhanced natural killer (NK) activity. Intrapleural (i.pl.) injection of OK432 induced or enhanced ATK and NK activities of effusion large granular lymphocytes (LGL) in patients who showed a reduction or disappearance of effusion tumor cells. No such increase in ATK and NK activities was seen with effusion LGL of patients who had no clinical benefit from i.pl. OK432 therapy. Blood ATK and NK activities were not consistently modified by the therapy. These results indicate that i.pl. administration of OK432 induces an augmentation of ATK activity of effusion LGL, which may be involved in the elimination of effusion tumor cells. The data also suggest that the host immune defense against tumor may be better represented by tumor-associated lymphocytes than by blood lymphocytes, and that it is important to examine the effect of biological response modifiers on ATK activity of tumor-associated effector cells.     

Augmented local immunity in the liver by a streptococcal preparation,OK432, related to antitumor activity of hepatic macrophages

The aim of this study was to investigate the augmentative effect of a streptoccal preparation, OK432, on the immunological competence of hepatic macrophages. We found that OK432 was distributed predominantly to hepatic macrophages after intravenous injection, and Northern blot analysis revealed that OK432 induced the gene expression of IL-1α, β, and TNFα in the liver. The induction of mRNAs was evident 1 h after the intravenous injection of OK432 and their accumulation reached a maximal level at 3 h. TNF production of hepatic macrophages was also increased by the intravenous injection of OK432. Furthermore, OK432 significantly increased the proportion of IL-2 receptor-positive hepatic macrophages. As for antitumor activity in the liver being augmented by OK432, the cytotoxic and cytostatic activity of hepatic macrophages from OK432-treated rats against tumor cells was significantly increased and OK432 markedly reduced the number of tumor nodules in the liver after the inoculation of tumor cells via the portal vein. These findings, which indicated that OK432 has various immuno-stimulating actions on hepatic macrophages, leading to the augmentation of antitumor activity in the liver, suggest that OK432 may be of some benefit in helping to prevent hepatic metastasis, at least in part, via its activation of hepatic macrophages.     

Effect of food and gastric acidity on absorption of orally administered ketoconazole

Effects of food and gastric acidity on the bioavailability of ketoconazole tablets were investigated in 12 volunteers using a six-treatment, randomized, Latin-square crossover design. All volunteers received all treatments, as follows: (A) ketoconazole 200 mg administered after a fast; (B) ketoconazole 200 mg with a standardized high-fat meal; (C) ketoconazole 200 mg with a standardized high-carbohydrate meal; (D) ketoconazole 200 mg after pretreatment with glutamic acid hydrochloride 680 mg as capsules; (E) ketoconazole 200 mg in a simulated achlorhydric state induced with cimetidine and sodium bicarbonate; and (F) ketoconazole 200 mg administered with glutamic acid hydrochloride in a simulated achlorhydric state. Ketoconazole concentrations were measured by high-performance liquid chromatography in plasma samples drawn immediately before and at various times over 24 hours after drug administration. Bioavailability variables, including natural logarithm transformation for area under the concentration-time curve (AUC), were subjected to analysis of variance followed by Duncan's Multiple Range testing. Treatments B and C significantly prolonged the times required to achieve the peak plasma ketoconazole concentration, and treatment C also significantly reduced the peak plasma ketoconazole concentration (Cmax) compared with treatment A. There was a trend toward increased AUC values with treatment B and decreased AUC values with treatment C. Treatment D produced a higher Cmax compared with treatment A, and treatment E produced large, significant reductions in Cmax and AUC values compared with treatment A. Treatment F significantly increased AUC values and Cmax compared with treatment E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of food and antacid on absorption of orally administered ticlopidine hydrochloride

Ticlopidine is a potent inhibitor of platelet aggregation. Absorption of ticlopidine after oral dosing is rapid and complete. Ticlopidine is extensively metabolized with a relative minor component of unchanged ticlopidine in plasma. The randomized crossover study described here was undertaken to examine the effect of food and antacid on the oral bioavailability of a single dose of ticlopidine (250 mg) in normal volunteers. After postprandial treatment the rate and extent of absorption of ticlopidine was earlier and greater relative to fasting treatment [tmax = 1.71 +/- 0.33 hr (fed) vs 1.92 +/- 0.56 hr (fasting) and AUC0-infinity = 2.164 +/- 0.813 micrograms X hr/mL (fed) vs 1.808 +/- 1.052 micrograms X hr/mL (fasting)]. The oral bioavailability of ticlopidine was increased by 20% when taken after a meal. In contrast, absorption of ticlopidine administered after antacid treatment was approximately 20% lower than under fasting conditions. Administration of ticlopidine with food is recommended to maximize gastrointestinal tolerance.     

A phase I clinical and pharmacokinetic study of CS-682 administered orally in advanced malignant solid tumors

Summary CS-682 (1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-N⁴-palmitoylcytosine) is a novel orally administered 2’-deoxycytidine-type antimetabolite, which has a wide spectrum of antitumor activity in human tumor xenograft models. We conducted a phase I study to define the toxicity, pharmacokinetics and antitumor activity of CS-682 in patients with advanced solid tumors. Forty patients were enrolled to receive escalating doses of CS-682. CS-682 was given orally, once daily three times a week (Monday, Wednesday and Friday), for four weeks consecutively, followed by a two-week rest period. Twenty-two men and 18 women, median age 63.5 (range 31 to 82) were treated. The most common tumor type was colorectal cancer with 15 patients. Others tumors occurring in 3 or more patients included prostate, breast and lung carcinomas. Sixty percent of the patients had received greater than 2 prior chemotherapy programs. Patients have been treated at each of the following dose levels (mg/m²/day): 1.5, 12, 20, 25, 30, 50, 67, 90, 120, 160 and 220. Non hematologic toxicities grade 3 [NCI Common Toxicity Criteria (version 2.0)] related to treatment included nausea in 2, vomiting in 1, anorexia and asthenia in 2, and dehydration in 1. Severe hematologic toxicities (grade 3–4) were seen more frequently with 10 patients experiencing grade 3–4 neutropenia, 2 with grade 4 thrombocytopenia and 2 with grade 3 anemia. Neutropenia requiring hospitalization occurred in 3 patients. Dose-limiting neutropenia was observed at 220 mg/m²/day. The maximum tolerated dose was determined to be 160 mg/m²/day. No tumor responses were observed in this study. Six patients experienced stable disease, including one who has stable disease after having received 34 courses of CS-682. After oral administration, CS-682 is rapidly absorbed and metabolized to CNDAC, which is further metabolized by cytidine deaminase to the inactive product CNDAU. Peak plasma concentrations of CNDAC were achieved 2.2 ± 0.9 h after drug administration and the terminal elimination half-life was 1.7 ± 1.5 h. Measurable concentrations of CNDAU were first seen 0.60 ± 0.31 h, peak plasma concentrations were achieved 3.1 ± 0.9 h after the CS-682 dose, and the terminal elimination half-life was 2.3 ± 1.7 h. The recommended phase 2 starting dose for the 3 days/week regimen of CS-682 is 160 mg/m²/day for 4 weeks repeated after a 2-week rest period. Supported by Sankyo Co., Ltd, and Cyclacel Ltd.     

The effect of metoclopramide and atropine on the absorption of orally administered mexiletine.

The effect of pretreatment with intravenous metoclopramide (10 mg) and atropine (0.6 mg), both separately and combined, on the absorption rate and relative oral bioavailability of the antiarrhythmic drug, mexiletinee (400 mg) was studied in eight fasting healthy males using a Latin Square design for order of pretreatment administration. The time (Tmax) of the maximum mexiletin plasma concentration (Cpmax) was reduced by metoclopramide (P < 0.001) and was increased by atropine (P < 0.01) compared with saline control. Tmax was not significantly altered by combined metoclopramide and atropine pretreatment. Atropine pretreatment was associated with a significant reduction of Cpmax (P < 0.05) and of elimination half-life (P < 0.05). The area under the mexiletine plasma concentration-time curve was not affected by any of the pretreatments. The results suggested that metoclopramide enhanced and atropine decreased the rate of mexiletine absorption without altering the relative oral bioavailability. When the pretreatments were administered in combination, metoclopramide reversed the delay in mexiletine absorption produced by atropine.     

Dose-finding and pharmacokinetic study of orally administered indibulin (D-24851) to patients with advanced solid tumors

Indibulin (ZIO-301/D-24851) is an orally applied small molecule with antitumor activity based upon destabilization of microtubule polymerization. The purpose of this phase I study was to determine the maximum tolerated dose (MTD) as well as the dose limiting toxicity (DLT), the pharmacokinetics, safety and tolerability of orally administered indibulin as capsule formulation in patients with advanced solid tumors. Patients received a single dose of indibulin. Seven dose-levels were evaluated: 100 mg, 150 mg, 250 mg, 350 mg and 600 mg once daily (QD), 450 mg and 600 mg twice daily (BID). After a washout period, patients received indibulin at the pre-defined daily dose for 14 days every 3 weeks (multiple dose part). A total of 28 patients entered the study. Indibulin administered as capsules was generally well tolerated. The MTD was not reached. There was a disproportionate increase of the area under the plasma concentration-time curve (AUC) with dose, with declining AUC corrected for dose starting at the 250 mg dose-level. There was no significant difference in AUC of indibulin after multiple dosing (day 1–14) compared to single administration (day-4). Inter-patient variability in AUC (102% CV) was high. A plateau in drug exposure was observed prior to reaching the MTD. Continued dose-escalation was unlikely to yield any increase in exposure of indibulin. The formulation needs optimization to increase the systemic exposure upon oral administration.     

Phase I and pharmacokinetic study of the orally administered farnesyl transferase inhibitor R115777 in patients with advanced solid tumors

R115777 is a novel selective inhibitor of farnesyl transferase, an enzyme that is involved in the proliferation of the malignant cell type. This study was designed to determine the toxicity, maximal tolerated dose and pharmacokinetics of R115777 when given orally b.i.d. for 28 days followed by 1-2 weeks of rest. Patients with advanced solid tumors for whom no standard therapy was available could enter the study. The starting dose of R115777 was 200 mg/dose and inter- as well as intra-patient dose escalations were performed with increments of 100 mg/dose. Nine patients entered the study and received in total 23 treatment cycles. A dose of 300 mg b.i.d. proved feasible with grade 4 neutropenia occurring in one of six patients who completed the first treatment cycle. Other toxicities were infrequent. Pharmacokinetic analysis demonstrated that peak plasma concentrations of 881+/-393 ng/ml were reached within 1-5 h. No accumulation of R115777 was observed over a 28-day period. The study was terminated based on these results together with the observation from a related phase I study in which higher doses of R115777 were associated with the frequent occurrence of grade 3-4 myelosuppression. We conclude that the recommended dose of R115777 given for 28 days followed by 1-2 weeks of rest is 300 mg b.i.d. Myelosuppression is the dose-limiting toxicity.     

The effect of intravenous cimetidine on the absorption of orally administered diazepam and lorazepam.

1 The effect of intravenous cimetidine 200 mg or 400 mg on the absorption of lorazepam 2.5 mg tablet and diazepam 10 mg tablet and capsule was studied. 2 Considerable individual variation in plasma concentrations was found with all preparations. 3 Cimetidine increased the absorption of diazepam and lorazepam. 4 Capsule preparations of diazepam generally produced higher drug plasma concentrations than the tablets.     

The influence of bacterial gut hydrolysis on the fate of orally administered isonicotinuric acid in man

Following oral administration to human subjects, isonicotinuric acid was hydrolyzed within the gastrointestinal tract to isonicotinic acid. This metabolism did not occur following intravenous administration. Evidence is presented indicating that most of the ingested isonicotinuric acid escaped absorption from the small intestine and passed into the large intestine, where gastrointestinal bacteria hydrolyzed it to isonicotinic acid. The latter compound was subsequently absorbed into the systemic circulation, where some was reconjugated with glycine, forming the originally administered compound, isonicotinuric acid.This work was supported in part by National Science Foundation Grant GY 10651 and National Institutes of Health Training Grant GM 00728, U.S. Public Health Service, Bethesda, Maryland.This paper was submitted to a Consulting Editor who served as the Journal Editor during its review process.National Science Foundation Undergraduate Research Participant, Summer 1973, under Grant GY 10651.