Evaluation of the systemic toxicity and pharmacokinetics of the immunoconjugate anti-B4-blocked ricin in non-human primates. Delivered by multiple bolus injections and by continuous infusion.

The systemic toxicity of an immunoconjugate of blocked ricin and the anti-CD19 monoclonal antibody, anti-B4, was studied in cynomolgus monkeys to evaluate its safety for use in humans. Anti-B4-blocked Ricin (Anti-B4-bR) is a highly cytotoxic immunoconjugate which can kill up to 5 logs of antigen positive target cells at concentrations easily achievable in blood. Subacute toxicity studies with Anti-B4-bR were performed in 20 cynomolgus monkeys and 4 rhesus monkeys, which, unlike humans, do not express the CD19 epitope recognized by the anti-B4 antibody on their B-lymphocytes. Anti-B4-bR was administered to cynomolgus monkeys by 5 daily intravenous bolus injections of 10 or 100 micrograms/kg/day, and non-conjugated blocked ricin was administered by 5 daily intravenous bolus injections of 30 micrograms/kg/day. Total doses of the conjugate of 200, 500, 1000 or 1500 micrograms/kg were also delivered to rhesus monkeys by continuous intravenous infusion over seven days. The clinical signs of toxicity, clinical pathology parameters, and gross and microscopic tissue changes associated with Anti-B4-bR were minimal to moderate where present, and primarily hepatic. In monkeys treated with 5 x 10 micrograms/kg of Anti-B4-bR, lesions were noticeable on day 7 after the start of the treatment but were less severe or absent on day 14, suggesting that the toxic effects were reversible. Clearance of the conjugate from the serum after bolus injections of Anti-B4-bR was evaluated by ELISA and demonstrated an initial t 1/2(alpha) of 1.4-2.0 h and a secondary t 1/2(beta) of about 14 h. Serum concentrations of Anti-B4-bR were about 10-20-fold lower at 24 h as compared to 1 h after each of the 5 bolus injections in monkeys. Continuous infusion of Anti-B4-bR in primates achieved plateau levels of the immunotoxin in blood for almost the entire duration of the infusion. The therapeutic utility of the Anti-B4-bR is currently being evaluated in patients with B-cell malignancies.

A 5-year safety and efficacy evaluation with fibrel in the correction of cutaneous scars following one or two treatments.

In a multicenter clinical trial 300 patients were treated with Fibrel, 4 weeks following a negative skin test, for the correction of cutaneous scars. Fibrel treatment was restricted to one or two implants in a maximum of four scars. The scar corrections were evaluated by the physician, the patient, and also via an objective photogrammetric method. At the end of 1 year the percentage of scars with moderate, marked, or complete correction were 65, 63.3, and 85.8% according to physician, patient, and photogrammetric evaluations, respectively. A cohort of 111 patients were followed for up to 2 years and 87 patients were followed up to 5 years postimplantation. The physician and patient evaluations showed 55.1 and 50.6%, respectively, of the scars in the moderate, marked, or complete correction category at the end of 5 years with only one or two treatments. Safety evaluations included tests for antinuclear antibodies, rheumatoid factor, and presence of antibodies to Fibrel and their crossreactivity to human collagen I and III. These tests did not show any causal relationship to Fibrel treatments and the patients did not have any untoward immunologic symptoms. The data from these patients demonstrate that one or two Fibrel treatments are effective in maintaining greater than 50% correction of depressed cutaneous scars up to 5 years with negligible adverse sequelae and no untoward immunologic symptoms.

Pharmacokinetics of sulfasalazine metabolites in rats following concomitant oral administration of riboflavin.

Sulfasalazine, 60 mg/kg, was administered orally to groups of rats (n = 4) along with 1, 5, or 10 mg/kg of riboflavin. Plasma and urine were assayed for 5-aminosalicylic acid, acetyl-5-aminosalicylic acid, sulfapyridine, and acetyl-sulfapyridine using an HPLC method. The mean percent of dose recovered as total metabolites in urine was significantly greater (alpha = 0.01) for the group receiving 10 mg/kg riboflavin compared to the controls or the group receiving 1 mg/kg riboflavin. Plasma AUC and Cmax values were also significantly greater (alpha = 0.05) for the 10 mg/kg riboflavin group. These results suggest that at higher doses, a significant fraction of riboflavin reaches the colon intact and stimulates more efficient reduction of the azo bond in sulfasalazine. Since the concentrations of 5-ASA achieved in the colon may be directly related to the efficacy of sulfasalazine in treating inflammatory bowel disease, concomitant administration of riboflavin may enhance sulfasalazine's efficacy in humans.

A simple and rapid liquid chromatographic method for the determination of major metabolites of sulfasalazine in biological fluids.

A simple and rapid assay for quantitation of sulfasalazine metabolites in rat urine and plasma was developed using high-performance liquid chromatography (HPLC). The method involves dilution of urine or plasma samples (0.1 mL) with methanol for protein precipitation, followed by mixing and centrifugation at 10,000 x g. Chromatography was accomplished with a reversed-phase ODS C-18 column (5 mu; 4.6 x 250 mm). The mobile phase consisted of 20% methanol in 5.0 mM phosphate buffer (pH 6.0), with 0.5 mM tetrabutylammonium chloride as an ion-pairing agent. The flow rate was 1.7 mL/min. An injection volume of 30 microL was used and the metabolites were quantitated by an ultraviolet detector at 254 nm. Benzamide was used as the internal standard. This method is linear in the range of 0.5 to 25 micrograms/mL for 5-aminosalicylic acid (5-ASA), acetylsulfapyridine (Ac-SP), and acetyl-5-aminosalicylic acid (Ac-5-ASA), and from 0.25 to 25 micrograms/mL for sulfapyridine (SP). The percent relative standard deviation ranged from 1 to 7.9% for the metabolite standard curves and precision studies. The limit of detection for 5-ASA, Ac-SP, and Ac-5-ASA is 100 ng/mL, and for SP is 50 ng/mL, in both urine and plasma. This method is rapid, precise, and accurate, and has been used to determine sulfasalazine metabolites in individual rat plasma and urine samples following an oral dose of 60 mg/kg of sulfasalazine.

Single-dose pharmacokinetics of clomiphene citrate in normal volunteers.

Twenty-four healthy adult female volunteers participated in a randomized, three-phase double-blind crossover trial comparing the single-dose (50 mg) pharmacokinetics of three formulations of clomiphene citrate (CC). Plasma levels of both the Z(cis) and E(trans) isomers of CC, as well as principal metabolites, were determined at periodic intervals; and no differences between formulations were observed. The active Z isomer attained peak blood levels later than the inactive E isomer and was eliminated much more slowly, significant plasma concentrations still being detected up to 1 month after treatment. The results of this study demonstrate that three commercially available formulations of CC are bioequivalent.

Dose-dependent absorption of disodium etidronate.

The gastrointestinal absorption of disodium etidronate (as [14C]disodium etidronate) was investigated in the rat proximal jejunum in-situ. Studies using various initial concentrations of the drug suggested that etidronate absorption occurred by passive diffusion at initial concentrations below 0.08 M. At initial concentrations above 0.08 M, the rate of absorption was significantly greater than would be expected if passive diffusion was the only mechanism responsible for absorption. Etidronate absorption is not mediated by the carrier mechanism responsible for phosphate ion absorption.

Competitive inhibition between folic acid and methotrexate for transport carrier in the rat small intestine.

Folic acid absorption from the lumen of the rat small intestine in situ obeyed Michaelis--Menten kinetics. The values of Vmax and Km for absorption were 4.63 x 10(-7) M/min and 1.21 x 10(-6) M, respectively. Folic acid and methotrexate were mutual competitive inhibitors of absorption. Their Ki values were 1.28 x 10(-6) and 1.9 x 10(-5) M, respectively.

Drug absorption VIII: Kinetics of GI absorption of methotrexate.

The absorption of methotrexate from the lumen of the rat small intestine, in situ, was found to obey Michaelis--Menten kinetics. The values of Vmax and Km for the absorption process were 4.78 X 10(-7) M/min and 1.49 X 10(-5) M, respectively.