Influence of immunogenicity on the pharmacokinetics of BMS-191352, a Pseudomonas exotoxin immunoconjugate, in rats and dogs.

BMS-191352 is an immunotoxin construct of modified Pseudomonas exotoxin conjugated to a fragment of the BR96 monoclonal antibody. We have investigated the potential for immunogenicity of BMS-191352 and its influence on the pharmacokinetics in rats and dogs. BMS-191352 was administered intravenously at doses of 0.75, 1.5, and 3 mg m(-2) once every two days for a total of five doses in rats, and 1.2, 2.4, and 4.8 mg m(-2) once every three days for a total of five doses in dogs. Blood samples were collected on days 1 and 9 in rats, and on days 1, 7, and 13 in dogs to monitor pharmacokinetics and anti-BMS-191352 immune response. Plasma concentrations of BMS-191352 and serum anti-BMS-191352 antibody titre were determined using ELISA assays. Pharmacokinetics were assessed using a non-compartmental method. Anti-BMS-191352 antibodies were not observed in rats within the drug administration interval. In all dogs, except one, markedly higher anti-BMS-191352 antibody titres were observed on day 13 compared with days 1 and 7, and its magnitude was independent of BMS-191352 dose. The single dose kinetics of BMS-191352 in rats and dogs were linear and the drug exposures were generally dose proportional. Mean half-life, total body clearance, and volume of distribution were 1.74 h, 3.35 mL min(-1) m(-2), and 0.27 Lm(-2) in rats, respectively, and 4.27 h, 6.28 mL min(-1) m(-2), 1.19 L m(-2) in dogs, respectively. The multiple-dose (day 9) kinetics in rats were similar to the single-dose kinetics. In dogs, the disposition of BMS-191352 on day 7 was similar to that on day 1; however, there was a precipitous reduction in the systemic drug exposure (by 5- to 110-fold) and marked increase in drug clearance on day 13. These changes in the kinetics of BMS-191352 were attributed to the generation of anti-BMS-191352 antibodies. In the one dog that did not develop anti-BMS-191352 antibodies, the pharmacokinetics were unchanged. The pharmacokinetics of BMS-191352 may be perturbed due to an immune response thus restricting the therapeutic utility of the immunotoxin.

BR96 sFv-PE40 immunotoxin: nonclinical safety assessment.

BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewis(y)-related antigens. Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans. Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40. In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative). Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats. These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts. In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas. Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis. After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative). No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewis(y)-expressing dogs. Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function. The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40.

Assessment of dose proportionality, absolute bioavailability, and immunogenicity response of CTLA4Ig (BMS-188667), a novel immunosuppressive agent, following subcutaneous and intravenous administration to rats.

PURPOSE: The objectives of this study were: to delineate the pharmacokinetics of CTLA4Ig in rats after single and multiple intravenous (IV) and subcutaneous (SC) doses; to assess the relationship of the pharmacokinetic parameters of CTLA4Ig vs dose; to calculate the SC absolute bioavailability; and to assess the antibody response of CTLA4Ig. METHODS: A total of 48 (24 male and 24 female) Sprague Dawley rats were divided into eight treatments with 3 rats per gender in each group: a single dose of 10, 80, or 200 mg/kg of CTLA4Ig given either IV or SC and a repeated dose of 10 mg/kg (once every other day for 7 doses over 13 days) given either SC or IV. Serial blood samples were collected up to 43 days after single dose administration and up to 50 days following the administration of the last multiple dose on day 13. The serum concentration of CTLA4Ig and anti-CTLA4Ig antibodies were measured using ELISA assays. RESULTS: After single IV doses, Cmax and AUCinf increased in a dose proportional manner; CL appeared to be dose independent, while both Vss and T1/2 increased as the administered dose increased. Following single SC doses, Cmax and AUCinf increased in a linear manner but not proportionally; mean Tmax values were prolonged but similar among the three dose levels, while T1/2 increased as the administered dose increased. The absolute SC bioavailability of CTLA4Ig decreased as the dose increased from 10 (62.5%), 80 (55.7%), and 200 mg/kg (41.1%). Comparison of the AUCtau values between the first and last doses suggested an accumulation (3.1-4.7) of CTLA4Ig. However, regardless of the route of dosing, AUCtau after the last dose were comparable to AUCinf values following the single dose. Anti-CTLA4Ig antibodies were detected at the 10 mg/kg dose level after single or multiple doses for both routes of administration. However, regardless of single or multiple doses, antibody titers were relatively greater for the SC compared to the IV administration. CONCLUSIONS: The key findings of this study were: (i) the elimination characteristics of CTLA4Ig were comparable between the SC and IV routes; (ii) the repeated dosing did not alter the pharmacokinetics of CTLA4Ig; (iii) the SC absolute bioavailability tended to decrease as the administered dose increased; and (iv) a greater formation of anti-CTLA4Ig antibodies was observed after SC compared to IV at a single 10 mg/kg dose level; however, after multiple dosing, the formation of antibodies from either of the two routes was relatively slower, and (v) during the study period, no antibodies were observed at either the 80 or 200 mg/kg dose levels regardless of the route of administration.

In vivo efficacy and toxicity of a single-chain immunotoxin targeted to CD40.

G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37 degrees C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and monkeys, with the maximum tolerated dose being 0.48, 1.0, and 1.67 mg/kg, respectively. Comparative immunohistology showed that the G28-5 specificity was qualitatively similar between human and monkey tissue. In summary, G28-5 sFv-PE40 was effective at inducing complete antitumor responses in lymphoma xenografted mice at doses that were well tolerated in mice, rats, and monkeys.

Didanosine (ddl) and stavudine (d4T): absence of peripheral neurotoxicity in rabbits.

Some 20 male New Zealand White rabbits (five/group) were given either didanosine (ddl) or stavudine (d4T) at 750 and 1500 mg/kg body weight/day by oral intubation for 24 wk. An additional group was given 300 mg/kg body weight/day zidovudine (AZT) as a negative control. After 13 weeks the high dose of ddl was lowered from 1500 to 1000 mg/kg body weight/day following the death of one rabbit and continued inappetence in the dose group. The rabbits were observed daily, plasma drug levels were monitored, and electrophysiological measurements of peripheral nerve conduction were performed during the study. Additionally, body weight and food intake were recorded, and clinicopathological parameters were evaluated. Sections of selected peripheral nerves, and dorsal and ventral spinal nerve roots were examined by light and transmission electron microscopy. Although peripheral neuropathy has been reported in rabbits with the nucleoside analogue zalcitabine (ddC), based on clinical observations, electrophysiological measurements, and light and electron microscopy, no evidence of peripheral neurotoxicity was observed in rabbits given either ddl of d4T.

BR96-doxorubicin conjugate (BMS-182248) versus doxorubicin: a comparative toxicity assessment in rats.

The toxicity of BMS-182248, an immunoglobulin (cBR96)-cytotoxic drug (doxorubicin) conjugate, was investigated in Sprague-Dawley rats at single intravenous doses of 508, 1,200, and 2,550 mg/m2 (conjugated doxorubicin doses of 14.7, 34.8, and 74 mg/m2, respectively) and compared to that obtained from administration of free doxorubicin at single doses of 33.6 and 72 mg/m2 (approximately equivalent to that contained in the 1,200- and 2,550-mg/m2 doses of BMS-182248, respectively). Necropsies were conducted on day 8, upon death/moribund sacrifice, or after an approximate 3-mo observation period following completion of treatment. Death/moribundity of all rats that received 72 mg/m2 and of 9 of 20 rats given 33.6 mg/m2 free doxorubicin were attributed primarily to delayed cardiotoxicity and glomerulonephropathy. With BMS-182248, death from glomerulonephropathy and cardiotoxicity occurred in only 4 of 20 rats given 2,550 mg/m2 (74 mg/m2 doxorubicin equivalent). No deaths or cardiotoxicity occurred in rats given 508 or 1,200 mg/m2 BMS-182248. Additional effects noted with either drug included testicular atrophy, axonal degeneration of sciatic nerve and nerve tracts of brain and spinal cord, teeth (incisor) abnormalities, thymic atrophy, bone marrow hypocellularity, splenic lymphoid and red-pulp depletion, and increased extramedullary hematopoiesis in the spleen and liver. Also noted were altered chief cells in the stomach, vacuolation of adrenal gland and corpora lutea in the ovary, uterine and seminal vesicle atrophy, ulceration and myocyte regeneration/degeneration in the tongue, increased osteoclasts and osteoblasts in bone, and lymphoid hyperplasia of mandibular lymph node. In general, these effects were more severe in doxorubicin-treated rats. All changes observed with BMS-182248 were considered primarily due to the effects of doxorubicin and were substantially less severe (most notably cardiotoxicity) compared to those produced by an equivalent amount of doxorubicin.

Acute cardiotoxicity of nucleoside analogs FddA and FddI in rats.

The acute cardiotoxic potential of single dosages of FddA (2'-fluoro-2',3'-dideoxyadenosine) and FddI (2'-fluoro-2',3'-dideoxyinosine) was investigated in 6- to 9-week-old rats. Both nucleoside analogs were administered orally at 1000 and 2000 mg/kg and intravenously at 500 or 1000 mg/kg. For comparative purposes, additional groups of rats received 2'-deoxyadenosine or the 2-fluororibose moiety common to both the FddA and FddI molecules. The effects of two adenosine receptor antagonists, caffeine and theophylline, on the cardiotoxicity induced by FddA were also investigated. Deaths occurred within a few hours to a few days in FddA-treated rats given 2000 mg/kg orally or 500 mg/kg intravenously and in FddI-treated rats given 1000 mg/kg intravenously. Microscopic examination of the hearts revealed myocardial degeneration and necrosis for all rats that died and myocardial fibrosis for many survivors. No deaths or cardiac lesions were observed after administration of 2'-deoxyadenosine or the 2-fluororibose moiety. FddA was more cardiotoxic than FddI in rats at equivalent dosages administered either orally or intravenously. Based on the anatomic findings, all deaths were attributed to cardiac lesions. The administration of high, oral dosages of caffeine and theophylline accentuated the acute cardiotoxicity of FddA in rats.

Comparability of the electrophysiologic responses and plasma and myocardial tissue concentrations of sotalol and its d stereoisomer in the dog.

The relative plasma, myocardial, and skeletal muscle concentrations as well as activities of racemic and d-sotalol were assessed in both anesthetized and conscious dogs. In acute anesthetized experiments, the agents were infused i.v. over a 15-min interval at doses of 1 and 4 mg/kg. Arterial blood samples and punch biopsy specimens from the left ventricular myocardium and skeletal muscle (gastrocnemius) were taken at the completion of each infusion and at periodic intervals for the ensuing 3 h. The drugs were also administered over a 2-week dosing interval to conscious dogs at a dose of 5 mg/kg given twice daily. ECG alterations and venous blood samples were withdrawn on the 1st, 3rd, 7th and 14th day of drug administration. Myocardial and skeletal muscle samples were taken at killing on day 14. In anesthetized dogs, both forms of sotalol decreased heart rate, lowered arterial pressure, prolonged ventricular refractoriness, and caused measurable increases in the PR, QT, and QTc intervals in the absence of any effect on QRS duration. Similar effects on heart rate and QTc and lack of influence on the PR and QRS interval were observed in conscious animals. Tissue drug concentrations were closely correlated with plasma drug levels. Comparable mean steady-state tissue/plasma ratios of 2.26-2.94 were attained immediately following acute i.v. drug infusions. These were larger than those observed following chronic oral drug administration for 14 days. The data, however, clearly demonstrated the equivalence of the plasma and myocardial drug levels obtained in dogs following i.v. infusion of 1 mg/kg or oral administration of 5 mg/kg of dl- or d-sotalol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal model for evaluating the convulsive liability of beta-lactam antibiotics.

The beta-lactam antibiotics imipenem-cilastatin, BMY-26225, and cefazolin significantly lowered the convulsive threshold of pentylenetetrazole in mice. In addition, imipenem-cilastatin and cefazolin were found to inhibit 3H-labeled gamma-aminobutyric acid binding to synaptic membranes from rat brains. Our results suggest that the pentylenetetrazole convulsive model may be useful in evaluating the proconvulsive liabilities of new carbapenems and other beta-lactam antibiotics and that the mechanism of imipenem-cilastatin and cefazolin toxicity may involve interaction with gamma-aminobutyric acid receptors.

Single-dose and multiple-dose intravenous toxicity studies of BMY-25282 in rats.

Single-dose and multiple-dose (daily X 5 and weekly X 5) intravenous toxicity studies in rats were conducted to determine the possible acute and delayed toxicity of BMY-25282 (7-N-(dimethylaminomethylene) mitomycin C), a potential anticancer drug. Rats in the single-dose study received either 0.05, 0.25, or 0.50 mg/kg (0.3, 1.5, or 3.0 mg/m2) of BMY-25282; rats in the daily X 5 multiple-dose study received doses of 0.005, 0.025, or 0.050 mg/kg (0.03, 0.15, and 0.3 mg/m2) of BMY-25282 once each day for 5 days; and rats in the weekly X 5 multiple-dose study received 0.05 mg/kg of BMY-25282. All doses were in 0.1% Pluronic F-68 diluent. Acute toxicities included gastrointestinal epithelial necrosis, myelosuppression, and splenic lymphoid depletion in the high and intermediate dose groups in the single-dose study and myelosuppression in the high dose group of the daily X 5 multiple-dose study. One death in a high dose male of the single-dose study was attributed to acute gastrointestinal and lymphoid toxicity. Between the interim necropsy on Day 5 or 9 and termination of the 9-week dose-free observation period, 9/20 rats of the high and intermediate dose groups of the single-dose study and 4/10 high dose rats in the daily X 5 multiple-dose study died, primarily due to hydrothorax and congestive heart failure caused by delayed, drug-related myocardial degeneration. The most prominent drug-related histopathologic changes observed in rats of both the single-dose study and the daily X 5 studies were myocardial degeneration (cardiomyopathy), glomerulopathy with tubular degeneration, and necrotizing arteritis. These three changes, observed at 0.5 and 0.25 mg/kg in the single-dose study and at 0.05 mg/kg/day in the multiple-dose (daily X 5) study, were delayed in onset and irreversible. Drug-related tubular degeneration and slight glomerulopathy were observed in male BMY-25282-treated rats in the weekly X 5 study, but cardiotoxicity, pulmonary arteritis, hydrothorax, and lethality were not observed. The diluent, Pluronic F-68, was not associated with any morphologic or clinico-pathologic changes. A single-dose of 0.05 mg/kg or 5 daily doses of 0.025 and 0.005 mg/kg of BMY-25282 were considered nontoxic doses in rats. A cumulative dose of 0.25 mg/kg, which caused cardiotoxicity in the daily X 5 study, was not cardiotoxic in the weekly X 5 study. These results indicate that the delayed cardiotoxicity of BMY-25282 is schedule dependent.

Testicular toxicity of N-methyltetrazolethiol cephalosporin analogs in the juvenile rat.

The testicular toxicity of 10 antibiotics was evaluated in juvenile rats. Three of the antibiotics, cefbuperazone, cefamandole, and cefoperazone, contain the N-methyltetrazolethiol group as the 3-substituent; ampicillin, cefazolin, cephalothin, cefoxitin, piperacillin, and ceforanide do not contain this moiety. Testicular degeneration, partially irreversible in nature, was observed with those antibiotics which contain the N-methyltetrazolethiol substituent. Further, free N-methyltetrazolethiol also produced testicular degeneration in the juvenile rat. This substituent is most likely responsible for the testicular toxicity observed with cefbuperazone, cefamandole, and cefoperazone in the juvenile rat. The implications of these findings to man are undetermined.

Pain on injection and muscle irritation: a comparison of animal models for assessing parenteral antibiotics.

Pain on injection due to parenteral administration of cephaloridine, cephalothin, and cefoxitin with or without 1% lidocaine was examined in a rat paw-lick model and the results compared with those obtained in a rabbit intramuscular model of irritation. In both animal models, cephaloridine caused similar or a slightly greater response than sterile water. Conversely, cefoxitin and cephalothin caused a much greater reaction than water in both models. The only major difference in the rankings by the two models was with formulations in which an anesthetic agent was incorporated into the diluent. As expected, the presence of a local anesthetic masked pain on injection but not muscle damage. The rat paw-lick model is an alternative to the traditional rabbit muscle irritation model for rapidly assessing both pain on injection and muscle irritation of parenteral formulations.

The toxicological evaluation of tallysomycin s10b--biosynthetic tallysomycin derivative.

Tallysomycin S10b, a biosynthetic derivative of tallysomycin B, was subjected to intravenous toxicologic studies in mice and dogs. LD50 and LD10 values from lethality studies in mice were utilized to establish dose levels for single and five daily dose toxicity studies in mice and dogs. Nephrotoxicity was the most consistent and prominent drug-related alteration in single and multiple dose studies in both species and was considered the dose limiting toxicity. Other toxicities included pulmonary toxicity, lymphopenia and necrosis of extremities in mice and dogs and testicular degeneration and focal vacuolation of adrenal cortical cells in the dog. Bleomycin was administered to dogs as a reference control agent at a single dose of 270 mg/m2 which was approximately equivalent to the highest tallysomycin S10b dose of 240 mg/m2. Tallysomycin S10b showed a greater nephrotoxic potential than bleomycin at earlier time periods. However, at termination there was no meaningful difference in the degree of chronic nephrotoxicity. The pulmonary toxicity of both drugs was comparable.

The toxicologic evaluation of marcellomycin--an antineoplastic anthracycline antibiotic.

Dose-related toxicologic effects of marcellomycin, an antineoplastic anthracycline antibiotic, were observed in single-dose studies in mice iv (43.05-67.65 mg/m2) and dogs iv (41.0-90.2 mg/m2), and in multiple-dose studies in dogs iv (9.8-29.6 mg/m2 2X/week for 6 weeks) and rats sc (9 weekly doses at 26.2-72.2 mg/m2). Toxicity was primarily manifested by suppression of myeloid tissue, especially the erythrocytic and thrombocytic series, and lymphoid tissues. Initially a neutrophilic leukocytosis was observed in dogs and rats, which was considered possibly to be due to mobilization of the marginal and bone marrow neutrophil pools. In dogs, this was followed by a marked, dose-related neutropenia; and, in rats that died, there was marked depletion of bone marrow cells. Other toxicities observed included enteropathy, severe subcutaneous serofibrinous inflammatory edema and necrosis at injection sites, prostate and seminal vesicle atrophy, uterine hypoplasia, testicular and pancreatic degeneration, thyroid follicular proliferation and hemorrhage in various organs. In general, these toxicities were reversible in surviving animals during recovery periods. Significant cardiotoxicity was not demonstrated.

Acute toxicity of micronized 8-methoxypsoralen in rodents.

The acute oral and intraperitoneal LD50 values of micronized 8-Methoxypsoralen were determined in rats and mice. The values obtained in the present study were lower than those previously reported. The suggestion is made that the discrepancy in the LD50 values may possibly be attributed to differences in compound particle size in the preparations used.