Nephrotoxicity studies of the immunosuppressants tacrolimus (FK506) and ascomycin in rat models.

The nephrotoxic potential of ascomycin, the C21-ethyl analogue of FK506, was defined and ways explored to enhance its detection. After 14-day dosing in the Fischer-344 rat, FK506 and ascomycin reduced creatinine clearance by >50% at doses of 1 and 3 mg/kg, i.p., respectively. Ascomycin also had a 3-fold lower immunosuppressive potency in a popliteal lymph node hyperplasia assay, resulting in an equivalent therapeutic index consistent with a common mechanistic dependence on calcineurin inhibition. Renal impairment with different routes of administration was correlated with pharmacokinetics. Sensitivity of detection was not adequate with shorter dosing durations in rats with unilateral nephrectomy or in mice using a cytochrome P-450 inhibitor, SKF-525A. In 14-day studies, nephrotoxicity was not induced by continuous i.p. infusion of ascomycin at 10 mg/kg/day or daily oral administration (up to 50 mg/kg/day) in rats on a normal diet, nor by continuous i.v. infusion (up to 6 mg/kg/day) in rats on a low salt diet to enhance susceptibility. The lack of toxicity at high oral doses of FK506 or ascomycin, and the finding of non-linear oral pharmacokinetics of ascomycin show that this drug class has an oral absorption ceiling. The negative results with continuous infusion suggest that ascomycin nephrotoxicity is governed by peak drug levels. In addition to defining ways to meaningfully compare the nephrotoxic potential of FK506 derivatives, these results have implications for overall safety assessment and improved clinical use.

Effects of intramedullary injection of methiodal and lidocaine on spinal cords of dogs.

A solution of methiodal sodium (20%) and lidocaine HCl (0.20%) was given by intramedullary injection into the lumbar spinal cords of 12 anesthetized dogs (group I). Two control groups of 12 dogs each were subjected to needle placement only or were given 5% dextrose. The results showed that both solutions given by intramedullary injection caused severe spinal cord malacia and cavitation in 2 group I dogs and in 1 group III dog.

Bulla curettage for chronic otitis media and interna in dogs.

In a series of 10 clinically affected and 6 clinically normal dogs, the technique of bulla curettage via the external ear canal, after lateral ear resection, was evaluated. In 5 of the 10 dogs with chronic otitis media and interna, the head carriage became normal 2 to 8 weeks after surgery and remained so for at least 18 months; 4 other dogs improved, but the improvement was either temporary or incomplete. In the clinically normal dogs, postsurgical complications were minimal; the tympanic membrane healed completely in 4 of the 6 dogs.

Innovar-Vet-induced pathologic changes in the guinea pig.

High doses of Innovar-Vet administered im induced pathologic changes in tissues at the site of drug deposition. The onset and extent of the changes were dose related. Distal self-mutilation occurred coincidentally to the peak of pathologic changes in the ischiatic nerve.

Specific in situ hybridization of Haemobartonella felis with a DNA probe and tyramide signal amplification.

Haemobartonella felis is an epierythrocytic bacterium suspected to be the causative agent of feline infectious anemia. Previous studies with a polymerase chain reaction assay have identified a mycoplasmal 16S rRNA gene sequence that coincides with clinical disease and the presence of organisms in the blood. Tissues from a cat experimentally infected with H. felis were used for in situ hybridization studies to physically link this 16S rRNA gene to the organisms on the red cells. A biotin-labeled probe was used in conjunction with tyramide signal amplification to visualize the hybridization signal. This study clearly demonstrates a specific hybridization signal on the red cells in the tissues of the H. felis-infected cat. This in situ hybridization study is the final step in fulfilling the molecular guidelines for disease causation and proves that H. felis, a mycoplasmal organism, is the causative agent of feline infectious anemia.

The Lathyrus sativus neurotoxin: resistance of the squirrel monkey to prolonged oral high doses.

Prolonged oral administration of the Lathyrus sativus neurotoxin, L-3-oxalylamino-2-aminopropionic acid (OAP), to three young male squirrel monkeys at dose rates of 0.6 to 6.0 mg/g body weight/day produced no neurologic signs and no adverse effects other than depressed activity and occasional foaming at the mouth. When the dose was increased to 8.0 to 8.5 mg/kg body weight/day in two animals, seizures and death occurred after 3 and 5 days. The signs were typical of acute OAP intoxication observed previously to occur in the squirrel monkey within an hour after a single ip dose (2 mg/g body weight) of OAP. Total cumulative dosage of OAP and duration of the experiments were as follows: Experiment 1, 9.5 g, 38 days; Experiment 2, 157 g, 178 days; Experiment 3, 64 g, 33 days. Histologic examination revealed no abnormalities in brain, spinal cord, or other organs. These experiments suggest that the adult squirrel monkey is highly resistant to chronic oral OAP intoxication. Under our experimental conditions, this species did not provide a satisfactory animal model for human neurolathyrism, a disease postulated to result from chronic OAP intoxication.

Comparative vestibular toxicity study with 3-O-demethylfortimicin A disulfate and gentamicin sulfate in cats.

3-O-demethylfortimicin A disulfate (ODMF), a novel aminocyclitol antibiotic, was administered subcutaneously for three months to groups of male and female cats at 15, 30 or 60 mg base/kg/day. Gentamicin sulfate (GS) at doses of 6 and 13 mg base/kg/day served as a reference compound. Signs of vestibular toxicity were considered to include persistent unsteady gait and stance, impaired righting reflex and abnormally diminished postrotatory vestibular nystagmus. Renal toxicities produced by ODMF and GS were also determined and compared. ODMF at 15 and 30 mg base/kg/day produced no signs of vestibular toxicity, while a dosage of 60 mg base/kg/day of ODMF produced vestibular toxicity in 7/10 cats. Three affected male cats died or were killed in moribund condition between study days 49 and 64. Vestibular toxicity was observed in 10/10 cats treated with GS at 13 mg base/kg/day and in 3/10 cats at 6 mg base/kg/day. All ten cats treated with GS at 13 mg base/kg/day died or were killed in moribund condition between study days 30 and 81. The deaths and moribundity in cats treated with ODMF or GS were attributed to renal toxicity. The vestibular toxicity and nephrotoxicity produced by ODMF and GS were more severe in male cats than in females. In conclusion, ODMF given at doses up to 60 mg base/kg/day for three months induced comparatively less vestibular toxicity and renal pathology than did GS at a dose of 13 mg base/kg/day.

The neurotoxicity of valine deficiency in rats.

When valine, an essential amino acid, was withdrawn from the diet of weanling rats, the animals rapidly developed a unique pattern of neurological symptoms characterized by head retraction, staggering and aimless circling. At necropsy degenerative changes were most prominent in the neurons of the red nuclei, brain stem structures which modulate motor function. To explore the pathogenesis of the neurotoxicity associated with valine deficiency, we fed rats purified diets deficient in valine alone or in valine plus other branched chain and neutral amino acids, and we examined brain tissues by light microscopy. Motor disfunction and red nuclei damage occurred only in rats fed diets lacking valine alone and not in rats fed diets lacking all three branched chain amino acids. These results suggest that the neurotoxicity of valine deficiency results from amino acid imbalance rather than from lack of dietary valine per se.

Terazosin: intravenous safety evaluation in rats.

Terazosin, an alpha-adrenergic antagonist, was administered as a 15 mg/ml solution to rats intravenously at a rate of 2 ml/min. Under these conditions the LD50 was 277 mg/kg for males and 293 mg/kg for females. When administered daily for 1 month at dosages of 0, 10, 40 or 150 mg/kg/day, the no-toxic-effect dosage was 40 mg/kg/day. Evidence of toxicity at 150 mg/kg included hypothermia and deaths. Death resulted from acute, exaggerated pharmacologic effects leading to cardiorespiratory failure. Evidence of sympatholytic activity observed at lower dosages included hypoactivity, blepharoptosis, ptyalism and splenic congestion.

Buflomedil: three month intravenous safety evaluation in rats.

Buflomedil was administered intravenously to rats at dosages of 1, 4, 12 or 30 mg/kg/day for up to three months. The no-adverse-effect dosage was considered to be 12 mg/kg/day. At 30 mg/kg/day several deaths and clinical signs, including ataxia, decreased activity, dyspnea and jerking movements after dosing, were observed. These were considered to result from the acute, exaggerated pharmacologic effects of buflomedil. Body weight gain and food consumption were decreased after six weeks in males at 30 mg/kg/day. Increases in the relative weights of the kidneys, brain and testes of males at 30 mg/kg/day were correlated with decreased body weight gain in this group. There were no effects on hematology or serum chemistry parameters, and no morphologic changes were found.

The effect of valine deficiency on neutral amino acid patterns in plasma and brain of the rat.

Valine deficiency in rats produced motor incoordination attributable to selective damage to the red nuclei, midbrain structures that modulate motor activity. Neither incoordination nor red nuclei damage occurs in rats deprived of valine, isoleucine and leucine, thus suggesting that valine neurotoxicity results from amino acid imbalance rather than from lack of valine per se. To explore this possibility, we compared neutral amino acid patterns in plasma and brain of rats fed for 7 days a complete diet fed ad libitum or pair-fed, a valine-free diet or a diet lacking in all three essential branched-chain amino acids (BCAA). Statistical evaluation showed that plasma valine in valine-deprived rats was lower (P less than 0.01) than in pair-fed and ad libitum-fed controls but did not differ from rats lacking BCAA. Brain valine in valine-deprived rats did not differ from ad libitum-fed controls and actually was higher (P less than 0.01) than in pair-fed and BCAA-deprived rats. The most striking changes seen in the amino acid pattern of valine-deprived rats as compared to all other groups were in the increased leucine:valine ratio (P less than 0.01 for plasma and brain) and in the increased leucine + isoleucine:valine ratio (P less than 0.01 plasma; P less than 0.001, brain). These results are consistent with the view that amino acid imbalance is a critical factor in the development of the neurotoxicity of valine deficiency.

Effects of a GnRH agonist on fertility following administration to prepubertal male and female rats.

Leuprolide, a GnRH agonist, was administered daily to male and female rats for 90 days. Animals were sexually immature (25 days old) at the outset. Dosages were 20 and 200 micrograms/kg/day. Five males and five females were euthanized on Day 91. Sex organs were weighed and evaluated for histopathologic changes. These procedures were repeated 140 days later. Following a recovery period lasting 45 days (onset of normal-appearing estrous cycles) in females and 140 days (two spermatogenic cycles) in males, the fertility of these rats was assessed by mating with untreated animals. Treated males gained less weight while treated females gained more weight than controls. Weights of primary and secondary sex organs were reduced below control, but returned to normal following 140 days of recovery. Treated males were fertile and produced normal litters. Reproductive performance of low-dosage (20 micrograms/kg/day) females was normal 45 days after treatment cessation, but half of the high-dosage (200 micrograms/kg/day) females failed to become pregnant. However, reproductive performance of this group compared well with control performance after an additional 6 weeks of recovery. Atrophic changes were noted in male and female sex organs. Following 140 days of recovery, ovaries, uterus, vagina, prostate, and seminal vesicle were normal. Although testes and epididymides showed partial recovery at this time, multifocal or segmental atrophy and mineralization were noted in portions of some seminiferous tubules.

Heinz bodies, methemoglobinemia, and hemolytic anemia induced in rats by 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline.

Sprague-Dawley CD strain rats were given 18, 35, 70, or 140 mg/kg/day of 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline by gavage for 2 weeks. Heinz bodies were seen in the erythrocytes of rats given 140 mg/kg/day. Dose-related increases in methemoglobin were found at 35 mg/kg/day or more. Hemolytic anemia was characterized by dose-related decreases in hematocrit, hemoglobin, and total erythrocyte count. Reticulocytosis, decreased myeloid:erythroid ratio, splenomegaly, extramedullary hematopoiesis, increased serum total bilirubin, and icterus were also observed. This compound was found to oxidize oxyhemoglobin to methemoglobin in vitro, suggesting that the parent compound is capable of causing the hematological changes observed in vivo without conversion to active metabolites.

Acute, subchronic, and chronic toxicity studies with 3-O-demethylfortimicin A disulfate, a new aminocyclitol antibiotic.

The acute LD50 for 3-O-demethylfortimicin A disulfate (ODMF) in mice and rats were 419 and 778 mg activity/kg (dosages are expressed in terms of antibiotic activity (potency), rather than on a weight basis) for single-dose im administration and, 90 and 96 mg activity/kg for single-dose iv administration, respectively. No drug-related gross or microscopic lesions were found in rabbits given single iv infusions of ODMF at dosages of 10 to 400 mg activity/kg. Minimal to mild muscle irritation was seen in rabbits given im concentrations of 3.8 or 7.5% ODMF at dosages of 48 or 93 mg ODMF activity/kg. In 1-month iv studies in dogs treated with ODMF at dosages of 0.4, 1, 4, or 8 mg activity/kg/day, and in concurrent studies in rats treated with ODMF dosages of 1, 3, 6, or 12 mg activity/kg/day, treated animals remained essentially free of adverse effects. In 1-month im studies in dogs treated with ODMF at dosages of 1, 4, 8, or 16 mg activity/kg/day, no renal lesions occurred after an ODMF dosage of 1 mg activity/kg/day. Concurrent im studies in rats treated with ODMF at dosages of 6, 12, 24, or 48 mg activity/kg/day showed that ODMF dosages of 6 and 12 mg activity/kg/day did not produce renal lesions. In 6-month chronic im studies in dogs with ODMF dosages of 0.5, 1, or 4 mg activity/kg/day or gentamicin sulfate (GS) dosages of 2 mg activity/kg/day, and in concurrent studies in rats treated with ODMF dosages of 0.5, 2, or 6 mg activity/kg/day or GS dosages of 3 mg activity/kg/day, less severe local irritation and nephrotoxicity occurred after treatments with ODMF than with GS. In both rats and dogs treated by either the iv or the im route of administration, higher concentrations of ODMF and GS were found in the kidneys than in the sera. Mean serum and tissue concentrations of GS were higher than those of ODMF. Local tissue irritation and nephrotoxicity were lower with ODMF than with GS on a milligram activity per kilogram basis.

Tulobuterol: one-month intravenous safety studies in rats and dogs.

Tulobuterol was given intravenously to rats and dogs at dosages of 1, 5, or 25 mg/kg/day and 0.6, 2, or 6 mg/kg/day, respectively. The no-toxic-effect dosages were 5 mg/kg/day in rats and 6 mg/kg/day in dogs. Two rats died at 25 mg/kg/day. Convulsions, jerking movements, hyperactivity, tremors, hypoactivity and ptyalism were observed in rats given 25 mg/kg/day. Restlessness, ptyalism and hypoactivity were also observed in dogs at 2 and 6 mg/kg/day. Cutaneous and/or mucosal erythema were observed in rats and dogs at all dosages. Increased body weight gain occurred in drug-treated rats and in mid- and high-dose female dogs. Slight elevations in serum creatinine and BUN were seen in rats and dogs at the highest dosages. Heart weights were increased in rats at all dosages after 1 month of treatment and in rats given 25 mg/kg/day after 2 weeks of recovery. There were no treatment-related morphologic changes in either species.