A four-hour topotecan infusion achieves cytotoxic exposure throughout the neuraxis in the nonhuman primate model: implications for treatment of children with metastatic medulloblastoma.

The purpose of this study was to define the length of topotecan (TPT) i.v. infusion necessary to attain a cytotoxic exposure for medulloblastoma cells throughout the neuraxis. In vitro studies of human medulloblastoma cell lines (Daoy, SJ-Med3) were used to estimate the length and extent of TPT systemic exposure associated with inhibition of tumor cell growth or the exposure duration threshold (EDT). We evaluated TPT systemic and cerebrospinal fluid (CSF) disposition in six male rhesus monkeys (8-12 kg) that received TPT 2.0 mg/m2 i.v. as a 30-min or 4-h infusion. Plasma and CSF samples were assayed for TPT lactone by high-performance liquid chromatography, and the CSF exposures were compared with the estimated EDT. Results of the in vitro studies defined an EDT as a TPT lactone concentration of > 1 ng/ml for 8 h (IC99) daily for 5 days. The mean +/- SD for systemic clearance (CL(SYS)), penetration into fourth ventricle (%CSF(4th)), and penetration into lumbar space (%CSF(LUM)) were similar for the 30-min and the 4-h infusions. At a TPT lactone systemic exposure (AUC(PL)) of 56.7 +/- 19.9 ng/ml x h, time above 1 ng/ml in the fourth ventricle was 1.4-fold greater for a 4-h infusion compared with a 30-min infusion. At a TPT lactone AUC(PL) of 140 ng/ml x h, the 4-h infusion achieved the desired TPT exposure throughout the neuraxis (lateral and fourth ventricles and lumbar space), whereas the 30-min infusion failed to achieve it in the lumbar space. In conclusion, prolonging TPT i.v. infusion from 30-min to 4-h at a targeted AUC(PL) achieves the EDT throughout the neuraxis and represents an alternative method of TPT administration that will be tested prospectively in patients with high-risk medulloblastoma.

The cat/feline immunodeficiency virus model for transmucosal transmission of AIDS: nonoxynol-9 contraceptive jelly blocks transmission by an infected cell inoculum.

OBJECTIVES: To develop an animal model to study transmucosal lentivirus transmission, and to determine whether topical application of contraceptive jelly can block transmission by an infected cell incoulum. DESIGN: Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an AIDS-like disease in domestic cats. HIV is transmitted primarily across mucosal surfaces, and infected cells may be important in this transmission. We tested the ability of FIV-infected cells to transmit infection across the vaginal, rectal and oral mucosa of the cat, and whether a vaginal contraceptive jelly could prevent such transmission. METHODS: An inoculum consisting of 2 million FIV-infected primary cat T cells was administered vaginally, rectally or orally to female cats that had received either no pretreatment or pretreatment with a contraceptive jelly containing the detergent nonoxynol-9 as spermicide. Transmission was detected by monitoring recipient animals for viral antibodies and by viral cultures of blood leukocytes. RESULTS: A single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina or rectum (10 out of 11 animals became infected). Pretreatment of the vagina (five animals) or rectum (four animals) with contraceptive jelly protected all animals from transmission by the highly infectious inoculum. CONCLUSIONS: The cat/FIV model provides an efficient means to study transmucosal transmission of lentivirus infections, and for assessing vaginal barrier methods that could block transmission. One such method, nonoxynol-9 contraceptive jelly, effectively prevents transmucosal transmission by an FIV-infected cell inoculum.

Interspecies comparison of platelet aggregation, LIBS expression and clot retraction: observed differences in GPIIb-IIIa functional activity.

We examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species' platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

Evaluation of dose-dependent pharmacokinetics of cocaethylene and cocaine in conscious dogs.

Cocaine use continues to be widespread in the United States. Most cocaine users co-ingest ethanol resulting in decreased elimination of cocaine and formation of the active cocaine metabolite, cocaethylene, by hepatic carboxylesterases. In a recent study from our laboratory in dogs to evaluate the cocaine-ethanol interaction, we demonstrated a similar ethanol-induced reduction in cocaine metabolism, although we were unable to detect cocaethylene when the two drugs were given together. This unexpected finding could be explained by ethanol-induced inhibition of cocaine metabolism via a pathway that does not involve hepatic carboxylesterases or formation of cocaethylene that inhibits cocaine metabolism and is then rapidly cleared. The purpose of the present study is to determine which of these mechanisms best explain our data by characterizing the pharmacokinetics of cocaine and cocaethylene over a range of doses in conscious dogs. Seven adult mongrel dogs received 1, 3, and 5 mg/kg cocaine and cocaethylene HCl base with each drug dose administered i.v. on a separate study day. Arterial blood samples were collected at various times after each dose and analyzed for cocaine and cocaethylene by HPLC. Cocaine clearance was dose-dependent with clearance decreasing from 1.53 +/- 0.31 to 1.09 +/- 0.11 l/min as the dose was increased from 1 to 5 mg/kg (p<0.05). Vmax x Vss and Km for cocaine were 0.95 +/- 0.40 l/min/kg and 11.2 +/- 6.2 mg/kg, respectively. Cocaethylene pharmacokinetics were similar to those of cocaine, but were not dose-dependent over the dose range of 1-5 mg/kg. These results suggest that cocaethylene is not formed and rapidly cleared after co-administration of cocaine and ethanol to the dog, but rather suggests that cocaethylene is not formed in appreciable quantities in the dog. Therefore, we conclude that the decrease in cocaine elimination in the dog associated with ethanol administration is due to ethanol-mediated inhibition cocaine metabolism, rather than inhibition by cocaethylene.

Comparative pharmacokinetics of submucosal vs. intravenous flumazenil (Romazicon) in an animal model.

PURPOSE: This study was performed to determine the bioavailability and local tissue toxicological safety of flumazenil (Romazicon) when administered by oral submucosal (SM) as opposed to intravenous (i.v.) injection. METHODS: Six dogs each received SM flumazenil (0.2 mg), and their serum was collected at predetermined time intervals (0-2 h) and frozen (-70 degrees C). Seven days later, the dogs received an identical dose of i.v. flumazenil, and serum samples were again collected, as above. Comparative quantitation of flumazenil levels (i.v. vs. SM) was made using a sensitive HPLC assay (UV detection). Direct/local drug toxicity was visually scored by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. An oral pathologist examined slides processed from control and treatment tissues (hematoxylin and eosin staining) taken (punch biopsy) 1 week following SM injection to compare with direct clinical scores. RESULTS: Serum flumazenil levels reached a plateau (8.5 +/- 1.5 ng/mL, mean +/- SD) within 4 min of SM drug injection and declined thereafter to -2 ng/mL by 2 h. Bioavailability of SM flumazenil was 101 +/- 14%, based upon measuring the area under the serum concentration-time curves over 1.5 h (AUC 0-1.5 h, SM vs. i.v. drug). Thus, serum drug levels following SM drug administration were broadly comparable to those obtained during the elimination phase of corresponding i.v. drug delivery. Regarding drug tissue toxicity, no evidence of direct drug toxicity was observed by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. Following pathologic review, no difference was seen in the degree of inflammation between treatment and control tissue. CONCLUSION: At the quantity and concentration used, SM drug flumazenil administration appears to be both a safe and a viable alternative to bolus i.v. drug delivery and worthy of further investigation.

Nonendoscopic placement and use of percutaneous gastrostomy tubes in pigs (Sus scrofa domestica).

Swine have supplanted dogs and other large animal species in many biomedical applications and have become the model of choice for numerous areas of research. Anatomic, behavioral, and handling concerns often require pigs to be surgically instrumented for many procedures. Oral dosing in pigs can be accomplished by feeding substances in a palatable substrate, temporarily placing an oral or nasal gastric tube, or permanent surgical placement of an esophagostomy or gastrostomy tube. Oral and nasal gastric tube placement is difficult in conscious, unrestrained animals and is suitable only for short-term enteral access. Placing esophagostomy and gastrostomy tubes is suitable for long-term enteral access and allows easy and rapid administration of substances directly into the stomach. However, these methods frequently require major surgical manipulation for placement. Nonendoscopic percutaneous gastrostomy tube placement is used in companion animals to establish permanent or temporary enteral access. We successfully have adapted this procedure in swine as a method for short-term enteral access. This technique has several advantages over the traditional surgical method for gastrostomy tube placement. It is minimally invasive, results in less animal pain and distress, requires less procedural and recovery time, and is inexpensive. Disadvantages include the potential for splenic entrapment, splenic penetration, perforation of the esophagus, and other complications related to misplacement of the tube or applicator. This paper describes the equipment, technique, and potential complications for placing a nonendoscopic percutaneous gastrostomy tube in swine.

Evaluation of five agents/methods for anesthesia of neonatal rats.

Although neonatal altricial rodents are frequently used in experimental projects in which they must undergo survival surgical procedures, there are few published guidelines for anesthetizing them. Many of the drugs and methods that are commonly used to anesthetize newborn rodents provide inadequate anesthesia or are associated with problems, such as excessively high mortality. A two-part study was undertaken with the intention of identifying agents or methods that can be used to provide humane, safe, and effective anesthesia for neonatal rats. In part I, 1- to 3-day-old rat pups were anesthetized with methoxyflurane or one of several injectable agents, or by induction of hypothermia (immersion in ice water). Times to loss of the righting reflex, surgical anesthesia (indicated by loss of the pedal reflex and failure to respond to a skin incision), and recovery of the righting reflex were measured, and heart rate, respiratory rate, and oxygen saturation were monitored. Ketamine (100 mg/kg of body weight, i.p.), pentobarbital (30 to 40 mg/kg, i.p.), and fentanyl-droperidol combination (0.16 mg of fentanyl and 8.0 micrograms of droperidol/g, i.p.) proved unsafe (> 50% mortality) and/or ineffective at inducing short-term surgical anesthesia. In contrast, methoxyflurane and hypothermia were safe and effective. On the basis of subjective observations that they struggled and vocalized less, pups placed in protective latex sleeves prior to immersion in ice water appeared to be less distressed than pups immersed unprotected. In part II of the study, it was possible to safely maintain 1- to 3-day-old pups at a surgical plane of anesthesia for 30 min with either methoxyflurane or hypothermia. Supplementation with pentobarbital (20 mg/kg, i.p.) offered no advantages but significantly prolonged time to recovery. Surviving pups in both studies grew at similar rates during the month after testing. It was concluded that methoxyflurane or hypothermia are good choices for short- or long-term anesthesia of neonatal rats, and that use of a protective sleeve appears to reduce distress associated with induction of profound hypothermia.

Water soluble propofol anesthesia: an effective and inexpensive alternative.

The authors discuss solubilized propofol, and demonstrate its acute use in guinea pigs as an effective, less expensive, easy-to-use alternative for laboratory animal anesthesia.

Characterization of dermatologic changes in geriatric rhesus macaques.

Nonhuman primates are frequently used for aging studies. We observed a high prevalence of skin disease among a group of geriatric rhesus monkeys (mean age = 25 years; n = 9) used in aging behavioral studies. Gross and histopathologic changes in the skin of these geriatric rhesus were compared with skin from control adult monkeys (mean age = 10; n = 4) and sun-exposed monkeys (mean age = 11; n = 4) to characterize age-related skin changes. Biopsy specimens were taken from four specified skin locations (lateral to bridge of nose, ventral midline, dorsal midline, perineal area) and from additional areas where skin lesions were present. Samples were routinely processed and evaluated by light microscopy. Blood samples were collected and tested for estrogen, thyroid-stimulating hormone, triiodothyronine thyroxine, and cortisol levels. The axilla was swabbed and samples were obtained for bacterial culturing. All nine of the geriatric monkeys had notable dermal lesions, while one of the control monkeys and one of the sun-exposed monkeys had abnormal findings. Major gross findings included increased areas of erythematous skin, wrinkling, focal skin scaling, thinning of hair, foot calluses, and exudative lesions. Histologic skin changes included subacute dermatitis, acanthotic dermatitis, and a lesion resembling an early solar lentigo in the sun-exposed animal. These changes were not associated with hormonal abnormalities or bacterial pathogens. Histologic changes are compatible with nonspecific skin changes observed in elderly humans. This study establishes a baseline of dermatologic changes of the aging rhesus macaque.

Relationship of serum iron and nonprotein-bound iron concentrations following administration of ferrous sulfate in pigs.

A prospective analytical study was conducted to determine the relationship between nonprotein-bound iron and serum iron concentrations following gastric instillation of ferrous sulfate. Four female pigs (20-22 kg) with indwelling central venous lines and gastrostomy tubes were studied. A 5% solution of ferrous sulfate (20 mg elemental iron/kg bwt) was administered through the gastrostomy tube over 1 to 2 min. Six hourly blood samples were collected, and serum samples were subjected to ultrafiltration with the filtrate representing nonprotein-bound iron. Iron concentrations were determined by atomic absorption spectrophotometry. Baseline (mean +/- SD) iron concentrations were 73 +/- 25 micrograms/dL as the serum total and 21 +/- 4 micrograms/dL as nonprotein-bound iron. The serum iron and nonprotein-bound iron concentrations achieved a peak of 191 +/- 66 and 23 +/- 10, respectively, at 2 h and declined to near baseline values at 6 h. The mean ratio of filtrate to serum iron concentration was 0.16 +/- 0.08 and ranged from 0.08 to 0.29. Nonprotein-bound iron did not increase as the serum iron concentration increased (r = 0.18) within the ranges achieved in the study. The absence of protein, particularly transferrin and albumin, was verified by electrophoresis. A form of apparent nonprotein-bound iron was isolated from serum by ultrafiltration and its concentration was relatively constant despite the rise and fall of total serum iron concentrations during the 6 h. These observations warrant further investigation to understand the development of toxicity in acute iron poisoning.

Correlation of drug pharmacokinetics and effectiveness of multiple-dose activated charcoal therapy.

STUDY OBJECTIVE: To evaluate an animal model of multiple-dose activated charcoal (MDAC) therapy and correlate the pharmacokinetic properties of four drugs with the efficacy of MDAC. DESIGN: Prospective, randomized, controlled, crossover design. SETTING: A university animal research facility. PARTICIPANTS: Seven female pigs (15 to 22 kg) with an indwelling central venous line and gastrostomy tube. INTERVENTIONS: Acetaminophen (30 mg/kg), digoxin (30 micrograms/kg), theophylline (8.9 mg/kg), and valproic acid (18 mg/kg) were simultaneously administered intravenously over 12 minutes. In the experimental arm, 25 g activated charcoal was administered at 0, 2, 4, 6, 12, 18, 24, and 30 hours through the gastric tube. In the control arm, an equal volume of water was given at the same times. Blood specimens were obtained over 36 hours to measure serum drug concentrations. RESULTS: Each drug exhibited enhanced elimination (P < .01) in the MDAC group except valproic acid. Lower intrinsic clearance was correlated (P < .05) with increased systemic elimination during the charcoal arm. Volume of distribution, half-life, and protein binding were not significantly correlated with charcoal-enhanced systemic drug elimination. CONCLUSION: The response of a drug to MDAC may be affected by its intrinsic clearance. The restrictive nature of the protein binding of valproic acid may be responsible for its lack of response. Results with the porcine model are consistent with the effects observed in human beings.

Changes in adrenal oxygen consumption during catecholamine secretion in anesthetized dogs.

Prior attempts to study adrenal medullary O2 metabolism during catecholamine secretion have been unsuccessful because venous blood from medulla mixes with venous blood from the much larger cortex. To circumvent this problem, eight adult mongrel dogs were pretreated for 5-6 wk with the adrenocorticolytic agent 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p'-DDD). Prednisolone (5 mg/day) and fludrocortisone (0.1 mg.10 kg-1.day-1) were administered orally to prevent adrenocortical insufficiency. Animals were then anesthetized with pentobarbital sodium and subjected to splanchnic nerve stimulation (NS) at 20 and 4 Hz to elicit catecholamine secretion. NS at 20 Hz increased epinephrine secretion from 1.6 +/- 0.7 to 1,780 +/- 762 ng.min-1.g medulla-1 but had no effect on medullary O2 consumption. Medullary blood flow (MQ) increased from 216 +/- 63 to 1,522 +/- 182 ml.min-1.100 g-1, and O2 extraction decreased from 2.7 +/- 0.7 to 0.8 +/- 0.2%. NS at 4 Hz increased epinephrine secretion from 3.1 +/- 1.4 to 76 +/- 17 ng.min-1.g medulla-1 and MQ from 226 +/- 66 to 649 +/- 122 ml.min-1.100 g-1 but had no effect on adrenal O2 consumption or extraction. Cortical blood flow was 342 +/- 98 ml.min-1.100 g-1 at baseline and was unaffected by NS. Gross weight of cortex was reduced by 80% in o,p'-DDD-treated animals, and histological examination of glands from three animals showed only rare islands of glomerulosa cells remaining. These data suggest that increases in MQ during NS do not occur in response to changes in O2 consumption.

Effects of ethanol and cocaethylene on cocaine pharmacokinetics in conscious dogs.

Coingestion of cocaine and ethanol is common among cocaine users, and this combination is reported to enhance the euphoric effects of cocaine. The cardiovascular effects of cocaine are increased in the presence of ethanol, although the mechanism(s) involved in this interaction are poorly understood. Recent studies suggest the enhanced cardiac effects may be caused by ethanol-mediated inhibition of cocaine metabolism leading to higher cocaine plasma concentrations. However, these studies were all performed in animals or humans that form cocaethylene when ethanol and cocaine are coadministered. Thus, it is also possible that cocaethylene could inhibit cocaine's metabolism. Preliminary studies in our laboratory indicate the dog does not form detectable quantities of cocaethylene after coadministration of cocaine and intravenous ethanol. Thus, the dog may be a useful model for isolating the individual contributions of ethanol and cocaethylene to this interaction. The purpose of the present study was to confirm this observation, and to determine the effects of ethanol and cocaethylene on cocaine pharmacokinetics in the conscious dog. Six dogs received cocaine (3 mg/kg i.v.) alone, ethanol (1 g/kg i.v.) followed by cocaine (3 mg/kg i.v.), and cocaine (3 mg/kg i.v.) + cocaethylene (3 mg/kg i.v.). Cocaethylene was not detected in any of the plasma samples from the six dogs after administration of cocaine and ethanol. Ethanol and cocaethylene reduced mean cocaine clearance by 47% and 26%, respectively. Inhibition of cocaine's metabolism by both ethanol and cocaethylene may play an important role in mediating the enhanced effects of cocaine in the presence of ethanol.