Personalized medicine: going to the dogs?

Interindividual variation in drug response occurs in canine patients just as it does in human patients. Although canine pharmacogenetics still lags behind human pharmacogenetics, significant life-saving discoveries in the field have been made over the last 20 years, but much remains to be done. This article summarizes the available published data about the presence and impact of genetic polymorphisms on canine drug transporters, drug-metabolizing enzymes, drug receptors/targets, and plasma protein binding while comparing them to their human counterparts when applicable. In addition, precision medicine in cancer treatment as an application of canine pharmacogenetics and pertinent considerations for canine pharmacogenetics testing is reviewed. The field is poised to transition from single pharmacogene-based studies, pharmacogenetics, to pharmacogenomic-based studies to enhance our understanding of interindividual variation of drug response in dogs. Advances made in the field of canine pharmacogenetics will not only improve the health and well-being of dogs and dog breeds, but may provide insight into individual drug efficacy and toxicity in human patients as well.

Evaluation of a hydrophilic gingival dental sealant in beagle dogs.

A liquid solution, gingival sealant containing polymers that form a barrier film upon application was evaluated in dogs. It is a non-toxic, low viscosity, hydrophilic barrier sealant that dries in approximately 10 to 15-seconds after subgingival application. It was designed as a preventative to be applied immediately following a professional oral hygiene procedure in order to block plaque and calculus formation in the sulcus and aid in the prevention of periodontal disease in companion animals. Additionally, the polymer was designed to promote an aerobic environment in the sulcus by oxygen and water transport through engineered pores within the polymer. A 30-day split-mouth, blinded study in two groups of 15 beagle dogs was used. Plaque was significantly (p < 0.05) reduced on the side receiving the sealant by 30.0% and 50.5% (average = 40.3%) in groups 1 and 2, respectively. Calculus was significantly (p < 0.05) reduced on the side receiving the sealant by 27.2% and 20.0% (average = 23.6%) in groups 1 and 2, respectively. Gingival inflammation was monitored to assess product safety. Sides receiving sealant showed no statistically significant differences in gingival index score. No adverse events were observed in the study. This study demonstrates that this gingival sealant can be used as another valuable tool for aiding in the prevention of periodontal disease in dogs.

Drug residues.

The use of veterinary drugs in animal production is necessary for the prevention and treatment of disease; however, such use may result in residues. Regulatory authorities administer legislative frameworks which ensure that foods derived from animals treated with approved veterinary drugs are safe for human consumption. A human food safety evaluation is conducted as follows: it estimates the risk to human health and safety--based on scientific assessment of the available information and data--formulates measures for controlling the risks identified, and communicates the findings and implications of the risk assessment to interested parties. Foods derived from animals are monitored for the presence of drug residues. The reported incidence of illegal residues from these programmes is very low. These findings reassure the public that veterinary drugs are effectively regulated and that food obtained from treated animals does not contain residues that might constitute a health hazard to consumers. Non-regulatory organizations, including the veterinary pharmaceutical industry, producer organisations, veterinarians and food processors, all contribute to a safe food supply. The food safety risk analysis framework is continually refined to ensure that the health of all consumers is protected.

Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 µm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 µg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 µg/mL, and oxypurinol at 10-200 µg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

Residue Distribution and Depletion of Ractopamine in Goat Tissues After Exposure to Growth-Promoting Dose.

The objectives of the present study was to investigated the ractopamine (RAC) distribution and depletion process in various tissues of goat including liver, kidney, spleen, lung, heart, fat, bile, brain and the eyes. The experiment was carried out on 21 goats (18 treated and 3 controls). Treated goats were orally administered RAC in a dose of 1 mg/kg body mass per day for last 28 days and randomly sacrificed on withdrawal days of 0.25, 1, 3, 7, 14 and 21. RAC in all matrices were determined by ultra-high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry. After 21 days treatment discontinuation, the levels of RAC in bile reached at 13.48 ± 3.36 mg/L, which was significantly higher than that in the other tissues. The concentrations of RAC were followed by kidney, the excretory organ and liver, the major metabolic organ (4.49 ± 0.16 mg/kg for kidney and 1.81 ± 0.11 mg/kg for liver, respectively). The residual concentration of the drug in the eyes of goat was less than that in bile, kidney, liver, lung and spleen on withdrawal days 0.25. RAC residues was higher than the limits of detection = 0.15 µg/mL in liver on Day 21. These findings demonstrated that liver can serve as an alimentary matrix and as a matrix for the control of RAC abuse hypothetically except for urine.

Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC-MS-MS.

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney.

Antibiotic use in critical illness.

OBJECTIVE: To provide a review on the current use of antimicrobials with a discussion on the pharmacokinetic and pharmacodynamic profiles of antimicrobials in critically ill patients, the challenges of drug resistance, the use of diagnostic testing to direct therapy, and the selection of the most likely efficacious antimicrobial protocol. ETIOLOGY: Patients in the intensive care unit often possess profound pathophysiologic changes that can complicate antimicrobial therapy. Although many antimicrobials have known pharmacodynamic profiles, critical illness can cause wide variations in their pharmacokinetics. The two principal factors affecting pharmacokinetics are volume of distribution and drug clearance. Understanding the interplay between critical illness, drug pharmacokinetics, and antimicrobial characteristics (ie, time-dependent vs concentration-dependent) may improve antimicrobial efficacy and patient outcome. DIAGNOSIS: Utilizing bacterial culture and susceptibility can aid in identifying drug resistant infections, selecting the most appropriate antimicrobials, and hindering the future development of drug resistance. THERAPY: Having a basic knowledge of antimicrobial function and how to use diagnostics to direct therapeutic treatment is paramount in managing this patient population. Diagnostic testing is not always available at the time of initiation of antimicrobial therapy, so empiric selections are often necessary. These empiric choices should be made based on the location of the infection and the most likely infecting bacteria. PROGNOSIS: Studies have demonstrated the importance of moving away from a "one dose fits all" approach to antimicrobial therapy. Instead there has been a move toward an individualized approach that takes into consideration the pharmacokinetic and pharmacodynamic variabilities that can occur in critically ill patients.

Consensus on the Rational Use of Antithrombotics in Veterinary Critical Care (CURATIVE): Domain 4-Refining and monitoring antithrombotic therapies.

OBJECTIVES: To systematically review the evidence for therapeutic monitoring of antithrombotic drugs in small animals, develop guidelines regarding antithrombotic monitoring, and identify knowledge gaps in the field. DESIGN: First, a standardized, systematic literature review was conducted to address predefined PICO (Population/Patient, Intervention, Control, Outcome) questions, with categorization of relevant articles according to level of evidence and quality. Preliminary guidelines were developed by PICO worksheet authors and the domain chair. Thereafter, a Delphi-style survey was used to develop consensus on guidelines regarding therapeutic monitoring of antithrombotics in dogs and cats. SETTING: Academic and referral veterinary medical centers. RESULTS: PICO questions regarding the utility of therapeutic monitoring were developed for 6 different antithrombotic drugs or drug classes, including aspirin, clopidogrel, warfarin, unfractionated heparin, the low molecular weight heparins, and rivaroxaban, The majority of the literature pertaining to therapeutic monitoring of antithrombotic drugs was either performed in experimental animal models of disease or involved studies of drug pharmacokinetics and pharmacodynamics in healthy laboratory animals. There was a paucity of high level of evidence studies directly addressing the PICO questions, which limited the strength of recommendations that could be provided. The final guidelines recommend that therapeutic monitoring should be performed when using warfarin or unfractionated heparin in dogs and cats at risk of thrombosis. There is insufficient evidence to make strong recommendations for therapeutic monitoring of aspirin or low molecular weight heparin in dogs and cats at this time. CONCLUSIONS: As in other CURATIVE domains, significant knowledge gaps were highlighted, indicating the need for substantial additional research in this field. Ongoing investigation of the role of therapeutic monitoring of antithrombotic therapies will undoubtedly facilitate improved outcomes for dogs and cats at risk of thrombosis.

Techniques for Monitoring Drug Efficacy.

The efficacy of drugs can vary greatly between species and individuals. Establishing efficacious drug doses for a species requires integration of population pharmacokinetic and pharmacodynamic data into a dose-response curve. Unfortunately, these data sets are rarely available for exotic species. The use of alternative monitoring techniques is required to determine drug efficacy and safety. This article discusses methods to integrate efficacy monitoring into clinical practice, including the use of diagnostic testing and therapeutic drug monitoring.

Simple and Fast Gas-chromatography Mass Spectrometry Assay to Assess Delta 9-Tetrahydrocannabinol and Cannabidiol in Dogs Treated with Medical Cannabis for Canine Epilepsy.

BACKGROUND: To date, an increasing number of pet owners, especially in the USA, are using cannabis-derived products containing generally delta 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) to help their animals' health. Unfortunately, studies on the clinical use of cannabinoids in veterinary medicine are still limited, and the application of analytical methodologies for the determination of cannabinoids in animal (especially dog) biological matrices such as plasma, is still missing. METHODS: A reliable, fast, accurate, simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the quantification of THC and CBD in plasma samples of eight dogs under therapeutic treatment for epilepsy and receiving oral administration of medical cannabis (Bediol). RESULTS: The method was linear for both the analytes under investigation with coefficients of determination (r2) of at least 0.99. Absolute analytical recovery (mean ± SD) ranged from 80.6 ± 6.2% for THC and 81.7 ± 4.3% for CBD. The matrix effect showed less than 10% analytical suppression due to endogenous substances for both the analytes. The intra-assay and inter-assay precision values ranged from 4.9% to 12.7%, and from 5.2% to 8.7% respectively. The intra-assay and inter-assay accuracy values ranged from 2.3% to 9.6% and from 3.4% to 13.0%, respectively. CONCLUSION: The validated method was successfully applied to real samples; moreover, to assess the potential of the method applicability and robustness in future veterinary clinical studies on cannabinoids therapy, we attempted to follow the kinetic of THC and CBD in the plasma of two dogs under therapy at different times after Bediol administration.

Hair Analysis to Monitor the Illegal Use of Salbutamol in Beef Cattle.

This study measured the concentrations of salbutamol residues in red and white hair of cattle during and after salbutamol administration. Three Chinese Simmental beef cattle received an oral administration of 150 µg/kg body weight/d salbutamol for 21 consecutive days. Salbutamol concentrations were determined on Days 1, 7, 14, and 21 of administration and on Days 7, 14, 28, 42, and 70 following the last administration dose using ultra-performance liquid chromatography-tandem mass spectrometry. The concentrations of salbutamol that eluted from hair were determined. The results revealed that salbutamol concentrations were higher in red hair than in white hair on the same sampling day (P < 0.01). In red hair, salbutamol concentrations increased from 29.82 ± 1.8 ng/g on Day 1 of administration to 442.55 ± 250.29 ng/g on Day 21 of administration, and decreased to 33.36 ± 19.22 ng/g on Day 70 after the last administration. In white hair, salbutamol concentrations changed from 4.25 ± 0.32 ng/g on Day 1 of administration to 33.81 ± 6.44 ng/g and 12.25 ± 2.51 ng/g on Days 14 and 70, respectively, after the last administration. The concentrations of salbutamol that eluted from white hair on Days 1 and 7 were 22.94 ± 2.00 ng/g and 92.94 ± 22.49 ng/g, respectively. Our findings revealed that hair is an appropriate biological matrix for assessing the illegal use of salbutamol in animal husbandry.

Hair analysis for veterinary drug monitoring in livestock production.

This review summarizes the basic information and applications concerning the use of hair analysis for the detection of misuse of therapeutic and anabolic agents in livestock animals. Hair biology, hair-shaft structure and the mechanisms of drug incorporation are described, considering the different factors which can affect the deposition. Sampling and extraction methods are reviewed with special attention to the particularities of this matrix, while the use of different analytical techniques is discussed, taking into account the concentration and the sensitivity required for drug detection. Advantages, drawbacks, promising prospects and possible applications of this technique in the future are also discussed.

Study of natural and artificial corticosteroid phase II metabolites in bovine urine using HPLC-MS/MS.

Corticosteroid compounds are widely used therapeutically for their anti-inflammatory properties and sometimes as growth promoters in food producing animals. In the field of drug residue analysis, knowledge of the main metabolic pathways of target analytes improves the efficiency of the corresponding control. Thus, phase II metabolism of corticosteroids, for which very little literature is available, was investigated in cattle. An LC-MS/MS detection method was developed for five commercially available conjugated corticosteroids, permitting direct monitoring during the development of their separation on anion exchange SPE. This separation method is further applicable to other potential urinary conjugated corticosteroids. Because our purpose was not to identify all the existing corticosteroid phase II metabolites, but to obtain their total relative proportions, enzymatic hydrolysis was optimized and performed on each separated fraction (glucuronides and sulfates). Finally, the phase II metabolic profiles of natural and artificial corticosteroids in bovine urine were studied and compared. LC-MS/MS detection with negative electrospray ionization appeared efficient for both glucuronide and sulfate conjugated corticosteroids, and quaternary ammonium stationary phase permitted their effective separation. The experimental design used for optimization of the enzymatic hydrolysis with a purified Helix pomatia preparation demonstrated optimal values for pH 5.2, temperature of 50 degrees C and incubation duration of 4h. Results on bovine urine samples collected on two animals before and after dexamethasone administration showed important differences regarding the proportion of total conjugated forms between endogenous cortisol, endogenous tetrahydrocortisol, and exogenous dexamethasone. This proportion appeared significantly higher for tetrahydrocortisol (40-65%) than cortisol (2-8%) or dexamethasone (4-27%). This innovative methodology demonstrates the suitability of anion exchange SPE and LC-MS/MS for the study of steroid hormones phase II metabolism, and appears promising to investigate metabolic profile differences linked to the hormone administration mode or origin, with direct application in the field of doping controls.

Validation and application of an enzyme-linked immunoassay for human erythropoietin to canine serum.

An enzyme-linked immunoassay (EIA) kit for measuring human serum erythropoietin was evaluated to determine its potential as an assay for canine erythropoietin. Dilutional parallelism was observed with the diluted human standard and pooled canine serum over a range of concentrations of erythropoietin, demonstrating cross-reactivity between human and canine erythropoietin. In canine samples the mean intra- and interassay coefficients of variation were 9.72 and 7.19 per cent, respectively. The theoretical minimum detectable concentration was 0.09 mIU ml-1. In 25 normal adult dogs the concentration of serum erythropoietin ranged from less than the minimum detectable concentration to 31.8 mIU ml-1, with a mean (SD) of 6.77 (6.82) mIU ml-1. The changes in serum erythropoietin could be monitored during the recovery of dogs from phlebotomy induced anaemia. These results suggest that the EIA kit can be used for the quantitative assessment of canine erythropoietin.

The monitoring of heparin administration by screening tests in experimental dogs.

The objective of this study was to investigate the relationship between different screening tests of haemostasis and amidolytic plasma activities of unfractionated (standard) heparin in dogs. Different doses of intravenous (i.v.) [25, 50 or 100 IU Kg(-1)bodyweight (BW)] and subcutaneous (s.c.) heparin (250, 500 and 750 IU kg(-1)) were given to groups each of five clinically healthy adult beagles. Measurements of heparin activity with a factor Xa-dependent chromogenic substrate, activated partial thromboplastin time (APTT) (two different reagents), thrombin time (TT, two different thrombin activities in the reagent: 3 and 6 IU ml(-1)) and the reaction time of the resonance thrombogram (RTG -r) with two different measuring devices were performed at different times. The relationship between ratio values (actual/baseline values) of the coagulation tests and heparin activity was analysed based on regression analysis and correlation coefficient. The greatest alterations were seen for the TT([3 IU ml(-1)])and the RTG -r which were near or exceeded the upper limit of measuring range, if 25 IU kg(-1)BW heparin were given i.v. at heparin plasma levels of 0.54 +/- 0.13 IU ml(-1). These results show, that only APTT and TT measured with high thrombin activity assay appear suitable for guiding high dose heparin therapy in dogs. Averaged alterations of APTT ratio in canine plasma were less than those observed in people for similar plasma heparin levels, indicating that the guideline extrapolated from people for monitoring high dose heparin therapy using APTT may not be valid for use in dogs. After coagulation times had been converted into ratio values, based on regression analysis and Wilcoxon's test, differences of heparin sensitivity were found not only for TT measured with different thrombin activities but also for different APTT reagents (P < 0.001). The correlation between amidylotic antifactor Xa activity and ratio of coagulation times was only moderate and found to be lower for RTG -r (instrument 1: r(s)= 0.711; instrument 2: r(s)= 0.573) than for the other coagulation tests (r(s)= 0.822 to r(s)= 0.890). This indicates a considerable variability of the ratio values of the screening tests at defined heparin plasma activities. These results show, that blood coagulation tests in general are little or unsuitable for heparin antifactor-Xa activity control.

Plasma adrenocorticotropin (ACTH) concentrations and clinical response in horses treated for equine Cushing's disease with cyproheptadine or pergolide.

Plasma ACTH levels have been variable in horses with a positive clinical response for therapy for equine Cushing's Disease (ECD). Therefore, our purpose was to determine the value of monitoring plasma adrenocorticotropin (ACTH) levels during treatment of equine Cushing's disease (ECD) with either cyproheptadine (n = 32) or pergolide (n = 10). First, we validated the chemiluminescent ACTH assay (specificity, precision, accuracy, intra-assay and interassay variations) and tested methods of handling the whole blood from the time of collection to when the ACTH was assayed. The sensitivity and specificity of high plasma ACTH levels for detecting ECD was determined in a retrospective study on hospitalised horses (n = 68). Surveys were sent to veterinarians who submitted equine ACTH levels that were high initially and had at least 2 ACTH samples to determine the value of monitoring ACTH levels during therapy of ECD. The ACTH chemiluminescent assay was valid. The ACTH was stable when whole blood was collected and held in plastic tubes for 8 h before separating the plasma. The sensitivity and specificity of plasma ACTH levels for detecting ECD were 84% (n = 19,95% CI 60,97) and 78% (n = 49,95% CI 63,88), respectively. Treated horses generally showed a decrease in plasma ACTH. Plasma ACTH levels may be helpful when monitoring therapy of ECD, although improvement in clinical signs should be considered most important. There were no differences between cyproheptadine and pergolide in terms of improvements in any of the clinical signs.

Pharmacokinetic/pharmacodynamic approach to assess irrelevant plasma or urine drug concentrations in postcompetition samples for drug control in the horse.

The current performance of analytical techniques used for drug control in horses lead the Regulatory Authorities to decide whether trace levels of drugs legitimately used for therapeutic medication should or should not be reported. Here, we propose a well-ordered and nonexperimental pharmacokinetic/pharmacodynamic approach for the determination of irrelevant drug plasma (IPC) and urine concentrations (IUC). The published plasma clearance is used to transform an effective (marketed) dose into an effective concentration (EPC). EPC is transformed into an IPC by applying a safety factor (SF). This method is based on several assumptions (eg, drug effects reversibly driven by plasma concentration, linearity of drug disposition). The suitability of the computed IPC and IUC can be checked by calculating the residual amount of drug at IPC and computing a minimal drug withdrawal time. It is concluded that controlling the drug effect (using drug or any analyte concentration as a marker) rather than the drug exposure will be more demanding and also makes urine a less than ideal matrix.

Development and use of an enzyme-linked immunosorbent assay to monitor serum and urine acepromazine concentrations in thoroghbreds, and possible changes associated with exercise.

OBJECTIVES: To develop an ELISA that is sensitive and suitable for measurement of immunoreactive acepromazine (ACP) in horse serum and urine and to determine the acute effects of exercise on immunoreactive ACP values in Thoroughbreds. ANIMALS: 12 healthy Thoroughbreds (5 mares, 5 geldings, 2 stallions), aged 2 to 8 years. PROCEDURE: A commercially available antibody and a horseradish peroxidase-conjugated oxime derivative of immunoreactive ACP were used to develop a one-step ELISA. Horses were used in a crossover design study to evaluate possible effects of treadmill exercise on serum and urine ACP concentrations after a single (25 mg) IM injection of the drug. RESULTS: Immunoreactive ACP was detectable at concentrations as low as 50 pg/ml in serum and 100 pg/ml in urine, with intra- and interassay variabilities of 1.1 and 5.2%, respectively. The antibody had some cross-reactivity with a limited number of other phenothiazines. After drug administration, serum ACP immunoreactivity achieved a peak concentration (10.5 ng/ml) within 30 minutes and could be measured up to 48 hours in serum and 120 hours in urine. Although exercise had no significant effect on serum drug concentration, immunoreactive ACP disappeared more quickly (by 48 hours) from the urine of horses in the exercised group. CONCLUSIONS: This one-step ELISA provides a simple and sensitive means to measure immunoreactive ACP in equine serum and urine. The ability to detect drug several days after administration of a low dose of ACP should augment efforts to control illicit use of this drug in performance horses. Potential changes in ACP kinetics after exercise warrant further study.

Effects of serum separation tubes on serum benzodiazepine and phenobarbital concentrations in clinically normal and epileptic dogs.

OBJECTIVE: To characterize the effects of serum separation tubes (SST) on serum drug concentrations. SAMPLE POPULATION: Clinically normal dogs (clorazepate, n = 7) or dogs with epilepsy (phenobarbital, n = 7) were studied in experiment 1, and samples submitted for therapeutic drug monitoring (n = 87) were studied in experiment 2. PROCEDURE: In experiment 1, blood containing either drug was placed in 2 types of 4-ml SST (SST-A and SST-B) and in nonserum separation tubes (non-SST [control]). Samples were processed, then stored at 20 to 22 C (both drugs) or 10 C (phenobarbital only). Aliquots were collected for 96 hours. The rate constant of disappearance and the percentage decrease of each drug over time were determined for each tube. For experiment 2, paired samples were collected in non-SST and SST and submitted by mail for therapeutic drug monitoring. The SST samples were either decanted from SST prior to shipment (group 1; n = 30) or mailed in SST with serum in contact with the silica gel (group 2; n = 57). Drug concentrations and drug elimination half-life were compared between groups. For both experiments, drugs were detected in samples, using polarized immunofluorescence. RESULTS: For experiment 1, the rate constant of drug disappearance for both drugs was greater in the 4-ml SST-A (P < 0.0001). This SST also caused the greatest percentage decrease (20% for phenobarbital and 35% for benzodiazepines) at 96 hours. Refrigeration reduced the mean decrease in phenobarbital at 96 hours to 11%. For experiment 2, phenobarbital concentration was lower for both SST, compared with non-SST (P < 0.0005). Phenobabital had decreased a mean 6.4 +/- 0.5% in group-1 and a mean 30.5 +/- 11.1% in group-2 (P < 0.0005) samples. CONCLUSION: The SST should be avoided when collecting serum for monitoring of either phenobarbital or benzodiazepines. CLINICAL RELEVANCE: The SST can falsely decrease serum drug concentrations and should be avoided when collecting blood for therapeutic drug monitoring.

Use of the urine cortisol-to-creatinine ratio for monitoring dogs with pituitary-dependent hyperadrenocorticism during induction treatment with mitotane (o,p'-DDD).

OBJECTIVE: To determine whether the urine cortisol-to-creatinine ratio (UCCR) could replace the ACTH stimulation test in monitoring effectiveness of mitotane induction treatment in dogs with pituitary-dependent hyperadrenocorticism (PDH). ANIMALS: 15 dogs with PDH. PROCEDURE: All 15 dogs were given an induction dose of mitotane (o,p'-DDD: 35 to 50 mg/kg of body weight/d) for 3 to 14 days. During the induction period, free-catch morning urine samples were collected for determination of UCCR, followed by ACTH stimulation testing, every other day. Treatment response was divided into 3 categories: well-controlled PDH (post-ACTH serum cortisol concentration > or = 28 nmol/L but < or = 138 nmol/L), deficient cortisol secretion (post-ACTH serum cortisol concentration < 28 nmol/L), and excess cortisol secretion (post-ACTH serum cortisol concentration > 138 nmol/L). RESULTS: The linear relation between UCCR and post-ACTH serum cortisol concentration was significant (P < 0.001); however, the prediction intervals surrounding the line were too broad to be clinically useful. The UCCR overlapped among the 3 categories of treatment response. Nevertheless, dogs with PDH receiving mitotane induction treatment and with UCCR > 79 x 10(-6) were always classified as having excess cortisol secretion. CONCLUSION AND CLINICAL RELEVANCE: The UCCR failed to predict post-ACTH cortisol concentration during mitotane induction treatment sufficiently close to be a clinically reliable indicator of treatment control. Seemingly, however, UCCR > 79 x 10(-6) obtained from a dog with PDH during mitotane induction would indicate inadequate adrenal cortex destruction and the need for continued mitotane induction; UCCR < or = 79 x 10(-6) would be inconclusive.