Mathematical modeling and simulation in animal health. Part III: Using nonlinear mixed-effects to characterize and quantify variability in drug pharmacokinetics.

A common feature of human and veterinary pharmacokinetics is the importance of identifying and quantifying the key determinants of between-patient variability in drug disposition and effects. Some of these attributes are already well known to the field of human pharmacology such as bodyweight, age, or sex, while others are more specific to veterinary medicine, such as species, breed, and social behavior. Identification of these attributes has the potential to allow a better and more tailored use of therapeutic drugs both in companion and food-producing animals. Nonlinear mixed effects (NLME) have been purposely designed to characterize the sources of variability in drug disposition and response. The NLME approach can be used to explore the impact of population-associated variables on the relationship between drug administration, systemic exposure, and the levels of drug residues in tissues. The latter, while different from the method used by the US Food and Drug Administration for setting official withdrawal times (WT) can also be beneficial for estimating WT of approved animal drug products when used in an extralabel manner. Finally, NLME can also prove useful to optimize dosing schedules, or to analyze sparse data collected in situations where intensive blood collection is technically challenging, as in small animal species presenting limited blood volume such as poultry and fish.

Individual prediction regions for multivariate longitudinal data with small samples.

Follow-up is more and more used in medicine/doping control to identify abnormal results in an individual. Currently, follow-ups are mostly carried out variable by variable using "reference intervals" that contain the values observable in 100(1-α)% of healthy/undoped individuals. Observations of the evolution of the variables over time in a sample of N healthy/undoped individuals, allows these reference intervals to be individualized by taking into account the possible effect of covariables and some previous observations of these variables obtained when the individual was healthy/undoped. For each variable these individualized intervals should contain 100(1-α)% of observable values compatible with previous observed values in this individual. General methods to build these intervals are available, but they allow only a variable by variable follow-up whatever the possible correlations over time between them. In this article, we propose a general method to jointly follow-up several correlated variables over time. This methodology relies on a multivariate linear mixed effects model. We first provide a method to estimate the model's parameters. In an asymptotic framework (N large enough), we then derive a (1-α) individualized prediction region. Sometimes, the sample size N is not large enough for the asymptotic framework to give a reasonable prediction region. It is for this reason, we propose and compare three different prediction regions that should behave better for small N. Finally, the whole methodology is illustrated by the follow-up of kidney insufficiency in cats.

CLIP Test: a new fast, simple and powerful method to distinguish between linked or pleiotropic quantitative trait loci in linkage disequilibria analysis.

An important question arises when mapping quantitative trait loci (QTLs) for genetically correlated traits: is the correlation due to pleiotropy (a single QTL affecting more than one trait) and/or close linkage (different QTLs that are physically close to each other and influence the traits)? In this article, we propose the Close Linkage versus Pleiotropism (CLIP) test, a fast, simple and powerful method to distinguish between these two situations. The CLIP test is based on the comparison of the square of the observed correlation between a combination of apparent effects at the marker level to the minimal value it can take under the pleiotropic assumption. A simulation study was performed to estimate the power and alpha risk of the CLIP test and compare it to a test that evaluated whether the confidence intervals of the two QTLs overlapped or not (CI test). On average, the CLIP test showed a higher power (68%) to detect close-linked QTLs than the CI test (43%) and a same alpha risk (4%).

Establishing bioequivalence of veterinary premixes (Type A medicated articles).

a) Key issues concerning Premix (Type A medicated articles) Bioequivalence evaluations: 1) This is a complex issue concerning both route of administration and formulation. 2) If the animal is not at the bunk/trough, the animal is not self-administering (eating medicated feed), thus there can be no drug absorption. b) Differing opinions among scientists and regulatory authorities/expert bodies regarding: 1) No harmonization on how to design, conduct, and interpret in vivo studies. 2) Applicability of biowaivers to Type A (premix) products. 3) Why are topdress and complete feed considered differently? Are they different formulations or different routes of administration? 4) Single dose vs. multi-dose studies. 5) What is the final formulation? c) What are the next steps: 1) Harmonize current bioequivalence guidelines through the VICH process. 2) Determine the applicability/non-applicability of the Biopharmaceutical Classification System (BCS). 3) Establish the Total Mixed Ration (i.e. formulation) effects. 4) Define the test subject (individual, pen, etc.).

Short course of immune-suppressive doses of prednisolone, evaluated through a prospective double-masked placebo-controlled clinical trial in healthy Beagles, is associated with sustained modifications in renal, hydration, and electrolytic status.

OBJECTIVE: To investigate the effects and duration of orally administered prednisolone on renal function evaluated by glomerular filtration rate (GFR) determination and creatinine (Cr) and symmetric dimethylarginine (SDMA) concentrations as well as on urinalysis, electrolytes, and hydric status in healthy dogs. ANIMALS: 14 healthy Beagles. PROCEDURES: In this prospective double-masked placebo-controlled study, dogs were randomized after baseline evaluation to receive a 7-day course of either prednisolone (1.5 to 2.0 mg/kg, PO, q 12 h) or a placebo. A repeated-measure design was performed, each dog participating in 4 successive sampling sessions. Clinical data, systolic blood pressure, CBC, and biochemical analyses including serum SDMA concentration, GFR determination, urine output quantification, and complete urinalysis were performed for all dogs the day before (D0) and at the end of steroid administration (D7) as well as 2 weeks (D21) and 4 weeks (D35) after the end of treatment. RESULTS: At D7, when compared with baseline, GFR increased significantly in treated dogs, whereas creatinine and SDMA concentrations decreased significantly. GFR and Cr but not SDMA modifications persisted significantly at D21. None of the variables differed significantly from baseline at D35. The OR of presenting an albumin band on urine electrophoresis was 2.4 times as high in treated versus control dogs (OR, 36; 95% CI, 1.8 to 719.4; P = 0.02). CLINICAL RELEVANCE: A short-term course of immune-suppressive prednisolone treatment in healthy dogs leads to a sustained but reversible renal hyperfiltration state. Modification in electrolytic variables can affect the clinical interpretation of blood work in such patients.

Hydroxychloroquine lung pharmacokinetics in critically ill patients with COVID-19.

Different dosage regimens of hydroxychloroquine (HCQ) have been used to manage COVID-19 (coronavirus disease 2019) patients, with no information on lung exposure in this population. The aim of our study was to evaluate HCQ concentrations in the lung epithelial lining fluid (ELF) in patients infected with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the virus that causes COVID-19. This was a retrospective, observational, multicentre, pharmacokinetic study of HCQ in critically ill COVID-19 patients. No additional interventions or additional samples compared with standard care of these patients were conducted in our teaching hospital. We included all intubated COVID-19 patients treated with crushed HCQ tablets, regardless of the dosage administered by nasogastric tube. Blood and bronchoalveolar lavage samples (n = 28) were collected from 22 COVID-19 patients and total HCQ concentrations in ELF were estimated. Median (interquartile range) HCQ plasma concentrations were 0.09 (0.06-0.14) mg/L and 0.07 (0.05-0.08) mg/L for 400 mg × 1/day and 200 mg × 3/day, respectively. Median HCQ ELF concentrations were 3.74 (1.10-7.26) mg/L and 1.81 (1.20-7.25) for 400 mg × 1/day and 200 mg × 3/day, respectively. The median ratio of ELF/plasma concentrations was 40.0 (7.3-162.7) and 21.2 (18.4-109.5) for 400 mg × 1/day and 200 mg × 3/day, respectively. ELF exposure is likely to be underestimated from HCQ concentrations in plasma. In clinical practice, low plasma concentrations should not induce an increase in drug dosage because lung exposure may already be high.

Breed dependency of reference intervals for plasma biochemical values in cats.

BACKGROUND: Reference intervals (RI) are pivotal in clinical pathology. The influence of breed on RI has been poorly documented in cats. HYPOTHESIS/OBJECTIVES: RI for plasma biochemistry variables are breed-dependent in cats. ANIMALS: Five hundred and thirty-six clinically healthy, fasted, client-owned cats from 4 breeds: Holly Birman (n=132), Chartreux (n=129), Maine Coon (n=139), and Persian (n=136). METHODS: Prospective observational study: Blood samples were collected from the cephalic vein into capillary tubes containing lithium heparin. Plasma glucose, urea, creatinine, total proteins, albumin, calcium, phosphate, sodium, potassium, chloride, and total CO2 concentrations and the activities of alanine aminotransferase and alkaline phosphatase were assayed with a dry slide biochemical analyzer. RI were defined as central 95% intervals bounded by the 2.5th and 97.5th percentiles. Data were analyzed by a linear mixed effects model with type I error rate of 0.05. RESULTS: A significant (P<.05) breed effect was observed for 9/13 variables. The magnitude of the differences between breeds could be clinically relevant for creatinine, glucose, and total protein. Age, body weight, sex, and housing conditions had significant (P<.05) breed-related effects on different variables. CONCLUSIONS AND CLINICAL IMPORTANCE: Breed-specific RI should be considered for cats.

A multicentric retrospective study of serum/plasma urea and creatinine concentrations in dogs using univariate and multivariate decision rules to evaluate diagnostic efficiency.

BACKGROUND: Urea and creatinine are the most frequently used indirect markers in plasma and serum of glomerular filtration rate in dogs. Both have been shown to lack sensitivity but their diagnostic efficiency for the diagnosis of kidney disease has been minimally investigated. OBJECTIVE: The purpose of this retrospective study was to investigate the influence of possible factors of variation on both analytes and to determine whether specific decision rules should be drawn up for subpopulations of dogs. METHODS: The results of urea and creatinine measurements, breed, sex, age, and health status (healthy, renal disease, or nonrenal disease) of 3822 dogs were collected from the archives of 5 veterinary clinics. Data were analyzed with univariate and multivariate decision rules with and without adjustment. RESULTS: There were significant effects and interactions of almost all of the sources of variation. Slight improvements in diagnostic efficiency were obtained by adjusting the decision rules to these sources of variations. Univariate decision rules gave approximately the same diagnostic efficiency for urea and creatinine concentrations, with sensitivity and specificity in the range of 70% and 90%, respectively, using the upper limit of the reference interval as the threshold value. Multivariate decision rules provided only minor improvements in diagnostic efficiency. CONCLUSION: Simultaneous measurement of both urea and creatinine is of limited diagnostic value over the analysis of a single variable. Creatinine is the preferred analyte as it is affected by fewer extrarenal factors of variation.

Comparison of routinely used intestinal biopsy forceps in dogs: an ex vivo histopathological approach.

OBJECTIVES: To compare the quality of duodenal and ileal samples obtained with different biopsy forceps. METHODS: Fifteen dogs were included in a prospective ex vivo study. After euthanasia, the duodenum and the ileum were sampled with four different forceps and evaluated according to a standardised scoring system. The biopsy forceps evaluated had alligator jaws or cups with smooth edge with or without a needle. RESULTS: The global quality of the biopsies was better in the ileum that in the duodenum regardless of the biopsy forceps. Biopsy forceps with smooth edge including a needle resulted in fewer artefacts than biopsy forceps with smooth edge but no needle in both sites and those with alligator jaws without a needle provided deeper biopsies than those with smooth edge without a needle only in the duodenum. There was no effect of the biopsy forceps type on the size of the biopsies. CLINICAL SIGNIFICANCE: Our findings may aid in choosing the appropriate type of forceps for intestinal biopsy.

The use of thermometer protective sheets provides reliable measurement of rectal temperature: a prospective study in 500 dogs.

OBJECTIVES: To measure and compare rectal temperature measurement with and without thermometer protective sheaths. To survey veterinary practices regarding thermometer use and disinfection. MATERIALS AND METHODS: Thermometers were validated with and without protective sheaths for accuracy and repeatability in a water bath with a high precision thermometer as reference. Then, the rectal temperature of 500 dogs was measured with and without protective sheaths in randomised order. The difference in temperature measured by the two methods was analysed with a generalised linear model with order of temperature measurement, life stage, dog size, body condition, level of consciousness, class of temperature and operator as explanatory variables. Practical thermometer use and disinfection was surveyed by online questionnaire. RESULTS: In the pre-clinical phase, study thermometers recorded average temperature differences of −0.05 ± 0.07°C (mean ± SD) and −0.03 ± 0.08°C with and without sheath, respectively and the coefficients of variation ranged from 0.1 to 0.5% with and without sheath. In the clinical study, temperatures measured with and without sheaths differed by less than ±0.2°C for 92% of the measurements. None of the explanatory variables were associated with the observed differences. Twenty-five percent of survey respondents were aware of the guidelines for disinfection of thermometers. CLINICAL SIGNIFICANCE: Thermometer protective sheaths do not affect the measurement of canine rectal temperature in the clinic.

Quantitative evaluation of an experimental inflammation induced with Freund's complete adjuvant in dogs.

A chronic inflammation model in dogs was induced by intraarticular injection of Freund's Complete Adjuvant in the stifle. After a primary, acute response during the first 24 hr, a secondary subacute response was observed after a delay of approximately 3 weeks and persisted for several weeks. To evaluate the time course of the inflammatory process quantitatively, we tested more than 100 different parameters. Finally, only four parameters were selected based on practicability and metrological properties, namely, the body temperature, difference in skin temperature, difference in stifle diameter and vertical force exerted by arthritic hind limb measured using a force plate. The main results of the experimentation were the demonstration that these four parameters were sufficiently repeatable, reproducible, and appropriate to be used for quantitative evaluation of the inflammatory process, and that training of both animals and investigators was required. Finally, it was illustrated that an adjuvant periarthritis in dogs can be used to carry out a pharmacokinetic/pharmacodynamic modelling of an antiinflammatory drug.

Neuroendocrine consequences of very early weaning in swine.

An experiment was conducted to investigate the consequences of very early weaning of piglets on neuroendocrine variables and growth. Sixty piglets from eight litters were either weaned on Postnatal Day 6 (early weaning, or EW piglets) or left with their dam until normal weaning at Day 28 (control piglets, or C). At Days 5, 7, 11, 14, and 19, urine was collected between 7:00 and 8:00 a.m. for the measurement of catecholamines, glucocorticoids, and creatinine. Compared with C, EW piglets displayed a transient increase in urinary cortisol on the day following separation from their dam (Day 7) (P<.05). Urinary norepinephrine (NE) was three times lower in EW compared to C piglets from Day 7 until Day 14 (P<.01) but there was no difference between the two groups on Day 19. Urinary epinephrine (EPI) did not differ between C and EW piglets on the day after weaning. Thereafter, EW piglets displayed a three times drop in urinary EPI as compared to C piglets until the end of the period (P<.01). Weaning induced an immediate reduction in food intake and growth rate and at Day 28, the body weight of EW piglets was 1.60 kg lower than that of C piglets (P<.0001). In conclusion, weaning of 6-day-old piglets results in a marked and prolonged suppression of the release of catecholamines. This result likely reflects physiological responses to insufficient energy intake after weaning, as reflected also by changes in thermoregulatory behavior. The transient increase in cortisol excretion in weanlings may be caused by both emotional distress and acute food deprivation.

Three-process model of supervisory activity over 24 hours.

OBJECTIVES: This study used endogenous and exogenous factors to develop a model for sampling supervisory activity that implied consulting numerous data on computer systems. METHODS: The study was carried out in an automated workshop of a chemical plant. Five crews worked on a 3x8 hour shift system, with changes at 0400, 1200 and 2000. Each team included 2 experienced controllers who supervised the process from computer systems in a control room. Starting and ending duty at the same time as the operators, the researchers used real time to code of all the screen pages selected on the computers by 8 controllers (from 4 teams) over 18 shifts. RESULTS: The time-of-day fluctuations of the call frequency confirmed the existence of endogenous factors (biological rhythms or fatigue), but they cannot explain all the variations observed. The modeling of the data yielded the following 3 explanatory factors: (i) the cognitive demands of the tasks, as an external factor, mainly concern the impact of shift changeover characterized by a strong peak of information gathering at the beginning of the shift, even at 0400; (ii) the shift duration appears as a "fatigue" factor and reflects a rapid reduction of information gathering over the shift; (iii) circadian rhythms are characterized by a minimum of activity at night and a maximum in the afternoon. CONCLUSION: In other similar work situations, in addition to an essential work analysis, our model could help the design of shift duration or schedules for shift changeover.

Stochastic modeling in toxicokinetics. Application to the in vivo micronucleus assay.

A stochastic model for the in vivo micronucleus assay is presented. This model describes the kinetic of the rate of micronucleated polychromatic erythrocytes induced by the administration of a mutagenic compound. For this, biological assumptions are made both on the erythropoietic system and on the mechanisms of action of the compound. Its pharmacokinetic profile is also taken into account and it is linked to the induced toxicological effect. This model has been evaluated by analyzing the induction of micronuclei is mice bone marrow by a mutagenic compound, 6-mercaptopurine (6-mp). This analysis enabled to make interesting remarks about the induction of micronuclei by 6-mp and to put to light an unsuspected wavy kinetic by optimizing the experimental design of the in vivo micronucleus assay.

Does the 1/29 Robertsonian translocation affect the fertility of Blonde d'Aquitaine breed bulls?

The effects of the 1/29 translocation upon male fertility were studied by analysis of the results of 1,350,385 first artificial inseminations with the semen of Blonde d'Aquitaine or Coopelso-93 bulls (n=220). A binomial logit model was designed, taking into account the translocation of sire, breed of sire, breed of dam, year, AI center, and all interactions between translocation, and breed of sire, and breed of dam. Male fertility was not affected by the 1/29 translocation, and the nonreturn rates at 60 to 90 days of Blonde d'Aquitaine females inseminated with the semen of carrier bulls (135,632 first AI) or noncarrier bulls (585,949 first AI) were 74.88% and 74.75%, respectively.

Flow cytometric analysis of bovine CD4 and CD8 lymphocytes: influence of blood sampling and processing methods.

Technical information to facilitate bovine blood treatment for optimum lymphocyte flow cytometry analysis is reported. Murine monoclonal antibodies CC8 and CC63 were used to identify phenotypes corresponding to bovine CD4 T cells and CD8 T cells. Blood samples collected in acid citrate dextrose (ACD) enhanced leucocyte subpopulation separation compared with ethylenediamine tetra-acetic acid, heparin and sodium citrate. To preserve bovine blood before immunophenotyping, samples collected in ACD may be kept at 22 degrees C or at 4 degrees C and should be analysed within 32 hours. For isolation of white blood cells, whole blood lysis was faster and gave the same results as Ficoll gradient separation 1.077 and Ficoll gradient separation 1.083. After immunophenotyping, blood could be stored at 4 degrees C if fixed with paraformaldehyde within seven days. Owing to diurnal variations, blood should be collected at a standard time of the day.

Creatine kinase in dog plasma: preanalytical factors of variation, reference values and diagnostic significance.

In the dog, plasma creatine kinase (CK) activity was stable up to one week at +4 degrees C and one month at -20 degrees C. Activity was higher in serum than in plasma due to interference by CK from the platelets. The reference values were determined in 232 dogs using the IFCC recommended method. There was a significant decrease in activity with age but no effect of sex. In adults, plasma CK exhibited a log-normal distribution ranging from 20 to 104 U per litre. In 510 dogs with various diseases, the overall sensitivity and specificity of CK determination were 40 per cent and 98 per cent, respectively. The numerous false negatives could result from the relatively short half-life of the enzyme, while the false positives could be due to secondary muscle damage.

Effects of renal impairment on the disposition of orally administered enalapril, benazepril, and their active metabolites.

The pharmacokinetics of benazepril, enalapril, and their active metabolites (benazeprilat and enalaprilat) were compared after a single administration of each product by the oral route at the recommended dosage (0.5 mg/kg for both drugs) in the dog before and after moderate experimental renal impairment. Ten dogs were randomly assigned to 2 groups of 5 animals in a 2-period crossover design for angiotensin-converting enzyme inhibitor administration. Renal failure was surgically induced by right nephrectomy and electrocoagulation of the remaining kidney. Renal mass reduction induced a significant decrease (P < .001) in glomerular filtration rate (GFR) (1.7 +/- 0.3 versus 3.3 +/- 0.7 mL/kg/minute). No significant differences before and after surgery were observed for enalapril and benazepril kinetics. The area under the curve (AUC) for enalaprilat increased after surgery from 23.6 +/- 14.7 to 42.4 +/- 20.9 micrograms.minute/mL (P < .01). Mean peak plasma concentration (Cmax) was increased in the impaired dogs (59.1 +/- 23.3 versus 43.9 +/- 32.9 ng/mL), but this variation was not significant (P > .05). Renal failure had no significant effect on AUC for benazeprilat (13.8 +/- 9.8 versus 14.9 +/- 5.0 micrograms.minute/mL) (P > .05), but Cmax decreased significantly (from 55.0 +/- 26.4 to 31.9 +/- 17.7 ng/mL) (P < .05). Multiple regression analysis showed that both GFR and AUC for enalapril were highly significant variables that explained the variation in AUC for enalaprilat (R2 = .86, P < .001) but not for benazeprilat (R2 = .12, P > .05). The results of this study indicate that exposure to enalaprilat, but not to benazeprilat, is increased in dogs with subclinical renal impairment.