A Bidirectional Permeability Assay for beyond Rule of 5 Compounds.

Bidirectional permeability measurement with cellular models grown on Transwell inserts is widely used in pharmaceutical research since it not only provides information about the passive permeability of a drug, but also about transport proteins involved in the active transport of drug substances across physiological barriers. With the increasing number of investigative drugs coming from chemical space beyond Lipinski's Rule of 5, it becomes more and more challenging to provide meaningful data with the standard permeability assay. This is exemplified here by the difficulties we encountered with the cyclic depsipeptides emodepside and its close analogs with molecular weight beyond 1000 daltons and cLogP beyond 5. The aim of this study is to identify potential reasons for these challenges and modify the permeability assays accordingly. With the modified assay, intrinsic permeability and in vitro efflux of depsipeptides could be measured reliably. The improved correlation to in vivo bioavailability and tissue distribution data indicated the usefulness of the modified permeability assay for the in vitro screening of compounds beyond the Rule of 5.

Effect of benazepril, robenacoxib and their combination on glomerular filtration rate in cats.

BACKGROUND: Combined use of angiotensin-converting enzyme inhibitors and nonsteroidal anti-inflammatory drugs may induce acute kidney injury in humans, especially when combined with diuretics. The objective of this investigation was to evaluate the effects of benazepril, robenacoxib and their combination in healthy cats. In each of two studies (study 1 followed by study 2), 32 healthy cats were randomised to one of four groups (n = 4 male and 4 female cats per group) in a parallel-group design. The groups received orally once daily for 7 days either placebo (control group), benazepril, robenacoxib or benazepril plus robenacoxib. In study 2, all groups received in addition 0.5 mg/kg furosemide twice daily by subcutaneous injection for 7 days. RESULTS: Benazepril, robenacoxib and their combination were well tolerated as evidenced from lack of clinical signs and no negative effects on body weight, feed consumption and clinical chemistry, haematology and urinalysis variables. The primary endpoint of the study was the glomerular filtration rate (GFR), which was estimated from the plasma clearance of iohexol. In the absence of furosemide, GFR was significantly higher in cats receiving the combination of benazepril plus robenacoxib compared to the other three groups, and was also significantly higher in females receiving only benazepril compared to the control. Administration of furosemide induced diuresis, reduced GFR and activated the renin-aldosterone-angiotensin system, evidenced from increased plasma renin activity and plasma aldosterone concentrations. Compared to the control group in cats treated with furosemide, GFR was increased by benazepril (females only) but decreased by robenacoxib (males only). Benazepril, robenacoxib and their combination significantly inhibited the increase in plasma aldosterone induced by furosemide. CONCLUSIONS: The combination of benazepril and robenacoxib was well tolerated and either increased or had a neutral effect on GFR in healthy cats without or with concomitant furosemide. The combination of benazepril and robenacoxib reduced plasma aldosterone concentrations increased by furosemide. It is recommended to test the efficacy and safety of the combined use of benazepril and robenacoxib in cats with clinical disease, notably proteinuric chronic kidney disease.

Influence of feeding schedules on the chronobiology of renin activity, urinary electrolytes and blood pressure in dogs.

The contribution of the renin-angiotensin-aldosterone system (RAAS) to the development of congestive heart failure (CHF) and hypertension (HT) has long been recognized. Medications that are commonly used in the course of CHF and HT are most often given with morning food for the sake of convenience and therapeutic compliance. However, biological rhythms and their responsiveness to environmental clues such as food intake may noticeably impact the effectiveness of drugs used in the management of cardiovascular disorders. Only sparse information about the effect of feeding schedules on the biology of the RAAS and blood pressure (BP) is presently available. Two studies were designed to explore the chronobiology of renin activity (RA), BP, renal sodium (UNa,fe) and potassium (UK,fe) handling in relation to meal timing in dogs. In a first experiment (Study a), blood and urinary samples for measurement of RA, UNa,fe and UK,fe were drawn from 18 healthy beagle dogs fed a normal-sodium diet at either 07:00, 13:00 or 19:00 h. In a second experiment (Study b), BP was recorded continuously from six healthy, telemetered beagle dogs fed a similar diet at 07:00, or 19:00 h. Data were collected throughout 24-h time periods, and analyzed by means of nonlinear mixed-effects models. Differences between the geometric means of early versus late time after feeding observations were further compared using parametric statistics. In agreement with our previous investigations, the results indicate that RA, UNa,fe, UK,fe, systolic, and diastolic BP oscillate with a circadian periodicity in dogs fed a regular diet at 07:00 h. A cosine model with a fixed 24-h period was found to fit the variations of RA, UK,fe and BP well, whereas cyclic changes in UNa,fe were best characterized by means of a combined cosine and surge model, reflecting a postprandial sodium excretion followed by a monotonous decay. Our data show that feeding time has a marked influence on the chronobiology of the renin cascade, urinary electrolytes, and BP. Introducing a 6- or 12-h delay in the dogs' feeding schedule caused a shift of similar magnitude (05:06 and 12:32 h for Studies a and b, respectively) in the rhythm of these biomarkers. In all study groups, RA and BP exhibited a marked fall just after food intake. The drop in RA is consistent with sodium and water-induced body fluid expansion, while the reduction of BP could be related to the decreased activity of renin and the secretion of vasodilatory gut peptides. An approximately 1.5-fold (1.2-1.6-fold) change between the average early and late time after feeding observations was found for RA (p < 0.0001), UNa,fe (p < 0.01) and UK,fe (p < 0.05). Postprandial variations in BP, albeit small (ca. 10 mmHg), were statistically significant (p < 0.01) and supported by the model-based analysis. In conclusion, the timing of food intake appears to be pivotal to the circadian organization of the renin cascade and BP. This synchronizing effect could be mediated by feeding-related signals, such as dietary sodium, capable of entraining circadian oscillators downstream of the master, light-dark-adjusted pacemaker in the suprachiasmatic nucleus.

Chronobiology of the renin-angiotensin-aldosterone system in dogs: relation to blood pressure and renal physiology.

The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in the regulation of blood pressure and volume homeostasis. Its contribution to the development of cardiovascular diseases has long been recognized. Extensive literature has shown that peptides of the RAAS oscillate with a circadian periodicity in humans, under strong influence of posture, sleep, and age. Although observations of time-variant changes in the renin cascade are available in dogs, no detailed chronobiological investigation has been conducted so far. The present studies were designed to explore the circadian variations of plasma renin activity (RA) and urinary aldosterone-to-creatinine ratio (UA:C) in relation to blood pressure (BP), sodium (UNa, UNa,fe), and potassium (UK, UK,fe) renal handling. Data derived from intensive blood and urine sampling, as well as continuous BP monitoring, were collected throughout a 24-h time period, and analyzed by means of nonlinear mixed-effects models. Differences between the geometric means of day and night observations were compared by parametric statistics. Our results show that variables of the renin cascade, BP, and urinary electrolytes oscillate with significant day-night differences in dogs. An approximately 2-fold (1.6-3.2-fold) change between the average day and night measurements was found for RA (p < 0.001), UA:C (p = 0.01), UK,fe (p = 0.01), and UNa (p = 0.007). Circadian variations in BP, albeit small (less than 10 mm Hg), were statistically significant (p < 0.01) and supported by the model-based analysis. For all variables but UNa and UNa,fe, the levels were higher at night than during the day. The data also indicate that blood pressure oscillates in parallel to the RAAS, such that, as opposed to healthy humans, BP does not drop at night in dogs. The postprandial decrease in RA is assumed to be related to body fluid volume expansion secondary to water and sodium intake, whereas the reduction of UA:C reflects aldosterone-stimulated secretion by the renin-angiotensin II pathway. UNa and UNa,fe peaked in the afternoon, about 7-8 h after food intake, which is consistent with the "impulse-response pattern" of sodium excretion described in previous publications. Finally, UK and UK,fe mirrored aldosterone-mediated potassium secretion in the kidney tubules. To describe the circadian variations of the various variables, two different mathematical representations were applied. A cosine model with a fixed 24-h period was found to fit the periodic variations of RA, UA:C, UK, UK,fe, and BP well, whereas changes in UNa and UNa,fe were best characterized by a surge model. The use of nonlinear mixed effects allowed estimation of population characteristics that can influence the periodicity of the RAAS. Specifically, sodium intake was found to interact with the tonic and the phasic secretion of renin, suggesting that varying feeding time could also impact the chronobiology of the renin cascade.

Growth hormone-releasing factor: structural modification or protection for more potent analogs.

Growth hormone-releasing factor was discovered in 1982 by Guillemin and has been subjected to intense investigations because of its huge potential applications. The major concerns encountered with the native molecules were their short half-lives in vivo in many species including man, precluding the practical use of these peptides for medical or production purposes. Many efforts to produce analogs of shorter length, more resistant to degradation and having higher affinity to the receptors have been made during the last decades. The present paper presents a quick review of the work done to produce such analogs.

Prevention and treatment of experimental osteomyelitis in dogs with ciprofloxacin-loaded crosslinked high amylose starch implants.

Crosslinked high amylose starch (CLHAS) matrix was used as a biodegradable drug delivery implant for the prevention and treatment of osteomyelitis. Thirty-two dogs underwent the femoral insertion of a screw inoculated with Staphylococcus aureus and were then randomly assigned to four groups: (A) prevention with ciprofloxacin-CLHAS implants, (B) surgical debridement (positive control), (C) surgical debridement and oral ciprofloxacin treatment and (D) surgical debridement and treatment with ciprofloxacin-CLHAS implants. At week 4 the osteomyelitis was confirmed, the infected site debrided and respective treatments initiated for groups B, C and D. Radiographs, macroscopic evaluations, bacterial cultures and histopathological examinations were used to evaluate the femora at week 10. Femora from preventive group A were almost normal. Dogs of both ciprofloxacin treatment groups C and D showed better bone healing, less periosteal reaction and less screw mobility than dogs from group B. Eradication of infection was observed at proximal/distal sites in B: 25%/12%, C: 37%/62% and D: 62%/75%. Both ciprofloxacin treated groups improved radiographically from week 4 to week 10. Periosteal and marrow neutrophilic and lymphoplasmocytic infiltrations were less severe in groups C and D versus group B. These data suggest that biodegradable ciprofloxacin-CLHAS implants are a safe and efficient modality for the prevention and treatment of osteomyelitis.

Tissue reaction and biodegradation of implanted cross-linked high amylose starch in rats.

The biocompatibility and degradation characteristics of cross-linked high amylose starch (Contramid were investigated in rats over 4 months. Contramid pellets (3-mm diameter and thickness) obtained by direct compression, were implanted subcutaneously and intramuscularly. On sequential time points, macroscopic observations of implantation sites were performed and tissue samples were removed, fixed, and histologically evaluated. No macroscopic inflammatory reaction was observed with Contramid.. Upon histologic examination, inflammatory reaction produced by Contramid was moderate and restricted to implantation sites. The sequence of inflammatory events with Contramid was similar regardless of implantation site. Degradation of Contramid pellets was characterized by fragmentation with formation of fibrovascular septa and phagocytosis by macrophages. Finally Contramid was mostly absorbed by the end of the 4-month period and substituted by adipocytes. It has been demonstrated that Contramid is a biocompatible and absorbable material.

Characterization of crosslinked high amylose starch matrix implants. 1. In vitro release of ciprofloxacin.

The objective of this study was to characterize in vitro the potential of crosslinked high amylose starch (CLHAS) as an implant matrix for the delivery of ciprofloxacin (CFX). Direct compression of dry blends of four different matrices: control CLHAS; CLHAS with 1% hydrogenated vegetable oil (HVO); and CLHAS with 10 or 20% hydroxypropylmethylcellulose (HPMC), each of them with three CFX loadings (2.5, 5.0 and 7.5%) was performed to prepare twelve implant formulations. All CLHAS implants were used for 24-h dissolution tests to evaluate swelling, erosion, water uptake and CFX release. Additionally, 1%-HVO- CLHAS implants were used for an extended dissolution test. The presence of HPMC in the matrix increased CFX release rate, swelling, erosion and water uptake in a concentration-dependent manner whereas HVO had no effect. With increasing drug loading, a decrease of cumulative CFX percent release was observed in both 24-h and extended dissolution tests. Of the different formulations tested, CLHAS implants with 1% HVO and 7.5% CFX provided the longest period of drug delivery without any initial burst effect.

Characterization of crosslinked high amylose starch matrix implants. 2. In vivo release of ciprofloxacin.

The purpose of this study was to develop a crosslinked high amylose starch (CLHAS) matrix implant as a sustained antimicrobial delivery system for local prevention and/or treatment of osteomyelitis. Implants (200 mg) of CLHAS containing 2.5% (5 mg), 7.5% (15 mg), 15.0% (30 mg) and 20.0% (40 mg) of ciprofloxacin (CFX), were prepared by direct compression of dry blends. Rabbits were administered six 2.5, two 7.5, one 15.0 or one 20.0%-CFX implants along the femur between the quadriceps and biceps femoris muscles to determine systemic (serum) versus local (muscle and bone) CFX concentrations over 1 month. Blood samples were taken throughout the study for CFX assay. Muscle and femur were collected at 3, 7, 14, 21 and 28 days after implantation for host response evaluation and CFX assay. Residual polymer was explanted to determine the remaining dose of CFX. All animals remained healthy during the study. Local tissue reaction was mild and limited to the implantation site. Serum CFX concentrations remained low regardless of implant loading. Increased drug loading resulted in a higher and longer release of CFX in muscle and in bone. Local CFX concentrations were detected largely in excess of the MIC over 28 days with 20.0%-CFX implants. More residual CFX in polymer was detected over a longer period of time at high loading. These results strongly support the development of CLHAS implants for local antibacterial therapy.

Effects of pentoxifylline on pulmonary function and results of cytologic examination of bronchoalveolar lavage fluid in horses with recurrent airway obstruction.

OBJECTIVES: To determine the effects of pentoxifylline (PTX) administration on lung function and results of cytologic examination of bronchoalveolar lavage fluid in horses affected by recurrent airway obstruction (RAO). ANIMALS: 10 RAO-affected horses. PROCEDURES: 6 horses were orally administered PTX (16 g) mixed with corn syrup, and 4 horses were administered corn syrup alone, twice daily for 14 days. Pulmonary function was evaluated before administration (day 0) and on days 8 and 15. Bronchoalveolar lavage (BAL) was performed on days 0 and 15. Reversibility of airway obstruction was assessed by measuring pulmonary function before and after administration of atropine (0.02 mg/kg, IV). Serum concentration of PTX was measured in 4 horses 30 minutes and 2 and 4 hours after administration of PTX on days 1, 2, 3, 7 and 14. RESULTS: Administration of PTX to BAO-affected horses resulted in a decrease in elastance value on day 8 and on elastance and resistance (RL) values on days 8 and 15. Results for cytologic examination of BAL fluid obtained on day 15 did not differ significantly, compared with values for day 0. Values of RL decreased in all horses following administration of atropine. When mixed in corn syrup and administered orally, PTX was poorly absorbed in horses, and there was noticeable variation in serum PTX concentrations over time and among horses. CONCLUSIONS AND CLINICAL RELEVANCE: Based on these results, it can be concluded that administration of PTX at high doses improved respiratory function of RAO-affected horses maintained in an unfavorable environment.

Characterization of subcutaneous Contramid implantation: host response and delivery of a potent analog of the growth hormone-releasing factor.

Cross-linked high amylose starch (Contramid) was investigated as a solid implant for evaluation of host response in mice and as a possible delivery system for a human growth hormone-releasing factor analog (Hex-hGRF) release profile in pigs. Seventy mice were administered subcutaneously one 3 mm diameter Contramid pellet and host reaction was evaluated over 6 months. Thirty pigs were divided into four groups. All animals of the three implanted groups were administered subcutaneously 15 mg Hex-hGRF, (1) one pure Hex-hGRF implant; (2) four 30/70 w/w Hex-hGRF/Contramid implants; or (3) eight 15/85 w/w Hex-hGRF/Contramid implants. The fourth group (n=6) was injected subcutaneously twice daily with 10 microg/kg of Hex-hGRF over 5 consecutive days. Serum insulin-like growth factor-I (IGF-I) was monitored over 1 month. In mice, no adverse reaction occurred after implantation. Macroscopic and microscopic inflammatory reactions were always localized. Polymorphonuclear cells (PMNs) and macrophages predominated within and around the pellets, respectively. Thin fibrovascular septas eventually subdivided Contramid pellets which were progressively phagocytosed by macrophages. In pigs, serum IGF-I concentrations were increased over a 10 day period in all implanted groups. The initial IGF-I peak observed in the daily injected group was avoided in both Contramid implant groups but not in the pure Hex-hGRF implant group. These encouraging results warrant the development of Contramid implants as a sustained delivery system for peptidic drugs.