Enhanced Precision of Circular Dichroism Spectral Measurements Permits Detection of Subtle Higher Order Structural Changes in Therapeutic Proteins.

Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.

Occupancy of Nociceptin/Orphanin FQ Peptide Receptors by the Antagonist LY2940094 in Rats and Healthy Human Subjects.

Therapeutic benefits from nociceptin opioid peptide receptor (NOP) antagonism were proposed for obesity, eating disorders, and depression. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) is a novel, orally bioavailable, potent, and selective NOP antagonist. We studied NOP receptor occupancy (RO) after single oral LY2940094 doses in rat hypothalamus and human brain by use of liquid chromatography with tandem mass spectrometry (LC-MS/MS) (LSN2810397) and positron emission tomography (PET) ([(11)C]NOP-1A) tracers, respectively. A bolus plus constant infusion tracer protocol with PET was employed in humans at 2.5 and 26.5 hours after administration of the LY2940094 dose. The RO was calculated from the change in regional distributional volume (VT) corrected for nondisplaceable volume using Lasson plots. The RO followed a simple Emax relationship to plasma LY2940094 concentration, reaching near complete occupancy in both species. For rat hypothalamus, the plasma concentration at half-maximum RO (EC50) was 5.8 ng/ml. In humans, LY2940094 was well tolerated and safe over the 4-40 mg dose range, and it peaked in plasma at 2 to 6 hours after a 1- to 2-hour lag, with approximate dose-proportional exposure. After 4-40 mg doses, NOP RO was similar across the prefrontal cortex, occipital cortex, putamen, and thalamus, with EC50 of 2.94 to 3.46 ng/ml, less than 2-fold lower than in rats. Over 4-40 mg doses, LY2940094 mean plasma levels at peak and 24 hours were 7.93-102 and 1.17-14.1 ng/ml, corresponding to the cross-region average NOP RO of 73%-97% and 28%-82%, respectively. The rat EC50 translates well to humans. LY2940094 readily penetrates the human brain, and a once-daily oral dose of 40 mg achieves sustainably high (>80%) NOP RO levels suitable for testing clinical efficacy.

Evaluation of hair loss in cats occurring after treatment with a topical flea control product.

BACKGROUND: A formulation containing 39.6% spinetoram resulted in a higher than anticipated number of reports of alopecia at the site of application in the first months following commercial product launch. HYPOTHESIS/OBJECTIVES: To determine the cause of the alopecia using histopathology, including assessment for inflammation, follicular findings of physical trauma (plucking/pulling behaviour) and changes in follicular cycling. ANIMALS: Twenty-four flea-free, male and female adult domestic short hair cats within a private research colony. METHODS: Cats were treated with a single application of 39.6% spinetoram on day 0; personnel were not blinded. Observations of the skin and hair coat began immediately and were repeated at 30 min and 1, 2, 3, 4, 6, 8 and 12 h post-application and then on subsequent days at the same time as initial dosing and at 2, 4, 6, 8 and 12 h after that time, until day 5. If hair thinning or loss was observed, a skin biopsy sample was collected. Two cats not exhibiting abnormalities were biopsied on day 6. RESULTS: Thirty-eight per cent of cats (nine of 24) developed hair thinning and alopecia of sufficient severity within 78 h post-application of the product to warrant skin biopsy. Abnormalities in the skin were limited to the application site and were consistent with physical trauma (pulling or plucking) to the hair. CONCLUSIONS AND CLINICAL IMPORTANCE: Microscopic changes in the hair follicles of affected cats were consistent with self-induced trauma or barbering behaviour. All changes were reversible and paralleled findings associated with well-established, topical flea control products.

Effects of orally administered spinosad (Comfortis) in dogs on adult and immature stages of the cat flea (Ctenocephalides felis).

The efficacy of spinosad against adult fleas (Ctenocephalides felis) on dogs was evaluated in three controlled, blinded studies. One study was conducted to determine speed of kill on experimentally infested dogs. Two additional studies were designed to assess the efficacy of spinosad in preventing environmental contamination with flea eggs (USA study and EU study). An additional objective of the USA study was to assess the effects of skin and hair-coat debris from spinosad-treated dogs on eggs and larvae of C. felis. Dogs were randomly allocated to treatment with beef-flavored spinosad tablets, administered orally at a minimum dosage of 30mg/kg, or placebo. In the first study, speed of kill was determined by flea comb counts performed at 0.5, 1, 2, 4, 8, 12, 24 and 48h after spinosad treatment. Reductions in geometric mean flea counts for spinosad-treated dogs, compared to placebo were 53.7% at 0.5h, 64.2% at 1h, 85.8% at 2h and 100% at 4 through 48h post-treatment (p<0.05 at 1h and beyond). In the 2 flea egg production studies, dogs were treated (spinosad or placebo) once on day 0, infested with 600 fleas approximately 3h post-treatment and reinfested with approximately 600 fleas at intervals over 1 month. Flea eggs were collected starting at approximately 72h after each infestation. Eggs were examined for any effects of spinosad on egg viability. Efficacy of spinosad was also evaluated against environmental eggs and larvae exposed to canine hair-coat debris collected on days 3, 7, 14, 21, and 30. Spinosad was highly effective in reducing flea egg production (>99.8% across the entire study period) compared to control dogs in both egg collection studies. Insufficient numbers of eggs were recovered from spinosad-treated dogs to determine the viability of those eggs. There was no evidence of any effect on environmental flea stages, indicating that spinosad was not present in the skin debris of spinosad-treated dogs. The capability of spinosad to quickly kill adult fleas, and to greatly reduce egg production following challenge with high numbers of adult fleas is important in breaking the flea life cycle and preventing the introduction and establishment of new flea infestations in the household.

Preliminary studies on the effectiveness of the novel pulicide, spinosad, for the treatment and control of fleas on dogs.

Spinosad is a novel mode-of-action insecticide produced from a family of natural products derived from fermentation of the actinomycete, Saccharopolyspora spinosa. Separate studies were undertaken to determine the minimum effective dose of spinosad given orally for the treatment of experimentally induced flea infestations (Ctenocephalides felis) on dogs, and to assess any potential impacts of feeding canned or dry food at the time of dosing. Both were randomized block (blocked by gender and pre-treatment flea counts), blinded parallel-arm studies, with dogs selected on health and ability to maintain pre-treatment flea populations. For dose selection, 48 dogs were allocated among six groups (8dogs/group; 4 males, 4 females): placebo-treated negative control, spinosad in gelatin capsules at 15, 20, 30 and 40mg/kg administered per os; and topical imidacloprid (10mg/kg) as a positive control. Placebo and spinosad treatments were administered on Days 0, 30 and 60, imidacloprid only on Day 0. In a second study to assess the impact of food type at the time of dosing, three groups were formed: placebo-treated control (8 dogs; 4 males, 4 females), spinosad (30mg/kg) administered with canned food (8 male dogs, 8 females); and spinosad (30mg/kg) with dry food (8 males, 8 females). Treatments were administered on Days 0 and 30. To assess post-treatment persistent efficacy, flea infestations were repeated at regular post-treatment intervals, beginning on Day 5 through Day 89 in the dose selection study and Day 58 in the impact of food type and dosing study. Flea counts were performed 48h post-infestation by study personnel who were blinded to treatments. In the dose selection study, compared to geometric mean live flea counts in the control group, each spinosad dose was highly effective (99.8-100%) at 7, 14 and 21 days after treatment. Only the 30 and 40mg/kg doses maintained high efficacy (97.2-100%) until 30 days after treatment, with no difference between the two. Imidacloprid was highly effective at Day 30, with significant difference only from the 15mg/kg spinosad group. Because there was no significant difference between the higher spinosad rates, 30mg/kg was selected as the optimal minimum effective dose. In the second study, spinosad was highly effective at all post-treatment flea counts (98-100%). Taken together, these studies demonstrate that repeated monthly oral treatments with spinosad at 30mg/kg provide sustained control of C. felis on dogs. There were no treatment-related adverse events in either study, indicating that spinosad has potential to be used monthly as a safe and effective flea adulticide, providing sustained activity that matches that of currently used topical products.

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions.

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.

Repellency and efficacy of a 65% permethrin spot-on formulation for dogs against Aedes aegypti (Diptera: Culicidae) mosquitoes.

A topically applied 65% permethrin spot-on (Defend EXspot Treatment for Dogs, Schering-Plough Animal Health) used for flea and tick control on dogs was evaluated for repellency and efficacy against the yellow fever mosquito, Aedes aegypti, a vector of canine filariasis. Six dogs were randomly assigned to receive a single application of 65% permethrin on Day 0 (n=3) or to remain untreated as controls (n=3). Dogs were anesthetized and exposed to 100 unfed, female mosquitoes in screened cages for 2 hours on Days -6, -4, -1, 0, 1, 7, 14, 21, and 28. Mosquito landing rates, engorgement rates, and mortality were determined for each mosquito challenge. Cages were thoroughly cleaned after each mosquito challenge. Treatment of dogs with 65% permethrin reduced the mosquito landing rates by 96.3% 6 hours after treatment and by 82.5% on Day 1. Mosquito mortality, relative to the control group, was 28.2% 6 hours after treatment, ranged from 84.0% to 90.9% through Day 21, and declined to 50.3% 28 days after treatment. Successful feeding by mosquitoes was significantly (P=.05) reduced on Days 1 through 28. The 65% permethrin spot-on treatment killed and repelled significantly (P =.05) more mosquitoes on treated dogs versus untreated dogs for 28 days after treatment.

Synthesis and in vivo evaluation of novel radiotracers for the in vivo imaging of the norepinephrine transporter.

The (R,R) and (S,S) enantiomers of 2-[(2-methoxyphenoxy)phenylmethyl]morpholine (MeNER) have been radiolabelled with carbon-11 in good yield and at high specific activity. These radiotracers are close analogues of reboxetine, a potent and selective ligand for the norepinephrine transporter (NET). They were examined as potential ligands for imaging NET in vivo by positron emission tomography (PET). The in vivo brain distribution of both [(11)C]-labeled enantiomers were evaluated in rats. Following tail-vein injection of the (R,R)-enantiomer regional brain uptake and washout of radioactivity was homogeneous at all time points examined (5-60 min). In contrast, administration of the (S,S)-enantiomer produced a heterogeneous distribution of radioactivity in brain with highest uptake in the hypothalamus, a NET rich region, and lowest uptake in the striatum, a brain region devoid of NET. Hypothalamus to striatum ratios of 2.5 to one were achieved at 60 min post injection of (S,S)-[(11)C]-MeNER. Pre-injection of the norepinephrine reuptake inhibitors, reboxetine or desipramine, reduced hypothalamus to striatum ratios to near unity while reuptake inhibitors of dopamine and serotonin had no significant effect on binding. In vitro autoradiography studies (rat brain slices) with (S,S)-[(11)C]-MeNER produced a regional distribution pattern that was consistent with the reported distribution of NET. (S,S)-[(11)C]-MeNER has the potential to be the first successful PET ligand to image NET.

Efficacy of three dose volumes of topically applied 65% permethrin against Ctenocephalides felis and Rhipicephalus sanguineus on dogs weighing 30 kg or more.

Twenty-five dogs were evenly and randomly allocated by weight to five treatment groups: untreated control, 2 ml 65% permethrin (dogs weighing 15 to 29 kg, "average dogs"), and 2, 3, or 4 ml 65% permethrin (dogs weighing > or = 30 kg, "big dogs"). Each dog was infested with 125 unfed, adult cat fleas, Ctenocephalides felis, and 50 unfed, adult brown dog ticks, Rhipicephalus sanguineus, on Days -3 (ticks only), -2 (fleas only), 3, 7, 14, 17, 21, 24, and 28. Fleas and ticks were counted 1 and 3 or 4 days after each infestation. The duration of efficacy (defined as >90%) against C. felis was 28 to 31 days. The efficacy against fleas 31 days after application of 2, 3, or 4 ml on big dogs ranged from 79.1% (2 ml) to 100% (4 ml). Big dogs that received either 3 or 4 ml of 65% permethrin had significantly (P < or = .05) fewer fleas at several evaluations between 15 and 31 days after treatment. The duration of efficacy against R. sanguineus was 15 (2 ml for big dogs) to 28 (2 ml for average dogs) days. The efficacy against R. sanguineus 28 days after treatment ranged from 79.1% (2 ml on big dogs) to 94.1% (2 ml on average dogs). Significantly (P < or = .05) fewer ticks were present at several evaluations after treatment on big dogs that received 3 or 4 ml than were present on big dogs treated with 2 ml. No significant differences were detected between the 3- and 4-ml groups from Days 10 to 31; however, the geometric mean number of ticks in the group treated with 4 ml was numerically lower than that for dogs treated with 3 ml on several occasions. These data indicate that a dose volume of 3 or 4 ml of 65% permethrin is needed to obtain an adequate level and duration of efficacy against both C. felis and R. sanguineus on dogs weighing 30 kg or more.