Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship.

Topical application of ectoparasiticides for flea and tick control is a major focus for product development in animal health. The objective of this work was to develop a quantitative structure permeability relationship (QSPeR) model sensitive to formulation effects for predicting absorption and skin deposition of five topically applied drugs administered in six vehicle combinations to porcine and canine skin in vitro. Saturated solutions (20 µL) of (14) C-labeled demiditraz, fipronil, permethrin, imidacloprid, or sisapronil were administered in single or binary (50:50 v/v) combinations of water, ethanol, and transcutol (6 formulations, n = 4-5 replicates per treatment) nonoccluded to 0.64 cm(2) disks of dermatomed pig or dog skin mounted in flow-through diffusion cells. Perfusate flux over 24 h and skin deposition at termination were determined. Permeability (logKp), absorption, and penetration endpoints were modeled using a four-term Abrahams and Martin (hydrogen-bond donor acidity and basicity, dipolarity/polarizability, and excess molar refractivity) linear free energy QSPeR equation with a mixture factor added to compensate for formulation ingredient interactions. Goodness of fit was judged by r(2) , cross-validation coefficient, coefficients (q(2) s), and Williams Plot to visualize the applicability domain. Formulation composition was the primary determinant of permeation. Compounds generally penetrated dog skin better than porcine skin. The vast majority of permeated penetrant was deposited within the dosed skin relative to transdermal flux, an attribute for ectoparasiticides. The best QSPeR logKp model for pig skin permeation (r(2) = 0.86, q(2) s = 0.85) included log octanol/water partition coefficient as the mixture factor, while for dogs (r(2) = 0.91, q(2) s = 0.90), it was log water solubility. These studies clearly showed that the permeation of topical ectoparasiticides could be well predicted using QSPeR models that account for both the physical-chemical properties of the penetrant and formulation components.

The pharmacokinetics of oclacitinib maleate, a Janus kinase inhibitor, in the dog.

The pharmacokinetics of oclacitinib maleate was evaluated in four separate studies. The absolute bioavailability study used a crossover design with 10 dogs. The effect of food on bioavailability was investigated in a crossover study with 18 dogs. The breed effect on pharmacokinetics was assessed in a crossover study in beagles and mongrels dogs. Dose proportionality and multiple dose pharmacokinetics were evaluated in a parallel design study with eight dogs per group. In all four studies, serial blood samples for plasma were collected. Oclacitinib maleate was rapidly and well absorbed following oral administration, with a time to peak plasma concentration of <1 h and an absolute bioavailability of 89%. The prandial state of dogs did not significantly affect the rate or extent of absorption of oclacitinib maleate when dosed orally, as demonstrated by the lack of significant differences in pharmacokinetic parameters between the oral fasted and oral fed treatment groups. The pharmacokinetics of oclacitinib in laboratory populations of beagles and mixed breed dogs also appeared similar. Following oral administration, the exposure of oclacitinib maleate increased dose proportionally from 0.6 to 3.0 mg/kg. Additionally, across the pharmacokinetic studies, there were no apparent differences in oclacitinib pharmacokinetics attributable to sex.

Pharmacokinetics of ceftiofur crystalline-free acid sterile suspension in the equine.

Absolute bioavailability and dose proportionality studies were performed with ceftiofur in horses. In the absolute bioavailability study, thirty animals received either an intravenous dose of ceftiofur sodium at 1.0 mg/kg or an intramuscular (i.m.) dose of ceftiofur crystalline-free acid (CCFA) at 6.6 mg/kg. In the dose proportionality study, 48 animals received daily i.m. ceftiofur sodium injections at 1.0 mg/kg for ten doses or two doses of CCFA separated by 96 h, with CCFA doses of 3.3, 6.6, or 13.2 mg/kg. Noncompartmental and mixed-effect modeling procedures were used to assess pharmacokinetics (PK). CCFA was well absorbed with a bioavailability of 100%. AUC(0-∞) and C(max) increased in a dose-related manner following administration of the two doses of CCFA at 3.3, 6.6, and 13.2 mg/kg. The least-squares mean terminal half-life (t(½) ) following the tenth daily i.m. injection of ceftiofur sodium at 2.2 mg/kg was 40.8 h, but the least-squares mean t(½) following the second i.m. injection of CCFA at 6.6 mg/kg was 100 h. The time that plasma ceftiofur equivalent concentrations remain above a threshold concentration of 0.2 µg/mL has been associated with efficacy, and following administration of two 6.6 mg/kg doses of CCFA, the mean time above 0.2 µg/mL was 262 h. Simulations with the nonlinear mixed-effect PK model predicted that more than 97.5% of horses will have plasma ceftiofur equivalent concentrations >0.2 µg/mL for 96 h after the second 6.6 mg/kg dose of CCFA.

Synthesis of homogeneous glycopeptides and their utility as DNA condensing agents.

Two glycopeptides were synthesized by attaching purified glycosylamines (N-glycans) to a 20 amino acid peptide. Triantennary and Man9 Boc-tyrosinamide N-glycans were treated with trifluoroacetic acid to remove the Boc group and expose a tyrosinamide amine. The amine group was coupled with iodoacetic acid to produce N-iodoacetyl-oligosaccharides. These were reacted with the sulfhydryl group of a cysteine-containing peptide (CWK18), resulting in the formation of glycopeptides in good yield that were characterized by 1H NMR and ESIMS. Both glycopeptides were able to bind to plasmid DNA and form DNA condensates of approximately 110 nm mean diameter with zeta potential of +31 mV. The resulting homogeneous glycopeptide DNA condensates will be valuable as receptor-mediated gene-delivery agents.

Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates.

The biodistribution, metabolism, cellular targeting, and gene expression of a nonviral peptide DNA gene delivery system was examined. (125)I-labeled plasmid DNA was condensed with low molecular weight peptide conjugates and dosed i.v. in mice to determine the influence of peptide DNA formulation parameters on specific gene targeting to hepatocytes. Optimal targeting to hepatocytes required the combined use of a triantennary glycopeptide (Tri-CWK(18)) and a polyethylene glycol-peptide (PEG-CWK(18)) to mediate specific recognition by the asialoglycoprotein receptor and to reduce nonspecific uptake by Kupffer cells. Tri-CWK(18)/PEG-CWK(18) DNA co-condensates were stabilized and protected from metabolism by glutaraldehyde crosslinking. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. Glutaraldehyde crosslinking of DNA condensates reduced the liver elimination rate from a t((1/2)) of 0.8 to 3.6 h. An optimized gene delivery formulation produced detectable levels of human alpha1-antitrypsin in mouse serum which peaked at day 7 compared to no expression using control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system.

Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides.

In a previous report (M.S. Wadhwa et al. (1997) Bioconjugate Chem. 8, 81-88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK18) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK18. Surprisingly, AlkCWK18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000-4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK18 and K20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK18 or K20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.

Stability of peptide-condensed plasmid DNA formulations.

Low molecular weight homogeneous peptides were used to form peptide/DNA condensates. A peptide possessing 18 lysines was found to protect plasmid DNA from serum endonuclease and sonicative-induced degradation whereas a shorter peptide possessing 8 lysines dissociated in 0.1 M sodium chloride and failed to protect DNA from enzymatic degradation. Peptide-condensed DNA showed no change in the ratio of supercoiled to circular DNA following 100 W sonication for up to 60 s and was able to transfect HepG2 cells with equivalent efficiency as untreated condensed plasmid DNA. Alternatively, uncondensed plasmid DNA was rapidly fragmented by sonication and serum endonucleases and resulted in negligible gene expression following condensation with peptide. Cationic lipid/DNA complexes were only partially effective at stabilizing DNA in serum compared to the complete stabilization afforded by peptide/DNA condensation. These results indicate that the stabilization afforded by condensation with a peptide protects DNA during formulation and preserves its structure in serum. These functions are important to achieve optimal gene expression from a nonviral gene delivery system.

Peptide-mediated gene delivery: influence of peptide structure on gene expression.

Cationic peptides possessing a single cysteine, tryptophan, and lysine repeat were synthesized to define the minimal peptide length needed to mediate transient gene expression in mammalian cells. The N-terminal cysteine in each peptide was either alkylated or oxidatively dimerized to produce peptides possessing lysine chains of 3, 6, 8, 13, 16, 18, 26, and 36 residues. Each synthetic peptide was studied for its ability to condense plasmid DNA and compared to polylysine19 and cationic lipids to establish relative in vitro gene transfer efficiency in HepG2 and COS7 cells. Peptides with lysine repeats of 13 or more bound DNA tightly and produced condensates that decreased in mean diameter from 231 to 53 nm as lysine chain length increased. In contrast, peptides possessing 8 or fewer lysine residues were similar to polylysine19, which bound DNA weakly and produced large (0.7-3 microns) DNA condensates. The luciferase expression was elevated 1000-fold after HepG2 cells were transfected with DNA condensates prepared with alkylated Cys-Trp-Lys18 (AlkCWK18) versus polylysine19. The gene transfer efficiencies of AlkCWK18 and cationic lipids were equivalent in HepG2 cells but different by 10-fold in COS 7 cells. A 40-fold reduction in particle size and a 1000-fold amplification in transfection efficiency for AlkCWK18 DNA condensates relative to polylysine19 DNA condensates suggest a contribution from tryptophan that leads to enhanced gene transfer properties for AlkCWK18. Tryptophan-containing cationic peptides result in the formation of small DNA condensates that mediate efficient nonspecific gene transfer in mammalian cells. Due to their low toxicity, these peptides may find utility as carriers for nonspecific gene delivery or may be developed further as low molecular weight DNA condensing agents used in targeted gene delivery systems.