Dosage forms of antimicrobials play a critical role in facilitating the attainment of pharmacokinetic-pharmacodynamic (PK-PD) targets as well as adherence in both veterinary and human medicine. The purpose of this study was to develop and evaluate a controlled-release subcutaneous amoxicillin implant for single-dose therapy of large ruminants such as goats, sheep, and deer. The degradable implant, designed to attain PK-PD targets following single administration, was evaluated for amoxicillin release rate and time-concentration profile. In vitro release studies demonstrated constant-rate release of approximately 40% of amoxicillin content within 96 h. In an in vivo study in goats, serving as a model for target animals, a serum concentration of approximately 0.4 mg/L was achieved within 8 h of implant insertion and maintained for >6 days. In comparison, in control goats given a standard single intramuscular amoxicillin dose of 15 mg/kg, amoxicillin peaked at 1.2 mg/L after 1 h, rapidly dropping to below detection level at 8 h. These results suggest that the proposed implant offers a unique modality for animal caregivers to conveniently administer a full antimicrobial course following a single dose of an efficient PK-PD-optimized dosage form. Furthermore, modifications of implant composition may allow for tailoring of its characteristics to various PK, PD, microbiological, and clinical requirements.

Beta-lactam antimicrobials, commonly used in both veterinary and human medicine, generally present short biologic half-lives, whereas their activity is enhanced as pathogen exposure is prolonged. These properties necessitate multiple-dose regimens of standard dosage forms, thereby hampering pet owner adherence, frequently resulting in therapeutic failure. This study presents a novel controlled-release gastroretentive oral drug delivery system for beta-lactams with which single-dose administration provides an effective antimicrobial course, optimizing pharmacokinetic (PK)-pharmacodynamic (PD) profiles, minimizing adverse effects and emergence of antimicrobial resistance and facilitating adherence. Our prototype sustained-delivery swelling-tablet (SDST), based on a degradable hydrophilic polymeric matrix, was designed to enable continuous input of these drugs to their absorption sites over several days. Several SDST formulations of the beta-lactam amoxicillin were evaluated in in vitro dissolution studies. Two formulations were selected for further in vivo canine studies, for determination of gastric retention and PK-PD profiling. Prolonged gastric retention times maintaining allowed for maintained effective drug concentrations against many clinically relevant pathogens for more than 48 h for one formulation and more than 5 days for the other. Both SDST formulations offer significant advantages over standard immediate-release therapy in achieving PK-PD goals and enhancing adherence. The prototypical formulations represent a novel platform which may be modified to meet various clinical requirements.

Nanospheres composed of the biocompatible and biodegradable polymer, poly-DL-lactide/glycolide and containing platelet-derived growth factor β-receptor antisense (PDGFβR-AS) have been formulated and examined in vitro and in vivo in balloon-injured rat restenosis model. The nanospheres (~300 nm) of homogenous size distribution exhibited high encapsulation efficiency (81%), and a sustained release of PDGFβR-AS (phosphorothioated). Cell internalization was visualized, and the inhibitory effect on SMC was observed. Partially phosphorothioated antisense sequences were found to be more specific than the fully phosphorothioated analogs. A significant antirestenotic effect of the naked AS sequence and the AS-NP (nanoparticles) was observed in the rat carotid in vivo model. The extent of mean neointimal formation 14 days after injection of AS-NP, measured as a percentage of luminal stenosis, was 32.21 ± 4.75% in comparison to 54.89 ± 8.84 and 53.84 ± 5.58% in the blank-NP and SC-NP groups, respectively. It is concluded that PLGA nanospheres containing phosphorothioated oligodeoxynucleotide antisense could serve as an effective gene delivery systems for the treatment of restenosis.

Background — Inflammation is critical to vascular repair after mechanical injury. Excessive inflammation enhances neointimal formation and restenosis. We examined whether transient systemic inactivation of macrophages at the time of vascular intervention could attenuate the degree of expected restenosis. Methods and Results — Liposomal clodronate (LC) inhibited the growth of cultured macrophages but had no effect on endothelial or smooth muscle cells and suppressed neointimal hyperplasia in hypercholesterolemic rabbits and rats after intravenous administration of LC, with no adverse effects. LC treatment reduced the number of blood monocytes and decreased macrophage infiltration in the injured arteries as well as smooth muscle cell proliferation, interleukin-1β transcription, and production and matrix metalloproteinase-2 activity. Conclusions — Macrophages play a pivotal role in vascular repair after mechanical arterial injury. Systemic inactivation and depletion of monocytes and macrophages by LC reduce neointimal hyperplasia and restenosis.

Poor drug residence in the arterial wall hinders clinical implementation of local drug delivery strategies for the treatment of restenosis. A rat carotid model of vascular injury and intraluminal delivery of tyrphostin-containing polylactic acid (PLA) nanoparticles (NPs) were used to determine the relationship between residence properties and biological activity of different formulations and administration modes. The effects of delivery modes (denudation and delivery time) and formulation variables (adsorbed vs encapsulated drug, and NP size) on arterial drug/NP retention were examined. Antirestenotic effects of large (160 nm) and small (90 nm) tyrphostin-containing NPs, surface-absorbed tyrphostin, and systemic treatment were compared. Fluorescent NPs were used to study the spatial distribution of the carrier in the arterial wall. The decrease in arterial tyrphostin level over time fitted a biexponential model. Delivery time and pressure, endothelium integrity, particle size, and drug-polymer association affected local pharmacokinetics and the antirestenotic results after 14 days. The PLA-based tyrphostin NP formulation ensured a prolonged drug residence at the angioplasty site after single intraluminal application. Several readily adjustable formulation and procedural factors considerably modified arterial ingress of the drug-loaded NPs and governed their subsequent redistribution, tissue binding, elimination, and ensuing antirestenotic effect.

The overall skin low molecular weight antioxidant (LMWA) capacity was evaluated during the aging process and following exposure to oxidative stress. Several invasive and non-invasive techniques were developed for evaluating total antioxidant activity. It was found that the skin possesses an extremely efficient and unique antioxidant activity that is better than other tissues. During the aging process a significant decrease in the levels and activity of the water-soluble LMWA was detected while no change and even a slight increase was recorded for the lipophilic LMWA. Similar results were obtained following exposure to oxidative stress. A significant decrease in the water soluble LMWA was recorded in all the stress induced procedures indicating a common mechanism of response. It has also been shown that along with the reduction in total water soluble antioxidant activity there is an accumulation of oxidized adducts. This was observed both on the surface of the skin and in deeper layers. It has been found that skin releases LMWA from its surface. This secretion phenomenon was found to be age dependent. Following exposure to oxidative stress of various kinds, the release of LMWA from the skin was significantly enhanced. This may suggest a physiological mechanism of the skin to cope with oxidative stress, which would open new possibilities for intervention.

It has been suggested that oxidative stress plays an important role in the chronic complications of diabetes. The experimental findings regarding the changes in tissue antioxidant enzymes and lipid peroxidation of diabetic tissues have been inconsistent. Previous studies in our laboratory demonstrated that the reducing power of a specific tissue correlates with its low molecular weight antioxidant (LMWA) capacity. In the present study, the overall LMWA capacity (reducing equivalents) of plasma and tissues of streptozotocin (STZ)-induced diabetic rats (1-4 weeks) and insulin treated diabetic rats were measured by cyclic voltammetry. Levels of water and lipid soluble LMWA capacity progressively decreased in the diabetic plasma, kidney, heart and brain, while the diabetic liver, at 2, 3 and 4 weeks after STZ injection, showed a significant increase in the overall lipid soluble LMWA capacity (p < 0.001). Subsequently, analysis of specific components by high pressure liquid chromatography (electrochemical detection) showed decreased levels of ascorbic acid in plasma, kidney, heart and brain of diabetic animals. The alpha-tocopherol level dropped in all tissues, except for the liver in which there was a significant increase (p < 0.01 and p < 0.001 at 2-4 weeks). Lipid peroxidation was assessed by conjugated diene levels, which increased significantly in all diabetic tissues except the liver. Insulin treatment that was started after 3 weeks of diabetes and continued for 3 weeks showed no change in the conjugated dienes and in the overall LMWA capacity in all organs. Our results suggest a unique behavior of the liver in the STZ-induced diabetic rats to the stress and indicate its higher capacity to cope with oxidative stress as compared to other organs.

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. 14C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[3H]Phe-[14C]pamidronate, and Pro-[3H]Phe-[14C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (FTIBIA) and 1.9 (FURINE) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.

A method has been developed for measuring and evaluating the overall antioxidant activity derived from the low–molecular weight antioxidants (scavengers). The principle governing this method is based on a common chemical characteristic of the scavengers, their reducing properties. It was hypothesized and then demonstrated that an evaluation of the overall reducing power of a biological sample correlates with the overall scavenging activity of the sample. In order to quantify the total reducing power, the cyclic voltammetry methodology was applied. The resulting measurements correlated with the antioxidant activity of both hydrophilic and lipophilic scavengers. The method is suitable for use in biological fluids and in tissue homogenates, and can supply information concerning the type of antioxidants and their total concentration without having to determine specific compounds. A noninvasive procedure for determining skin overall scavenging activity is also described. This method is based on a well containing an extraction solution that is attached to the skin’s surface. Following incubation time the extraction solution is analyzed using the cyclic voltammeter instrument and other methods. We have found these methods suitable for evaluating the reducing capacity status in various clinical conditions such as diabetes, ionizing and nonionizing irradiation, brain degenerative diseases, head trauma, and inflammatory bowel diseases. This method is also an efficient tool for evaluating the overall antioxidant capacity of mixtures of antioxidant preparations in vitro. The measurements themselves are simple and rapid. Furthermore, they do not require manipulation of the samples.

Restenosis, the principal complication of percutaneous transluminal coronary angioplasty is responsible for the 35–40% long-term failure rate following coronary revascularization. The neointimal formation, a morphological substrate of restenosis, is dependent on smooth muscle cells (SMC) proliferation and migration. Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptors system is involved in the process of post-angioplasty restenosis. The unsuccessful attempts to control restenosis by systemic pharmacological interventions have prompted many researchers to look for more promising therapeutic approaches such as local drug delivery. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the release kinetics and in vivo effects of nanoparticles containing PDGF-Receptor β (PDGFRβ) tyrphostin inhibitor, AG-1295. AG-1295-loaded poly(dl-lactide) (PLA) nanoparticles were prepared by spontaneous emulsification/solvent displacement technique. In vitro release rate and the impact of drug/polymer ratio on the nanoparticle size were determined. The degree of tyrosine phosphorylation was assessed by Western blot with phosphotyrosine-specific antibody in rat SMC extracts. Several bands characteristic of PDGF BB-stimulated SMC disappeared or weakened following tyrphostin treatment. Local intraluminal delivery of AG-1295-loaded PLA nanoparticles to the injured rat carotid artery had no effect on proliferative activity in medial and neointimal compartments of angioplastisized arteries, indicating a primary antimigration effect of AG-1295 on medial SMC.