Download Pharmacokinetics and bioavailability of three promising tilmicosin

Pharmacokinetics and bioavailability of three promising tilmicosin-loaded lipid
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nanoparticles in comparison with tilmicosin phosphate following oral
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administration in broiler chickens
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Ali RASSOULI1,*, Alwan AL-QUSHAWI1, Fatemeh ATYABI2, Seyed Mostafa
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PEIGHAMBARI3, Mehdi ESFANDYARI-MANESH4, Gholam Reza SHAMS1
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Department of Pharmacology, Faculty of Veterinary Medicine, University of Tehran,
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Tehran, Iran.
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Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical
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Sciences, Tehran, Iran.
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Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran,
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Tehran, Iran.
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Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran,
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Iran
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*Correspondence: [email protected]
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Abstract: Tilmicosin (TLM) is a semi-synthetic antimicrobial agent used mainly in
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poultry and cattle but it has relatively poor oral bioavailability. This study was
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conducted to compare the bioavailability (BA) and main pharmacokinetic (PK)
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parameters of TLM after oral administration of tilmicosin phosphate (TLM-PH) and
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three newly prepared lipid nanoparticles (LNPs) of TLM including solid lipid
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nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and lipid-core nanocapsules
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(LNCs). Sixty broiler chickens were divided into eight groups. In four treatment groups
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(n = 10), each bird was given a single oral dose (20 mg/kg) of a TLM formulation after
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an overnight fasting and in four control groups (n = 5), the vehicles of those
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formulations or distilled water were given. Plasma TLM concentrations were analyzed
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using an HPLC method and the related PK parameters (Cmax, Tmax, AUC0–∞, t1/2, kel,
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ClB/F, MRT and Vd/F) were obtained by non-compartmental analysis. The relative
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bioavailability (Rel. BA) of TLM-SLNs, TLM-NLCs and TLM-LNCs were 1.7, 2.7
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and 3.6 times, respectively, more than BA of TLM-PH. Mean Cmax values were 1.21,
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1.58, 1.76 and 2.17 μg/ml for TLM-PH, TLM-SLN, TLM-NLC and TLM-LNC,
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respectively. In conclusion, TLM-LNPs improved drug BA and PK parameters
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especially TLM-LNC formulation which suggests an efficient delivery system for
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TLM.
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Key words: Tilmicosin, lipid nanoparticles, pharmacokinetics, bioavailability, chicken
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1. Introduction
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Tilmicosin (TLM) is a broad-spectrum macrolide antibiotic derived from tylosin which
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is used in animals only. It has been approved for the treatment and control of
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respiratory diseases associated with Mycoplasma gallisepticum, Mycoplasma synoviae,
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and various bacteria such as Staphylococcus spp. and Pasteurella multocida in broiler
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chickens. Generally, gram negative Enterobacteriaceae are resistant to TLM (1-4).
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TLM is poorly water-soluble especially in basic mediums. At present, soluble TLM-PH
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is used in veterinary medicine as oral solution but this form has problems of low
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potency and low BA. High doses of TLM may enhance its efficacy but posing the risk
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of acute cardiac toxicity since the severity of TLM toxic effects is dose-dependent (5,
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6). Given these disadvantages, studies on new delivery systems for TLM are warranted.
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Oral administration of drugs is considered as the easiest and the most practical route
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but the gastrointestinal (GI) epithelium acts as a physical barrier and may reduce drug
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absorption and produce poor oral BA. To overcome these problems, a number of new
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delivery systems have been developed (7, 8). Oral BA of drugs is highly effected by
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their solubility and permeability, the most important physicochemical parameters
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which determine drug absorption (8). On the other hand, the BA of a drug usually
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determines its therapeutic efficacy because it may affect the onset, intensity and
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duration of action of the drug (1).
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Lowering particle size has revealed promise for increasing the dissolution of drugs as
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well as their BA since it can facilitate the delivery of drugs at the right place and time.
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Nanoparticle (NP) delivery systems can be prepared by using biodegradable materials
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such as lipids (5). These nanoparticles which should be stable and non-toxic can
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improve the efficacy and safety of loaded drugs (8). The size and surface properties of
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NPs highly affect their cellular internalization (9). In recent decades, the lipid-based
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nanoparticles (LNPs) have got special interests due to the use of natural or synthetic
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lipids in their formulation which demonstrate high drug biocompatibility and controlled
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release characteristics.
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Nanoparticles can augment drug absorption by enhancing its dissolution, decreasing
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gastric emptying rate and improving drug intestinal permeability. Lipids are recognized
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as agents which increase lymph formation and encourage lymph flow (10). Basically,
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the body takes up the lipid and solubilized drug at the same time. Therefore, it can be
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considered as a kind of “Trojan horse” effect (11).
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Oral LNPs are able to assist drug dissolution and solubilization because of their ability
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to protect drugs in a solubilized condition and facilitate their mixing with GI
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solubilizers such as bile acids. Furthermore, the protective effect of LNPs along with
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their sustained release properties save drugs from degrading conditions and improve
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their stability in the gastrointestinal tract. The nanoscale range of LNPs facilitates their
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absorption into microfold cells (M cells) of Peyer’s patches and eventually into the
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lymphatic system, so contributes to bypass the first-pass metabolism (12).
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Lipid nanoparticles including SLNs, NLCs and LNCs are colloidal carriers composed
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of a lipid matrix that is solid or liquid at body temperature. The efficiency of LNPs is
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highly influenced by their composition which are usually composed of lipids,
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surfactants as well as their structures which affect the release properties (13).
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Solid lipid nanoparticles were incorporated as a novel oral drug delivery system in the
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1990s. Loading poorly water-soluble drugs into SLNs can improve their GI
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solubilization, absorption, and BA and provide controlled release property as well (9,
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14).
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Nanostructured lipid carriers are considered as second generation of LNPs which
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incorporate biocompatible solid lipid matrix and oily lipid (15). These carriers have
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demonstrated high BA with various routes of administration such as oral route (7).
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Lipid-core nanocapsules are a hybrid nanocarrier system with vesicular structure
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composed of polymer and lipid. These nanocapsules have great qualities as drug
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delivery systems for oral route and are able to increase solubility of lipophilic drugs,
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shield them from enzymatic degradation, enhance drug BA and decrease their side
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effects (16, 17). In addition, LNCs are more appropriate for prolonged release (18).
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Lipid-core nanocapsules are different from other formulation by their composition
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since LNCs have three main components including drug, lipid, and polymer. Proper
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interactions between these components have a vital role in successful manufacturing
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and efficacy of LNCs (17, 19).
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The objective of the present study was to perform a PK analysis and to compare the BA
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of newly designed oral TLM-LNP formulations as potential new delivery systems for
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TLM in chickens. The main PK parameters of these LNP formulations were compared
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with the conventional TLM-PH formulation and the possible mechanisms were
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discussed.
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2. Materials and Methods
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2.1. Chemicals and drug formulations
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Tilmosin® (tilmicosin phosphate aqueous solution, 250 mg/ml) were provided from
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Rooyan Darou, Pharmaceutical Company (Semnan, Iran) and tilmicosin standard (TLM
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content 97.3%) was kindly donated by Razak Pharmaceutical Company (Tehran, Iran).
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TLM-SLN, TLM-NLC and TLM-LNC powders re-dispersed in distilled water to reach
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TLM concentrations at 250 mg/ml of TLM. These TLM-LNP formulations were
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prepared and their physiochemical properties and antibacterial activities were evaluated
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at nanotechnology center in the Faculty of Pharmacy of Tehran University of Medical
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Science (TUMS). In vitro antibacterial testing was carried out in Laboratory of Avian
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Microbiology and Laboratory of Pharmacology in the Faculty of Veterinary Medicine
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(FVM). The LNP preparations were in nanoscale range with suitable properties as
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shown in Table 1 (the original article is in press).
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TLM-PH and TLM-LNP formulations were diluted with distilled water to a final
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concentration of 25 mg/ml prior to oral administration to chickens.
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2.2. Experimental Animals
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Sixty apparently healthy broiler chickens, aged 35 days and 1.0-1.2 kg B.W., were
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obtained from the poultry farm of the Faculty of Veterinary Medicine (FVM),
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University of Tehran. The broilers were housed in cages with at 12 h dark/light cycle.
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Temperature was maintained at 25 ± 2 °C and humidity at 45 – 65% with free access to
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a balanced feed and water. The birds were monitored for one week for any apparent
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clinical signs and adaptation to study area before administration of the drugs. This
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study was approved by the ethics committee of FVM, Project Ethics No. 7506006-6-10.
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.
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2.3. Drug Administration
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The chickens were randomly divided into eight groups, four test groups (n = 10) and
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four control groups (n = 5). Birds were deprived from feed for 12 h prior to drug
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administrations and for 6 h after drug dosing but with free access to water. The test
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groups were given a single oral dose of 20 mg/kg of TLM-PH and TLM-LNP
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formulations equivalent to 20 mg/kg of TLM for TLM-SLN, TLM-NLC and TLM-
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LNC, respectively. Meanwhile, the control groups received equal volumes of distilled
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water or SLN, NLC and LNC vehicles (blanks). Oral administration was done directly
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into the middle of esophagus using a gavage attached to a syringe following the zero
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time-point blood sampling.
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2.4. Blood sampling
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Blood samples (about 1.5 ml) were collected from the brachial or jugular vein into
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sterile heparinized tubes prior to administration of different formulations (0 h) and at 1,
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2, 4, 8, 12, 24, 48, 72, 96, and 120 h post administration. Within 1 h after sample
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collection, the blood samples were centrifuged at ∼3500 rpm for 5 min. The harvested
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plasma samples were stored at − 20°C until further use.
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2.5. Sample preparation
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To prepare plasma sample, 50 μl of perchloric acid was added to 950 μl of each chicken
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plasma, vortexed for 30 seconds and centrifuged at ∼3500 rpm (Eppendorf Centrifuge,
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Model 5810 R, Germany) for 5 min. The supernatant was transferred into special glass
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tube and 20 𝜇l of each sample was injected into the HPLC system for analysis (1).
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2.6. HPLC analysis
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TLM concentrations in plasma were measured by using an HPLC system (Waters,
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USA) which consisted of a multi-solvent pump, solvent degasser, UV detector,
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autosampler, interface and Chromate software. The HPLC column was C18 (5 μm
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particle size, 125× 4.6 mm). The modified methods of Clark et al. (2009) and Eraslan
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(2007) were used for determination of TLM concentrations in plasma. HPLC analysis
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was conducted using a mobile phase consisting of 0.2 M ammonium acetate (pH 5),
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water, acetonitrile, and methanol (20:32:24:24). Mobile phase were filtered under
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vacuum through a 0.45 μm membrane filter. Chromatographic separation was achieved
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at a flow rate of 1ml/min using UV detection at 291 nm (3, 20).
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TLM stock solution of 1.0 mg⁄ml was prepared by adding 10 mg of TLM standard in 10
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ml of acetonitrile: water (1:1, v/v). Then it was further diluted in chicken plasma to
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yield 0.01, 0.05, 0.1, 0.5, 1 and 5 μg/ml.
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The HPLC method for TLM in chicken plasma was validated by assessing the linearity,
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accuracy, precision, recovery, selectivity and sensitivity according to performance
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criteria for method validation (3). Standard calibration curve was provided by using six
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concentrations of TLM (0.01 - 5 μg/ml) and it was used for calculation of TLM levels
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in plasma samples.
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2.7. Pharmacokinetic Analysis
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TLM plasma concentration data of each bird were used to depict its concentration-time
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profile. The maximal plasma concentration of drug (𝐶max) and the time to reach 𝐶max
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(𝑇max) were directly obtained from the observed concentration versus time profiles.
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Non-compartmental analysis was used to estimate the PK parameters (AUC0–∞, t1/2,
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Vd/F, ClB/F, kel, and MRT). The linear trapezoidal rule was used to calculate areas
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under concentration-time curves from 0-120 h (AUC0-120) and from 120 h to infinity
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(AUC120–∞) using the following equation: AUC120–∞ = last Cp/ kel. The Rel. BA
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calculated by using the following equation and PK parameters obtained using Excel
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2013.
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𝑅𝑒𝑙. 𝐵𝐴 =
AUC (0 − ∞) of TLM − LNP formulation
AUC (0 − ∞) of TLM − PH formulation
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2.8. Statistical Analysis
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Data were expressed as mean±SD and analyzed with SPSS software (Ver. 19). The
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differences in PK parameters were analyzed by using one-way ANOVA and Tukey’s
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test (P < 0.05).
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3. Results
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The calibration curve for HPLC analysis of TLM was linear over the range of 0.01– 5
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μg/ml as indicated by R² = 0.999. The calculated limit of detection (LOD) in chicken
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plasma was 0.005 μg/ml and the limit of quantification (LOQ) was 0.015 μg/ml. At
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TLM concentrations of 0.1, 1 and 5 μg/ml, the recovery rates were 99.6 ± 9.8%, 101 ±
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7.5% and 100 ± 3.7%, respectively, and the precision of the method as expressed by
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RSD% of inter-day and intra-day assays were (4.08, 3.30 and 3.46) and (3.06, 4.05 and
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3.74), respectively.
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TLM was well tolerated by the chickens without any noticeable event. The major PK
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parameters and mean concentration-time profiles for TLM-PH, TLM-SLN, TLM-NLC
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and TLM-LNC formulations were shown in Table 2 and Figure, respectively. There
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was no detectable peak corresponding TLM retention time in the plasma samples of
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control groups.
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The Cmax mean value of TLM-LNC and other TLM-LNPs formulation was significantly
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higher than that of TLM-PH (P < 0.05). In general, the mean values of AUC0–∞, t1/2, kel,
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ClB/F, Vd/F and MRT for various TLM-LNP formulations were significantly different
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from those of TLM-PH (P < 0.05). The AUC0–∞ values after oral administration of
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TLM-LNPs were significantly higher than TLM-PH (P < 0.05).
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4. Discussion
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In the present study, we compared TLM-loaded NPLs with its conventional
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formulation, TLM-PH. It was found that all TLM-loaded NPLs had significantly higher
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systemic BA than the TLM-PH.
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There are many possible mechanisms for increased oral BA of TLM by using LNPs
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formulations. In general, LNPs are incorporated solid or liquid lipids similar to the fat
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existing in food. Lipids can stimulate secretion of gastric-pancreatic lipases and co-
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lipases. Consequently, according to their residence time, a large amount of ingested
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lipids is already analyzed in GI (15). The absorption of fatty acids or mono and
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diacylglycerides which are available in LNP formulations or made following digestion
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by GI lipase may facilitate oral absorption of drugs (21). Besides, the release of biliary
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lipids and salts is stimulated, which in turn enhance the production of mixed micelles,
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which include solubilized drug molecules. As a result, by participation in mixed
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micelles, TLM-LPNs could be better absorbed through lymphatic system (15). The
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relatively lower absorption of TLM-SLN and TLM-NLC formulations in comparison
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with TLM-LNCs may be referred to lower ability of young chickens to digest long
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chain fatty acid (C14 to C24) existing in hydrogenated castor oil and Compritol 888
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ATO. In contrast, the higher BA of TLM-LNC formulation may be due to higher
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affinity of GI lipases in broilers for short and medium-chain fatty acids (C6 to C12),
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which are the main components of its lipid, coconut oil (22).
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The oral BA of TLM increased from 1.66 folds in TLM-SLNs to 3.61 folds in TLM-
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LNCs, which correlated inversely with particle size of LNPs as it decreased from 193.0
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± 2.64 nm (SLNs) to 116.6 ± 7.63 nm (LNCs). The nanoscale range of LNPs
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formulations leads to decrease in particle size and highly increases their surface area
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(15). In addition, the reduction in particle size is positively related to an adequate and
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steady absorption of TLM in GI. In general, the increase in the bio-adhesion of these
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LNPs formulations to the GI wall leads to prolong their residence time and their contact
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with epithelial membranes, which improve their absorption (21).The reduction in
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particles size, may also lead GI system to uptake them by other routes such as entry to
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sub-mucosal tissues through intracellular pathways. The process of GI uptake may
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include diffusion of particles through mucus and being more accessible to enterocyte
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surface, epithelial interaction and cellular trafficking (7).
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Negative surface charges (zeta potential) of TLM-LNP formulations can be considered
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as another possible mechanism for enhancement of TLM oral bioavailability. In
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general, glycocalix renders the intestinal mucosa a negative charge, which it will attract
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positively charged nanoparticles. Therefore, the intestinal mobility of particles seems to
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be highly related to their surface charges which is inversely related to particle surface
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charge potentials. With negatively charged particles, it can be expected a higher
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transport rates in comparison to near neutral or positively charged particles, whose
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transport will be highly limited because of particle aggregation and electrostatic
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adhesive interactions with intestinal mucin fibers. Increasing the efficiency of the
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penetration through intestinal mucosa is important to improve oral delivery system.
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Nanoparticles should be sufficiently small to keep away from severely steric inhibition
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and adhesion to the intestinal fiber mesh. On the other hand, NPLs should have some
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mucoadhesion to prolong their retention time and contact with intestinal mucosa (8). In
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addition, surface charge of NPs also plays an important role in M cells uptake. For
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instance, it has been reported that negatively charged NPs had higher M cell uptake
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than positive charged NPs (23). However, the TLM-NLC formulation did not achieve
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higher AUC values compared to TLM-LNC formulation in spite of showing higher ZP
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(zeta potential) value (- 23.5 ± 1.13 mv), because the particles size and their surface
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charge are working together in intestinal absorption process, and TLM-LNC had more
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ideal characteristics with regard to its smaller particles size (116.6 ± 7.63 nm) with
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optimal ZP value (- 16.3 ± 2.51 mv).
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LNPs can also enhance absorption of TLM through lymphatic flow. They can induce
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lipoprotein production and lymphatic lipid flux to augment the level of lymphatic TLM
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transportation, which is significantly affected by the lipid and surfactants type (21). In
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addition, the absorption by M-cells of Peyer’s patches is an additional way for
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lymphatic transport of LNPs (12). The small size of LNPs allows more efficient
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absorption particularly in the lymphoid system; consequently bypass the possible liver
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first-pass metabolism (19). Although highly lipophilic compounds such as long chain
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triglyceride lipids can easily reach systemic circulation by the lymphatic vessels,
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nevertheless, particles size persists as the most vital factor in lymphatic absorption (24).
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Particles with larger sizes may last longer in the Peyer’s patches, whereas smaller NPs
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are transported directly to the thoracic duct, especially when the NPs coated by
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polymers leading to be easily captured by lymphatic vessels (24). Therefore, it seems
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that small particles size of TLM-LNCs with their polymeric structure are responsible
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for enhancing lymphatic uptake and increasing their BA.
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Furthermore, non-ionic surfactants such as Poloxamer 407 and Tween 80 are the other
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factors which may increase the BA of TLM-LNC and TLM-NLC formulations due to
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the ability of these surfactants to improve their intestinal permeability by disturbing the
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cellular membranes and opening the tight junctions of intestinal epithelial cell (15) and
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facilitating paracellular transportation of LNPs (10). Surfactants can also contribute to
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the improvement of the affinity between LNPs and the intestinal epithelial membranes,
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and enhance their bio-adhesion to the GI wall (19). These surfactants are favored for
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oral formulations and efficiently reduce the degradation of LNPs in GI. The
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polyethylene oxide (PEO) chains in these surfactants hamper the anchoring of the
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lipase/co-lipase complex that is in charge of lipid degradation. By providing sterically
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stabilizing layers with different thicknesses of PEO chains on LNPs surfaces, the in
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vivo degradation rate of lipid matrix can be adjusted and slowed down and given time
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for the LNPs to be absorbed (13). In addition, the Tween 80 can increase the intestinal
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uptake due to its ability to inhibit the p-glycoprotein efflux pump (25). The results of
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the present study regarding the higher efficiency of LNCs in improvement PK
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properties are in line with findings of Bendera et al. (2012) who reported that Tween 80
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can be used to deliver drugs efficiently to the brain and to inflamed tissues (16).
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The presence of both surfactants, Poloxamer 407 and Tween 80 as ingredients of TLM-
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LNCs and TLM-NLCs may have contributed to the quality of these formulations, and
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as a result, increased their AUC and Cmax values much more than those of TLM-SLNs
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and TLM-PH. These surfactants may be acted by decreasing the degradation of these
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formulations, prolonging TLM release and enhancing their crossing through the
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intestinal barrier.
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Indeed, Eudragit S 100, which was used to coat TLM-LNCs, is a polymer with a pH-
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dependent solubility. It releases the drug in the regions of GI with pH > 7 like large
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intestine and colon (8), where it gets gradually soluble. Eudragit S 100 has been used to
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entrap insulin to protect it from degradation by GI juices and to permit it to be released
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in regions of the GI with pH > 7, where proteolytic enzymes are in low levels (26).
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Therefore, Eudragit S 100 served as a potential oral carrier in TLM-LNC formulations
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in the present study. In addition, Mohammadzadeh et al. (2014) demonstrated the
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efficiency of Eudragit S 100 in decreasing P-glycoprotein activity and efflux process. It
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seems that, this polymer improved the BA of TLM by dual actions (27). Generally, the
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presence of a polymeric coating wall provides a protective layer against harsh
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environment of the GI such as proteolytic enzymes and may prolong the exposure of
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TLM-LNCs with intestinal epithelial cells, and consequently it may enhance BA of
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TLM (17).
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The relatively high BA of TLM-NLCs in the present study is in accordance with
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findings of Aburahma and Badr-Eldin (2014), since a sustained release with little
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degradation/aggregation behavior had been demonstrated by using Compritol 888 ATO
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because of its long-chain fatty acids (23, 28). On the other hand, Severino et al. (2012)
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reported that medium-chain triglycerides lipids are more effective than long-chain
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triglycerides with regard to sustained release (11), which it seems more closely to the
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results of the present study especially with regard to TLM-LNCs, in which coconut oil
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comprising its oily core.
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Although hydrogenated castor oil in TLM-SLNs was an effective nanoparticle system
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for controlled release and improvement of PK characteristics of loaded drugs (14), it
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achieved the least optimal PK parameters values. This may be due to its more rapid
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degradation in GI which leads to an increased rate of TLM release (6). Many other
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factors may have also contributed to decreased TLM-SLNs absorption such as their
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relatively higher particles size (193.0 ± 2.64 nm), and less zeta potential (- 15.6 ± 3.21
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mv). However, Han et al. (2008) suggested that the high initial release rate of TLM
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from SLN might be helpful because it gets therapeutic level soon.
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The TLM release by TLM-LNPs was slowed down just to reach therapeutic serum
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levels so that the blood concentrations did not reach toxic levels and obviated adverse
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effects such as cardio-toxicity induced by high doses of conventional TLM (6). Using
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TLM-LNPs decreased TLM plasma elimination rate as indicated by higher elimination
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of t1/2 and increased MRT, which caused a longer stay for TLM in blood circulation.
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Abu-Basha et al. (2007) also studied the BA and PK parameters of TLM in chickens
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using Provitil orally at 30 mg/kg B.W. The average AUC0−72 was 24.2 ± 3.9 (μg.h/ml),
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Cmax (2.09 ± 0.37 μg/ml), Tmax (3.99 ± 0.84 h). In spite of using 1.5 fold higher dose,
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the values reported for PK parameters are much lower than those of TLM-LNC
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formulation especially with regard to AUC values (1).
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Keles et al. (2001) also investigated the PK and tissue concentrations of TLM after oral
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administration of a single dose (50 mg/kg, B.W.) in fowl. TLM was slowly eliminated
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from the serum and lung with t1/2 of 30.2 ± 2.4 and 75.7 ± 3.7 h, respectively. The mean
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Cmax was 6.2 times greater in the lung (7.96 ±0.30 μg/ml) than that in serum (1.28 ±
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0.04 μg/ml) with Tmax at 4.66 ± 2.0 and 17.78 ± 7.51 h, respectively (29)
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It is expected that TLM-LNCs followed by TLM-NLCs can demonstrate better in vivo
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antibacterial activity due to their higher AUC, Cmax and sustained release properties
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(14). In spite of their sub-MIC plasma concentrations against E. coli, it seems that
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TLM-LNPs may be more active than TLM-PH, since clinical efficacy of TLM
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formulations not only was affected by plasma drug levels but also it was related to
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intracellular TLM penetration which usually tends to be more accumulated within avian
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phagocytic cells (30), like macrophages, monocytes, and heterophils. Furthermore,
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TLM has a high post-antibiotic and sub-MIC effects, which could slow disease
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development by allowing immune system to eliminate bacterial infection (30). In
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general, TLM Cmax values of all tested formulations which were detected in chicken
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plasma after oral administration were higher than the MIC for M. gallisepticum and M.
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synoviae (0.0125-0.1 μg/ml). These values were also higher than the MIC of plasma
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TLM in cattle against Corynebacterium pyogenes (0.04 μg/ml) and S. aureus (0.78
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μg/ml) (1). Consequently, the clinical efficacy of tested TLM-LNP formulations is
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expected to be highly satisfactory with TLM-LNC formulation due to their better PK
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parameters followed by TLM-NLC and TLM-SLN formulations.
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In conclusion, systemic BA of TLM was significantly enhanced by using oral LNPs
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(TLM-SLNs, TLM-NLCs and TLM-LNCs) in comparison to conventional TLM-PH in
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broiler chickens. Newly formulated TLM-LNPs increased the AUC0–∞, Cmax, Tmax and
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MRT values depending on their particle size, particle surface charges, as well as lipid
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and surfactant compositions. The best BA results achieved by LNC formulation (3.6
10
fold) followed by NLCs (2.71 fold) compared to TLM-PH. It seems that the hybrid
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delivery system (LNCs) is more promising to achieve a sustained release TLM
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formulation with a higher antibacterial activity and lower drug toxicity.
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Acknowledgement
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The authors wish to thank the Nanotechnology Research Center of TUMS; A. Yazdani
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in Avian Microbiology Laboratory of FVM, University of Tehran for her technical
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assistance. They are also grateful to Razak Pharmaceutical Co. and Bachter
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Pharmaceutical Co. for donating TLM standard and Eudragit S 100, respectively.
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This study was partially supported by the Research Council of University of Tehran
22
(Grant No., 7506006-06-10).
23
24
16
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1
TLM
TLM-SLN
TLM-NLC
TLM-LNC
standard
Composition
TLM
Hydrogenated
Compritol 888 ATO
Eudragit S 100 as
(97.3%)
castor oil as a solid
as a solid lipid
polymer, Coconut
lipid matrix and
matrix, sesame oil as
oil as oil core lipid
Poly Vinyl
a liquid oil and
and Span 80,
Alcohol 5% (PVA) Poloxamer 407,
Tween 80 as
as a surfactant
surfactants
Tween 80 as a
surfactant
66.3 ± 2.67
86.5 ± 2.17
94.0 ± 3.60
Zeta potential mv
- 15.6 ± 3.21
- 23.5 ± 1.13
- 16.3 ± 2.51
Particle
193.0 ± 2.64
156.6 ± 7.63
116.6 ± 7.63
Entrapment
efficiency (%)
size
(mm)
In vitro release 97% drug
Initial burst
Initial burst release
Initial burst release
(h)
release
release (18%)
(15%) within first 2 h
(13%) within first 2
at pH 7.4
within 12 h
within first 2 h
followed by a
h followed by a
followed by a
constant sustained
constant sustained
constant sustained
release for 200 h
release for 200 h
release for 120 h
MIC
S. aureus
μg/ml E. coli
1
1
0.5
0.5
4
4
2
2
21
Table 1: Preparation, physicochemical properties and MIC against S. aureus and E.
1
coli of TLM-SLN, TLM-NLC, TLM-LNC formulations and TLM standard.
2
lipid
3
nanoparticles; NLC: nanostructured lipid carriers and LNC: lipid-core nanocapsule.
4
TLM-PH:
conventional
tilmicosin
phosphate
solution;
SLN:
solid
5
6
7
8
22
PK parameter
AUC0–∞(μg.h/ml)
TLM-PH
TLM-SLN
TLM-NLC
TLM-LNC
43.0 ± 2.5 d
71.5 ± 5.8 c
116.4 ± 17.8
155.1 ± 8.6 a
b
t1/2 β(h)
29.3 ± 2.6 c
39.8 ± 3.9 b
46.5 ± 5.0 a
41.3 ± 3.4 b
Vd/F (L/kg)
20.4 ± 2.0 a
10.3 ± 2.7 b
11.9 ± 1.5 b
7.84 ± 2.6 b
MRT (h)
42.0 ± 3.8 b
50.3 ± 5.8 a
67.2 ± 8.8 a
59.6 ± 0.1a
ClB/F (ml/(min.kg))
0.48 ± 0.10 a
0.29 ± 0.04 b
0.19 ± 0.05 c
0.14 ± 0.04 d
kel(h−1)
0.03 ± 0.01a
0.03 ± 0.01a
0.01 ± 0.01 c
0.02 ± 0.01 b
Tmax(h)
2.40 ± 0.24 c
5.60 ± 0.67 b
4.80 ± 0.80 b
12.00 ± 0.00 a
Cmax(μg/ml)
1.21 ± 0.09 b
1.58 ± 0.22 a
1.76 ± 0.38 a
2.17 ± 0.30 a
100
166.2
270.7
360.7
Rel.BA (F) %
1
2
3
Table 2: Pharmacokinetic parameters of TLM in chickens after oral administration of a
4
single dose of TLM-PH, TLM-SLN, TLM-NLC and TLM-LNC formulations (20
5
mg/kg body weight). Data are expressed as mean ± SD (n = 10). Data with different
6
letters (a, b, c, d) differ significantly (P < 0.05).
7
8
9
10
11
23
1
3
plasma concentration (μg/ml)
TLM-PH
2,5
TLM-SLN
TLM-NLC
2
TLM-LNC
1,5
1
0,5
0
0
20
40
60
80
100
120
140
Time (h)
2
Figure. Plasma concentration vs. time curves of TLM after a single oral administration
3
of TLM-PH, TLM-SLN, TLM-NLC and TLM-LNC formulations (20 mg/kg B.W) in
4
broiler chickens. Each value represents the mean and SD (n = 10)
5
6
7
8
24