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RESEARCH ARTICLE ISSN: 2468-1873 eISSN: 2468-1881
Surface Modified Self-Nanoemulsifying Formulations (SNEFs) For Oral Delivery of Gentamicin: In Vivo Toxicological and PreClinical Chemistry Evaluations
Chukwuebuka Umeyor1,*, Anthony Attama2, Franklin Kenechukwu2, Thaddeus Gugu3 and Jane Ugochukwu4 1
Pe N rs ot on Fo al rD U s is e tri O bu nl tio y n
Nanomedicines and Drug Delivery Research Group, Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria; 2Drug Delivery and Nanomedicines Research Group, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Enugu State, Nigeria; 3Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Anambra State, Nigeria; 4Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Enugu, Nigeria
ARTICLE HISTORY
Current Nanomedicine
Received: February 06, 2017 Revised: April 05, 2017 Accepted: April 15, 2017
DOI: 10.2174/2468187307666170512124452
Abstract: Objective: This study aims to investigate the in vivo toxicological profile and pre-clinical safety of surface modified self-nanoemulsifying formulations (SNEFs) for oral delivery of a broad spectrum, anti-bacterial agent, gentamicin. Method: SNEFs which were surface modified using PEG 4000, were formulated through water titration method using appropriate mixtures of soybean oil, a combination of Kolliphor® EL and Kolliphor® P188 as surfactants, and Transcutol® HP as co-surfactant, and encapsulating gentamicin. SNEFs were characterized by measuring the droplet sizes, size distribution and surface charges using a Zetasizer. The effects of the SNEFs on body weight, haematological, biochemical, and histopathological factors of rats after oral administration were determined. Results: Physicochemical characterization showed that the nanoformulations had droplet sizes ranging from 96 - 121 nm with surface charges of -32 to -36 mV. SNEFs did not show net suppression of body weights of rats. There were no clear indications of haematologic, hepatic and renal injuries in the study rats due to flip-flops in the levels of haematologic, hepatic and nephritic biomarkers evaluated. Histopathological organ examinations corroborated findings from the effect of SNEFs on the liver and kidney but revealed possible induction of astrocytosis in the cerebral cortex of rats. Conclusion: Results of the study indicate that surface modified SNEFs of gentamicin have a promising pre-clinical safety potential with minimum toxicity effects.
Keywords: Pre-clinical chemistry, gentamicin, self-nanoemulsifying formulations, toxicological, haematological, histopathological. 1. INTRODUCTION The introduction of self-nanoemulsifying formulations (SNEFs) as an important drug delivery *Address correspondence to this author at the Nanomedicines and Drug Delivery Research Unit, Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria; Tel: +234 806 3299850; E-mail:
[email protected] 2468-1873/17 $58.00+.00
strategy has revolutionized nanomedicine because of the propensity of this drug carrier system to alter the pharmacokinetic (PK) and biodistribution (BD) profiles of drugs. Essentially, SNEFs are isotropic mixtures of oil, surfactants and co-solvents which spontaneously emulsify to form oil-in-water nanoemulsions in the presence of gastrointestinal fluid. This colloidal system has been used to improve the oral solubilization and permeation properties of gentamicin due to its ability to increase © 2017 Bentham Science Publishers
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the active surface area of the drug [1, 2]. Gentamicin is an aminoglycoside anti-bacterial agent originally obtained from the microorganism, Micromonospora purpurea, and used for the treatment of infections caused by Gram-positive and Gram-negative bacteria. Gentamicin acts by binding very strongly to the 30s ribosomal sub-unit and inhibit bacterial protein synthesis [3]. Administration of gentamicin in the form of injection has been reported to cause nephrotoxicity characterized by anaemia, polyuria, decreased urinary osmolality, increased plasma urea nitrogen and creatinine. Furthermore, histopathological examinations showed gentamicin-induced tubular necrosis in the renal cortex and cast formation in the medulla [4]. Due to their very small size, SNEFs droplets are in constant close contact with biological tissues and this could increase the magnitude of injury inflicted on the tissues in the event of any toxicity reaction. Debates as to whether nanoformulations are toxic or non-toxic and the level of toxicity which should be tolerated have been ongoing among formulation scientists [5-7]. The rate of transport, permeation and docking of SNEFs nanoparticles in major tissues implies that adequate toxicological and safety evaluations of SNEFs need to be carried out to prevent toxicity reactions upon administration. In doing this, the initial protocol might be to develop in vitro tests to ascertain the safety of the starting materials for the formulations, but since most of the starting materials used in the development and preparation of SNEFs are assigned ‘GRAS’ (generally regarded as safe) status by appropriate regulatory bodies, it becomes imperative to apply standardized in vivo approaches for determining the clinical safety or otherwise of such colloidal systems [8].
(KP188, Ph. Eur., USP/NF, FDA IIG) (BASF SE, Ludwigshafen, Germany), Transcutol® HP (TrHP, Diethyleneglycol monoethyl ether, Ph. Eur., USP/ NF, FDA IIG), (Gattefossé, Saint-Priest Cedex, France), PEG 4000 (Carl Roth GmbH and Co. KG, Karlsruhe, Germany). Water was obtained from an all-glass still. All other reagents were of analytical grade and were used without further purification.
Since available research reports lack any information on the in vivo toxicological profile and preclinical chemistry evaluations of SNEFs for oral delivery of gentamicin at standard therapeutic doses, this study aims to fill this knowledge gap. To achieve this, we carried out physical, haematological, biochemical, and histopathological studies on gentamicin-loaded SNEFs so as to determine their safety potentials in vivo.
The droplet size and droplet size distribution (polydispersity index, PDI) of the gentamicinloaded and unloaded SNEFs were determined using photon correlation spectroscopy (PCS) and their surface measurements were evaluated using a Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). Before measurements, each SNEF sample was diluted with purified water (1:1000, v/v) and measurements (n = 5) were taken at a measuring angle of 173 ° at 25 ± 2 °C.
2.1. Formulation of Optimized GentamicinLoaded SNEFS
Pe N rs ot on Fo al rD U s is e tri O bu nl tio y n
Optimized gentamicin-loaded SNEFs were formulated following pre-formulation (gentamicinoil-surfactants solubility and gentamicin-oil-surfactants compatibility) studies as reported earlier [1, 2]. Briefly, pre-determined amounts of gentamicin (0.1 and 0.4 g) were dissolved in surfactant and co-surfactant mix at 50°C in a thermostated water bath for 10 min. The oil of choice, soybean oil, was then added and the mixture was vortexed and stirred using a magnetic stirrer at 500 rpm. PEG 4000 (0.5 g) was dispersed in a portion of the co-surfactant, stirred for 5 min and introduced into the nanoemulsion and left undisturbed for 30 min. The final mixture was shaken and stirred using a magnetic stirrer at 500 rpm for 10 min until gentamicin was completely dissolved and a clear solution obtained. The formulations were equilibrated at 25 ± 2°C for 48 h and examined for the signs of turbidity or phase separation. The formulations were stored for further use. The compositions of the gentamicin SNEFs are shown in Table 1.
2. MATERIALS AND METHODS Gentamicin (Juhel Parenterals, Awka, Anambra State, Nigeria), soybean oil (Aromachem, Essex, UK), Kolliphor® EL (KEL, Polyoxyl 35 castor oil, Ph. Eur., USP/NF, FDA IIG), Kolliphor® P188
2.2. Droplet Size and Surface Charge Measurements
2.3. Animal Care and Use Protocols Mature white albino rats (Wistar strain) (weight, 185-205 g) were obtained from the animal-breeding centre, Department of Animal
Surface Modified Self-Nanoemulsifying Formulations
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Table 1. Composition of gentamicin SNEFs (% w/w). Batch
Gentamicin
Soybean oil
Kolliphor® EL + Kolliphor® P188
Transcutol® HP
PEG 4000
A
0.1
37.0
31.5
31.5
0.5
B
0.4
42.6
28.7
28.7
0.5
UNL
-*
50.0
25.0
25.0
-#
UNL – unloaded SNEF, ‘*’ indicates ‘no drug’, and ‘#’ means ‘unpegylated’.
weights of animals were adapted for this study. On day ‘0’, intraocular blood samples were collected from the animals to determine basal levels of haematological factors. After this, experimental groups of rats, A and B were administered gentamicinloaded SNEFs at doses of 5 and 7 mg/kg orally by gavage. Aqueous solution of gentamicin (7 mg/kg) was administered to the positive control group (FD) orally, and the negative control group (UNL) was administered 10 ml/kg of unloaded SNEF orally. Drug administration took place on days 0, 2, 4, and 6 respectively, while intraocular blood samples were drawn for haematology into transparent plastic tubes on days 1, 3, 5, and 7 respectively. Parameters assayed include red blood cell (RBC) count, white blood cell (WBC) count (and its differentials), haematocrit (HCT), and haemoglobin (Hb) concentration using an auto-analyzer (Siemens Healthcare Diagnostics, USA). Furthermore, RBC indices such as mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) were calculated.
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Sciences, Faculty of Agriculture, University of Nigeria, Nsukka. Animals were maintained and treated according to the National Institute of Health (NIH) guidelines. All animal protocols were approved by the Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka in compliance with the EU Directive 2010/63/EU for animal experiments. The animals were housed in rat cages with three rats per cage after randomization, fed standard rodent diet (Vital Feeds Ltd., Nigeria) and allowed free access to clean, fresh water in glass water bottles ad libitum. They were acclimatized for 1 week prior to study, and a 12 h day/night cycle was maintained. 2.4. Effect on Body Weight
Randomly selected mature white albino rats of four groups (n = 7 per group) comprising two control and two treated groups respectively, were used for this study. Body weights (BW) of the animals were measured twice (every 2nd day) using a scale (Avery Berkel, Fairmont, USA) a week prior to study. Thereafter, gentamicin-loaded SNEFs at 5 and 7 mg/kg were administered orally by gavage to the treated groups (A and B) of rats. These doses were chosen since the daily dose recommended for gentamicin in normal renal function is 3-6 mg/kg body weight. The positive control group (FD) received 7 mg/kg of an aqueous solution of gentamicin orally, while the negative control group (UNL) was administered 10 ml/kg of unloaded SNEF orally. Immediately after each administration, individual rat BW was determined three times every week for two weeks, and cage-side clinical observations were made severally on a daily basis during study and changes were recorded. 2.5. Effect on Haematological Parameters Equal groups and numbers of mature white albino rats used to study the effect of SNEFs on body
2.6. Determination of Serum Clinical Chemistry Following bleeding of animals for haemotology, blood samples were collected into plain, transparent tubes and allowed to stand for 5 min for clotting to take place. Clotted blood was centrifuged at 4,000 rpm for 10 min for separation of plasma to obtain clear serum stored at 20°C until analysis. Clinical chemistry parameters analyzed include total bilirubin (BIL), creatinine (CREA), albumin (ALB), and liver enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), using automated clinical chemistry analyzer (Vital Scientific, Netherlands). 2.7. Histopathological Investigation At the end of the study, white albino rats in each group were humanely euthanized, and the
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kidney, liver and brain were collected for histopathological examination. These vital organs were fixed in 10 % formaldehyde and dehydrated in ascending grades of ethanol. Thereafter, the tissues were cleared in chloroform overnight, infiltrated and embedded in molten paraffin wax. The blocks were later trimmed and sectioned at 5-6 µm in thickness. The sections were deparaffinized in xylene, taken to water and subsequently stained with Haematoxylin and Eosin (H and E) for histopathological examination using light microscope (Leica, Wetzlar, Germany). 2.8. Statistical Analysis
3. RESULTS
aqueous solution of gentamicin, and 10 ml/kg of unloaded SNEF respectively, showed decreased BW from 0.205 ± 0.05 kg in week ‘0’ to 0.203 ± 0.10 kg in week 1 for group B, from 0.198 ± 0.08 kg in week ‘0’ to 0.196 ± 0.23 kg in week 1 for group FD, and from 0.185 ± 0.03 kg in week ‘0’ to 0.183 ± 0.23 kg in week 1 for group UNL. This was followed by an increase to 0.206 ± 0.67 kg for group B, 0.199 ± 0.45 kg for group FD, and 0.185 ± 0.04 kg for group UNL, after 2 weeks of study. However, the recorded increases in BW in week 2 were not significant (p>0.05) when compared with the basal body weights of the animals. Furthermore, cage-side clinical observations of the rats did not record any adverse behavioural changes. Table 2. Physicochemical properties of gentamicin SNEFs.
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Results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and student t-tests (using Graphpad Prism version 4.0 for Windows) were used for the analysis of data sets generated from the test gentamicin-loaded SNEFs groups and the unloaded SNEF/free drug solution control groups with a level of significance set at p < 0.05.
Umeyor et al.
3.1. Droplet Size and Surface Charge Measurements
1
2
3
Batch
Droplet size (nm)
Surface charge (mV)
Droplet size distribution
A
96.0 ± 0.0
-32.4 ± 1.7
0.249 ± 2.1
B
*96.0 ± 0.0
-36.7 ± 1.1
0.647 ± 0.5
UNL
*121.7 ± 1.0
-36.7 ± 1.1
0.240 ± 0.5
1,2,3
Table 2 shows the result of the physicochemical characterization of gentamicin-loaded and unloaded SNEFs. The table shows that gentamicinloaded SNEFs A and B had equal and the same droplet size of 96.0 ± 0.0 nm with droplet size distributions (polydispersity indices) of 0.249 ± 2.1 and 0.647 ± 0.5 for SNEFs A and B respectively. Surface charge measurement for SNEF A was 32.4 ± 1.7 mV, while SNEF B had -36.7 ± 1.1 mV. Conversely, the unloaded SNEF recorded droplet size of 121.7 ± 1.0 nm with a droplet size distribution of 0.240 ± 0.5, while the surface charge measured was equal with that of SNEF B at -36.7 ± 1.1 mV. 3.2. Effect on Body Weight
Result of the effect of SNEFs on body weights of rats is shown in Table 3. The table shows that group A rats administered with 5 mg/kg gentamicin-loaded SNEF orally recorded a steady, insignificant (p>0.05) increase in BW from 0.195 ± 0.25 kg recorded in week ‘0’ to 0.199 ± 0.01 kg recorded after 2 weeks. Groups B, FD and UNL rats administered 7 mg/kg gentamicin-loaded SNEF, 7 mg/kg
Mean ± SD (n = 5), * represents statistically significant sets of data.
3.3. Effect on Haematological Parameters Result of the effect of SNEFs on haematological factors is shown in Figs. (1-3). The result showed a steady significant (p
