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A new lipid-based oral delivery system of erythromycin for prolong sustain release activity
Materials Science & Engineering C 97 (2019) 245–253
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A new lipid-based oral delivery system of erythromycin for prolong sustain release activity
T
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Mumuni A. Momoha, , Emmanuel C. Ossaib, Omeje E. Chidoziea, Omenigbo O. Precscilaa, Franklin C. Kenechukwua, Kenneth O. Ofokansia, Anthony A. Attamaa, Kunle O. Olobayoc a
Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Nigeria c National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipid microparticles Erythromycin stearate Prolong release Pharmacokinetics
Erythromycin-loaded solid lipid microparticles (SLM) based on solidified reverse micellar solution (SRMS) as an oral delivery formulation was studied. Hot homogenization technique was employed to prepare erythromycin stearate-loaded SLMs using blends of Softisan® 154 and Phospholipon® 90H or beeswax in the ratio of 1:2, and characterized in vitro. Antibacterial evaluation of the formulations was carried out by agar diffusion technique against some selected clinical isolates of bacterial. Preliminary pharmacokinetic study was performed after oral administration in male Albino rats. The results of matrix contain Softisan® 154 and phospholipon® 90H (1:2) showed that erythromycin-loaded SLM was smooth; particle size ranged from 10.3 ± 11.24 μm to 18.1 ± 10.11 μm and maximum encapsulation efficiency and loading capacity were 95.11 ± 0.3% and 43.22 ± 0.1 mg, respectively. While that of beeswax- containing matrix showed maximum particle size of 18.9 ± 21.10 μm, maximum encapsulation efficiency of 89.01 ± 0.11% and loading capacity of 39.02 ± 0.12 mg. All the formulations had prolonged release and antibacterial activity. Significantly (p > 0.05), prolonged plasma erythromycin concentration was obtained in the optimized formulation (> 14 h) compared with commercial sample of erythromycin tablet (10h). Erythromycin stearate-loaded SLMs formulation could serve as an alternative to conventional oral formulation of erythromycin.
1. Introduction In recent times, formulation scientists have discovered that the poor aqueous solubility and dissolution rate of active pharmaceutical ingredients (APIs) is one of the biggest challenges in pharmaceutical development. The poor solubility often results in low bioavailability of orally administered drugs, and thus results in poor therapeutic efficacy [1]. According to the Biopharmaceutics Classification System (BCS), a drug is poorly soluble if the highest dose strength is not soluble in 250 ml aqueous media over the entire pH range at 37 °C (FDA, 2000). The candidate drugs of these nature are mostly considered as Class II (IIa or IIb), which are poorly soluble and highly permeable according to the pH of the gastrointestinal fluid and tend to present solubility or dissolution rate-limited absorption [2]. Despite their high permeability, these molecules often have low oral bioavailability because of their slow and limited release of drug in gastrointestinal fluid thereby posing big challenges in clinical practice on the utilization of these molecules [3]. Therefore, one of the major tasks that the pharmaceutical industry
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need to overcome is how to develop strategies that could improve the dissolution and/or apparent solubility of these drugs/compounds into orally bioavailable and therapeutically effective drugs for the benefit of patients in clinical settings [2,4]. Several approaches to overcome the poor aqueous solubility and it improve absorption after oral administration of such drugs have form the major research among the formulation scientist and their findings are well documented [5]. In comparison with other delivery system, such as liposomes, microemulsion, prodrug formation and microparticles that have been studied in the controlled release of incorporated drugs and in enhancing the solubility, improve absorption of drugs, solid lipids microparticles (SLMs) have been considered as one of the most successful strategies to maintain high degree of dispersity of drug profile of poorly soluble drugs. SLMs a new entrance in as drug delivery system and have so far been considered a promising drug carrier system, especially with a view to giving the incorporated active substance a sustained-release profile and improving the dissolution profile of poorly soluble drugs [5,6]. In contrast to polymeric drug
Corresponding author. E-mail address: [email protected] (M.A. Momoh).
https://doi.org/10.1016/j.msec.2018.12.041 Received 27 May 2018; Received in revised form 9 November 2018; Accepted 12 December 2018 Available online 13 December 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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delivery, SLMs as drug carrier has been considered as very safe and are said to be physicochemically stable as in some cases no organic chemicals is employ in its preparation [5]. Additionally, asides from its ease of preparation and scaling-up, it's have an edge over other delivery systems in the following areas; stability, low production cost and high encapsulation efficiency for lipophilic compounds [7]. The utilization of mixtures of lipids as solidified reverse micellar solutions (SRMS) in the preparation SLMs further strengthened the grip of SLMs as the last bus stop for the poorly aqueous soluble drug [8]. Formulations based on SRMS have been investigated and successfully employed to achieve controlled release and enhanced absorption of drugs [8–10]. For instance, SRMS consisting of phospholipids and solid lipids such as hydrogenated palm oil, which transform into a lamellar mesophase after melting on contact with water have widely been employed in nano-suspension [10], and in the delivery of diabetes drugs [5]. Consequently, this transformation enables controlled release of solubilized drugs, prevents drug expulsion and increase the drug encapsulation efficiency [10]. Interestingly, SRMS carriers have recently been investigated as a sustained release matrix for both hydrophilic and hydrophobic drugs; it combined several advantages of other known lipid carriers [11–13]. The recent discovery on the utilization of SLMsbased SRMS in the delivery of sensitive material against degradation and excellent in vivo tolerability is unparallel to the other system [14]. In view of these aforementioned advantages, it was the objective of this study to formulate and characterize, and in vitro erythromycinloaded – SRMS based SLMs from mixtures of lipids composed of mixture of Softisan® 154 and Phospholipon® 90H or beeswax for maintain high degree of dispersity of drugs and facilitate absorption after oral administration.
Table 1 Quantities of starting materials for the formulation of different batches of unloaded and erythromycin-loaded microparticles. Batch
Lipid matrix (g)
Sorbic acid (mg)
PVA (mg)
Sorbitol (mg)
Drug Conc. (mg)
Distilled water, q.s (% w/w)
B1 B2 B3 B4 C1 C2 C3 C4
20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0
30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0
600.0 600.0 600.0 600.0 600.0 600.0 600.0 600.0
600.0 600.0 600.0 600.0 600.0 600.0 600.0 600.0
500.0 250.0 100.0 – 500.0 250.0 100.0 –
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
Key: B1 – B3, contain 500, 250 and 100 mg of erythromycin respectively, C1 – C4 contain 500, 250 and 100 mg of erythromycin respectively, while batches B4 and C4 contains no drug.
Germany) at 8000 rpm for 4 min to produce the hot primary emulsion, which was collected in hot containers and allowed to recrystallize at room temperature. By adding decreasing concentrations of erythromycin (500, 250 and 100 mg) to the SRMS and following the above mentioned procedure, erythromycin-loaded SLMs were obtained and coded as B1, B2, and B3 respectively. Similarly SLMS based Softisan154 and Beeswax were also prepared using same drug concentration as above to obtained erythromycin loaded SLMs and coded as C1, C2, and C3 respectively. Drug free (unloaded SLMs) were similarly prepared (Table 1) and coded B4 and C4. 2.3. Characterization of SLMs
2. Materials 2.3.1. Differential scanning calorimetry (DSC) Melting transitions and changes in heat capacity as a function of the degree of crystallinity in the. physically structured lipid matrices were determined using a calorimeter (Netzsch DSC 204 F1, Germany). Approximately 5 mg of each SLMs was weighed into an aluminum pan and sealed hermetically, and the thermal behavior was determined in the range of 10 to 125 °C at a heating rate of 5 °C per min. Baselines were determined using an empty pan.
The following materials were used: Softisan® 154 (Schuppen, Condea Chemie GmbH, Germany), Phospholipon® 90H (Phospholipid GmbH, Köln, Germany), and beeswax® (Carl Roth GMbH, Germany). Erythromycin stearate was a gift from AC Drugs Ltd., Enugu State, Nigeria, and distilled water was obtained from Lion water (UNN, Nigeria). All other reagents and solvents were of analytical grade and were used as supplied. 2.1. Preparation of lipid matrix
2.3.2. Morphology and particle size analysis The particle size of the microparticles was analyzed periodically so as to know the effect of time on the formulation. Briefly, 3–5 mg from each batch of the SLMs was dispersed in distilled water and smeared on a microscope slide using a glass rod. The mixture was covered with a cover slip and viewed with a binocular microscope (Lieca, Germany) attached with a digital camera (Moticam, China) at a magnification of ×1000. The photomicrographs of the particles were also observed and taken.
The lipid matrix based on the binary mixture of 1:2 ratio of Softisan® 154 and Phospholipon® 90H (P90H) or beeswax was prepared by fusion. The ratio used in this study was based on our preliminary evaluation during the selection and screening of the lipids mixture to establish the best combination. In brief, a 10 g quantity of P90H or beeswax was carefully weighed and transferred into a crucible containing 5 g of Softisan® 154 and the mixture was heated at temperature of 60 °C on a hot plate (SR1 UM 52188, Remi Equip., India) to melt. The molten lipid mixture was then stirred thoroughly until solidification to get the solidified reverse micellar solution (SRMS).
2.3.3. Time-dependent pH stability studies The pH of the formulations, drug-loaded and unloaded (drug free) SLMs were determined in a time-dependent manner using a pH meter (Suntex TS-2, Taiwan) after 24 h, 4 weeks, and 12 weeks of storage condition in order to ascertain the stability of the formulations especially in the area of product degradation during storage.
2.2. Formulation of SLMs The following compositions for material were used in the preparation of SLM, 6.7% w/w of SRMS, 2% w/w polyvinyl alcohol (PVA), 0.1% w /w sorbic acid, 2% w/w sorbitol and enough distilled water to make 100% w/w. The hot homogenization method was adopted according to earlier work [17], with slight modifications. In each case, the lipid matrix was melted at 60 °C in a crucible, and PVA, sorbitol, and sorbic acid were dispersed in the appropriate concentration in sufficient volume of distilled water and brought to the same temperature with the lipid matrix. The hot aqueous phase was poured into the molten lipid matrix with gentle stirring with a magnetic stirrer and the mixture was further dispersed with a homogenizer (Ultra-Turrax T18, Basic, Ika,
2.3.4. Encapsulation efficiency (EE%) Approximately, 0.5% w/v dispersion of each batch of the SLMs in distilled water was prepared in a 100 mL volumetric flask, allowed to equilibrate for 48 h at room temperature, shaken and filtered. The filtrate was adequately diluted and analyzed for erythromycin content using a spectrophotometer (Unico 2102 PC UV/Vis Spectrophotometer, USA) at 214 nm. The amount of drug encapsulated in the microparticles was calculated with reference to a standard Beer's plot for erythromycin 246
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2.6. Pharmacokinetics study
to obtain the EE% using the formula below.
EE =
Actual drug content X 100 Theoretical drug content
The batches of the SLMs that show highest in recovery, loading capacity, encapsulation efficiency and release were selected and were used for the pharmacokinetic study. Following the set out criteria, batch B1 and C1 of the preparation were selected for the evaluation. Adult albino male rats weighing between the range of 255–270 g were used in the investigation. They rats were kept at the ambient temperature of 26 ± 1 °C, 12 h light/dark cycle, fed standard rat feed and had free access to drinking water. The animals were fasted for 12 h before experiment. Animal experiment in this research was in line with general standard as stipulated by our Institution's Animal Ethics Committee and in compliance with the European Community Council Directive of November 24, 1986 (86/609/EEC). Three groups (I, II and III) of five rats per group were used in this investigation. The formulations (B1 and C1) and the reference sample were orally administered to the rats. Rats in Group I received erythromycin-SLM (batch B1), rats in Group II received (batch C1), while rat in Group III received the reference sample of erythromycin (MS) tablet (10 mg/kg of body weight) in the form of suspension in 2.5 ml sterilized distilled water. Blood samples (about 0.5 ml each) were collected from the orbital plexus at 0, 1, 2, 4, 6, 8, 10, 12 h and immediately extracted in acetonitrile at blood to acetonitrite ratio of 1:3 and was subjected to low-speed centrifugation, and the supernatant collected were analyzed using a HPLC.
(1)
2.3.5. Loading capacity (LC) LC expresses the ratio between the entrapped drug and the total weight of the lipids. It was determined using Eq. (2):
LC =
Qd − Qs × 100 ql
(2)
where ql is the weight of lipid added in the formulation, Qd is the weight of erythromycin added to the formulation, and Qs is the amount of drug determined in the supernatant after separation of the lipid and aqueous phases.
2.4. In vitro release study on SLMs In vitro release was evaluated using a dialysis membrane technique as described in a previous study [9]. The polycarbonate dialysis membrane (MWCO 8000–10,000 Spectrum Labs, Netherlands) used as release barrier was pre-treated by soaking it in the dissolution medium for 24 h prior to the commencement of each release experiment. SLMs equivalent to 50 mg were placed in a dialysis membrane containing 5 mL of the dissolution media. The dialysis membrane was tied at both ends and was suspended in the basket of USP Type I dissolution apparatus (Electrolab, Mumbai, India). The baskets were immersed in 250 mL phosphate-buffered, pH 7.2, maintained at 37.0 ± 0.5 °C and was agitated at 75 rpm. At regular intervals, 5.0 mL of dissolution medium was withdrawn and was replaced with fresh buffer at the same temperature. The withdrawn samples were filtered through a 0.22 μm filter (Millipore®, USA) and analyzed for erythromycin content by HPLC method as described elsewhere [9].
2.6.1. Pharmacokinetic analysis Pharmacokinetic parameters were investigated and calculated using a computer program Calc 2002 (Korea Food & Drug Administration, Korea). The elimination rate constant (Ke) was obtained from the leastsquare fitted terminal log linear portion of the serum concentrationtime profile. The erythromycin Cmax and the corresponding Tmax were evaluated from the serum concentration-time profiles of the drug. The elimination half-life (T1/2) was calculated as 0.693/Ke. The trapezoid rule was used to investigate the area under the curve (AUC). Maximum plasma concentration (Cmax) and the time needed to reach the maximum plasma concentration (Tmax) were determined directly from the concentration-time data for the optimized SLM (B1) and the commercial sample (MS).
2.5. Antimicrobial susceptibility activity of SLMs using agar diffusion method The biological activities of erythromycin-loaded SLMs were measured by bacteria growth inhibition assay using an agar diffusion method. In another words, the antimicrobial activities of the SLM were evaluated using agar diffusion method previously reported (18). This method depends on the diffusion of antibiotics from holes on the surface of the microbial seeded agar. The test organisms (Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Salmonella paratyphii (S. paratyphii) and Staphylococcus aureus (S. aureus) as tests organisms) were standardized using 0.5 MacFarland standards which is equivalent to 1 × 10−8 colony forming unit (CFU). In brief, Mueller - Hinton agar (MHA) was prepared according to manufacturer's instruction, sterilized at 120 °C, 15 psi for 15 min, and allowed to cool to 45 °C. Thereafter, the molten MHA maintained at 45 °C was aseptically seeded with 0.1 ml of the standardized test organisms and poured into sterile plates. The set up (agar and test organism) was mixed together and was allowed to gel. Sterile cork borer was used to make holes/wells on the gel agar plate at equal distances from each other and thereafter sterile applicator was used to withdraw 0.1 ml of a batch of the SLM formulation and was added into each well (B1, B2, B3 for the loaded-SLMs), and was allowed to stand for diffusion of the antimicrobial agents. The seeded agar plates were incubated at (37.0 ± 0.5) °C for 24 h. All the tests were done in triplicates and growth was examined after incubation, with the average inhibition zone diameter (mm) of each sample determined. The same procedure was used for batch C1– C3 and a commercial sample of erythromycin (MS) was similarly evaluated and compared to the formulated erythromycin-loaded SLMs.
2.7. Statistical and data analysis Data were analyzed using SPSS Version 16.0 (SPSS Inc. Chicago, IL). All values were expressed as mean ± SD. Differences between means were assessed using one way ANOVA and student's t-test. p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of lipid matrices and SLMs The DSC results showed that the hybridization of Softisan® 154 with P90H or beeswax resulted in matrices with low enthalpies compared with the individual lipid material. The system displayed several prominent transitions upon heating which clearly showed the interaction of the lipid mixture as well as the various heat capacities of the formulation. The melting endothermic of Softisan® 154 was 61.4 °C with an enthalpy of - 41.99 mW/mg (Figure not shown). However, when the lipid was used in the formulation of SRMS matrix, the SRMS gave a melting endotherm value of 61.1 °C and an enthalpy of - 6.999 mW/mg. This value of enthalpy indicated that the matrix was less crystalline than the bulk lipid due to its lower enthalpy value and suggests that mixture of lipids can produce matrices of low crystallinity. Thus, the SRMS may have generated an imperfect matrix resulting from distortion of crystal arrangement of the bulk lipid after melting and solidification. This distortion of the lipid crystal may be an advantage in drug 247
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Fig. 1. (a, b). (a) Thermograph of unloaded SLM (4), pure erythromycin(12), batch B1 (16), B2(17) and B3(18); (b) Thermograph of unloaded SLM (4), pure erythromycin (9), C1(10), C2(11) and C3(12).
Batches B4 and C4 showed the least particle sizes throughout the period of evaluation, probably because the batch contained no drug, which could aid the growth of the particles during encapsulation. Particle size may be a function of either one or more of the following: formulation excipients, degree of homogenization, homogenization pressure, rate of particle size growth, crystal habit of the particle [11,18,21]. Alternatively, this observation may be due to sintering or Ostward ripening as postulated by the DLVO theory [23]. The particles tend to migrate toward each other and subsequently formed aggregates due to the kinetic advantage of gain by larger crystal associated with increase in the drug concentration. Similarly, larger crystals are more favoured thermodynamically and difficult to nucleate when compared with small crystals. According to the DLVO theory, there are forces that interacting between particles existing in colloidal dispersions these include electrical repulsion forces (VR) and the van der Waals attraction (VA), these parameters are said to complement each other [6]. In this context, it is very obvious that the particle size after drug incorporation was large compared with unloaded particles such that the interparticles distance was appreciably small thereby allowing the predomination of the van der Waals universal attractive forces to be predominant giving rise to larger particles on aggregation. This explanation was in agreement with earlier researcher who observed that particle in lipids formulation depend on the concentration of the incorporated drug, and it increase as concentration of the drug increases. However, in the unloaded formulation, it was observed that less particles sizes were maintained, which could be attributed to the larger inter-particulate distance such that the repulsion between the particles exists. Molecularly, the large inter-particulate distance brings about the setting of the secondary minimum. Over time, the particle tends to break-up into smaller particles thereby increasing further the inter-particulate distance. Consequently, the double-layer repulsion predominates giving a primary maximum in which case at a corresponding large thermal energy (kT) of the particles, colloidal systems thus become well dispersed and deflocculated [24].
encapsulation and entrapment [5,9,18]. However, the DSC thermogram of erythromycin shows a peak at 167.2 °C with an enthalpy of −2.661 mW/mg (Fig. 1a). When the SRMS -154 was employed to formulate SLMs, the DSC thermogram of the formulations showed different peak characteristics according to drug concentration. It was observed that B1 of the SLM formulated with 500 mg gave the minimum melting endotherm of 59.7 °C with an enthalpy of −7.623 mW/mg, whereas the unloaded (no drug) SLMs (B4) showed maximum melting endotherm (61.6 °C) together with maximum enthalpy value of −12.03 mW/mg (Fig. 1a). There was a significant difference (p < 0.05) between the SLM loaded with 500 mg of erythromycin (B1) compared to batches loaded with 250 mg (B2) and 100 mg (B3) of erythromycin. DSC thermograms of lipid carrier, beeswax, and the lipid matrix showed a sharp endothermic peak corresponding to melting transitions at 73.0, 61.4 and 66.5 °C with corresponding enthalpies of −55.7, −41.99 and − 14 mW/mg (Figure not shown), respectively. Thermograms of the erythromycin-loaded SLMs C1, C2 and C3 showed sharp endothermic peaks at 70.8, 64.8 and 66.9 °C with a corresponding transition enthalpies of –40.26, −16.03 and − 30.44 mW/mg, respectively (Fig. 1b), where the various batches are compared. However, lower enthalpy suggests less crystallinity and the possibility for retention of an entrapped drug over time, whereas high enthalpy means highly ordered crystalline arrangement (perfect crystals) which leads to drug expulsion upon crystallization of previously molten matrices [9,16,18]. 3.2. Morphology and particle size of SLMs Representative photomicrographs of batches of the SLMs containing decreasing amount of erythromycin (B1 - B3 and C1 - C3) with their corresponding unloaded batches or batches containing no erythromycin, B4 and C4 respectively, after four weeks of preparation are shown in Fig. 2. From the photomicrographs it could be seen that the SLMs were well formed, smooth, and non-porous, they were also stable and did not show sedimentation even after centrifugation at 2000 rpm for 20 min. The particle sizes within one week of formulation were small and ranged from 13.23 to 17.15 μm, and increased dose-dependently according to the concentration of entrapped erythromycin as shown in Fig. 3a. The particles sizes followed a decreasing order of magnititude: B1 > B2 > B3 > B4. Similarly, the sub-batches of C showed not appropriate increased particle size compared to the batches in B, but the particles increased as the concentration of drug increased and showed similar trend in increase in the order of C1 > C2 > C3 > C4.
3.3. Time-dependent pH stability studies on the formulations The pH of the different batches of SLMs were measured 24 h, 1 week, and 1 month after preparation to ascertain the variation of pH with time, which could be a function of degradation of the API or excipients (Fig. 3b). There was a slight decrease in the pH of the formulations within the storage period. This was also applicable to unloaded formulation (drug free) samples whose pH continued to decrease throughout the period of observation. The shifting of the pH toward 248
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Fig. 2. Photomicrographs (within 24 h) of SLMs formulated with SRMS containing (B1) 500, (B2) 250, (B3) 100; and (B4) 0 mg of erythromycin . While, C1, C2, C3, and C4 containing 500, 250, 100, and 0 mg or erythromycin, respectively.
acidity is an evidence of probably the degradation of the lipid components of the formulation and release of more free fatty acids [9,19,20]. However, the decrease in the pH of the formulation loaded with drug is not significant (p > 0.05) as compared to the decrease observed in the unloaded (drug free) formulation. This indicates that the decrease in the pH may not be enough to cause a decrease or change the expected activity of the formulation. In an earlier study, the author observed that, although, there was a tremendous improvement in the use of either synthetic or natural lipids in drug delivery, the decrease in the pH of the loaded formulation did not alter the activity of the antibiotic used in the formulation [21]. It was further suggested that such decrease or changes in the pH toward acidity could be overcome by the addition of stabilizing agent during the formulation. Similarly, previous researchers observed that Phospholipon® 90G or 90H could be used to stabilize SLM formulations and prevent product degradation [18,22]. However, since the level of decrease in pH of the loaded SLMs formulation was less than the unloaded formulation, it could be argued that the loaded formulation gave enough stabilization as compared to the unloaded; an indication that drug in the formulation did not degrade but rather improved the stability of the formulation. In other words, we can vividly argue that the changes in the pH was not associated with the drug incorporated into the lipid matrix, since the unloaded batches showed more in the decreased than the loaded sample. Scientifically, the particle surface pH in some systems may show some level of variation in the range of 2–5 units different from the pH of the bulk material because of the forces of attraction/adsorption coupled with the electrical double layer effect of other ions in the continuous phase of the same system [23].
Table 2 Result of some physical parameters of the SLMs. Formulation code
B1 B2 B3 B4 C1 C2 C3 C4
Parameters Percentage yield
EE (%)
LC (mg API/100 mg lipid)
96.10 91.12 86.23 75.50 92.10 81.12 81.23 73.50
95.11 92.23 89.85 – 89.01 79.12 78.11 –
43.22 27.42 33.26 – 39.02 29.12 31.11 –
± ± ± ± ± ± ± ±
0.11 0.31 0.01 0.12 0.41 0.31 0.01 0.12
± 0.13 ± 0.19 ± 0.01 ± 0.11 ± 0.11 ± 0.03
± 0.13 ± 0.20 ± 0.15 ± 0.12 ± 0.00 ± 0.11
Batches B1- B3 and C1-C3 contain erythromycin while batch B4 and C4 contain no drug.
3.4. Percentage yield, loading capacity and encapsulation efficiency The percentage yield or recovery rates of the formulation have a direct relationship to the methodology. As shows in Table 2, It was observed that maximum and minimum yield were observed in subbatch B1 (96.23%) and B3 (89.10%) for the loaded formulation. The sub-batch C1 and C3 showed maximum and minimum yields of 92.10 ± 0.11 and 81.23 ± 0.01% respectively. The unloaded (drug free, batch B4 and C4) showed the least recovery rate (75.50 and 73.50%, respectively) when compared to the loaded batches. In all cases, the percentage recovery across the batches increased with increase in drug loading. High values (> 70%) of the percentage of the SLMs recovered from the formulations are a strong indication that the formulation technique adopted was reliable. Fig. 3. (a, b). (a) Bar chart representing the particle size of erythromycin-loaded (B1-B3, C1-C3) and unloaded (B4 and C4) SLMs over time, were formulated with the SRMS lipid matrix. (n = 3) and (b) Timeresolved pH analysis of erythromycin-loaded (B1-B3, C1-C3) and unloaded (B4 and C4) SLMs formulated with the SRMS lipid matrix.
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3.6. Antimicrobial susceptibility activity
The role of any drug delivery system (DDS) is to deliver the incorporated drug to the target tissues intact with little or no toxic effect to other organ or system. Thus, the ability of the SLMs to accommodate active molecules is an important property. It can be expressed by the encapsulation efficiency (EE %) and loading capacity (LC). EE % defines the ratio between the weight of entrapped active pharmaceutical ingredient (API) and the total weight of API added to the dispersion, while LC expresses the ratio between the entrapped API and the total weight of the lipids [25]. As shows in Table 2, the EE% and the LC obtained for the various batches of SLMs after 4 weeks of the formulation. Both EE% and LC are dependent on factors such as the nature of the lipid, the formulation technique used and the lipophilic nature of the active pharmaceutical ingredient (API). The EE% ranged from 89.85 ± 0.4 to 95.11 ± 0.2% and 78.11 ± 0.03–89.01 ± 0.11 for batches B1 to B3 and C1 to C3, respectively. The varying EE% may be as a result of the concentration of the API used in the formulation. All the same, higher EE% values were obtained in all the batches of the formulation. However, there was no significant difference (p > 0.05) in the EE% obtained for batches B1, B2, and B3 SLMs. On the other hand, the batches in C, showed significant difference (p < 0.05) among the sub-batches in Cs. Similarly, the formulation showed high loading capacity ranging from 27. 22 ± 0.0–43.22 ± 0.1 and 31.11 ± 0.11–39.02 ± 0.12 mg for sub-batch of B and C, respectively.
The antimicrobial susceptibility testing using agar plate method was based on the diffusion of an antibiotic agent or formulation thereof through a solidified nutrient agar. The zone size around each antimicrobial disk was interpreted as size of areas of inhibition of growth (mm). The results of the antimicrobial activity of erythromycin loaded SLMs are shown in Fig. 5(a–d). From the results, the erythromycinloaded microparticles exhibited good anti-bacterial activity significantly different from the commercial sample against all the microorganisms used for the study (p < 0.05). The isolates (micro-organisms) showed sensitivity of all tested erythromycin-loaded SLM at varying degrees and are concentration dependent. The SLM-based SRMS formulations gave zones of inhibition in the following decreasing order of magnitude: B1 > B2 > B3 for SLMs for the entire test organisms. Similar results was obtained when beeswax was added to the formulation; the order of zone of inhibition in all cases was C1 > C2 > C3. The IZD was somehow low in batch B3 and C3, probably because the concentration of erythromycin contained in these batches were too low to yield concentrations equal to or above the minimum inhibitory concentration (MIC). MS, which served as the control, showed certain levels of inhibition, but somehow low when compared to the formulated SLMs at p < 0.05. Interestingly, several literatures reports the enhanced therapeutic effect, with reduced cytotoxicity, of several antimicrobial drugs when loaded in micro/nanoparticles as compared to the traditional formulation. Furthermore, when the different lipid combination (matrices) was compared, it was observed that combination of beeswax in the formulation generally produced formulation with lesser activities as compared to when P90H was used in all the batches of the formulations (B1- B3). This indicates that P90H was activity determinant in the formulation. Additionally, the formulations exhibited capacity-limited antimicrobial activity in a time-dependent, manner as observed in the IZD against the organisms tested. The antibacterial activities show timedependent increase in IZDs within 240 min implies that the formulated SLMs had potential for sustained drug release. The activity of the formulations suggests that SLMs increased the antibacterial activity of erythromycin. These results are similar to those obtained in related studies [4,5]. Earlier investigators have suggested several mechanisms for the enhanced activities of SLMs including fusion of the lipid component with the bacterial membranes [5,7,20]. Due to membrane merging, the antibiotic could easily penetrate inside the bacteria allowing the increased bactericidal efficacy observed with the drug, thus circumventing the normal pathway of penetration [21]. Generally, it's arguable that SLMs potentiate the activity of the incorporated drug either through high pay-load or inhibit drug expulsion during formulation and storage stage and the release of the drug in a controlled manner.
3.5. In vitro release The results of the in vitro release of erythromycin from the SLMs presented in Fig. 4(a, b) showed that all the erythromycin-loaded SLMs exhibited higher prolonged release properties significantly different from the reference erythromycin tablets (p > 0.05). The formulations gave a gradual and more sustained release of erythromycin over the study period. The results showed that erythromycin-loaded SLMs could be used once daily or at most twice daily as compared to the conventional formulation that must be taken thrice or more before attaining the required plasma concentration. More so, the formulations showed good encapsulation of the incorporated drug as there was no burst effect or dose dumping. The results revealed capacity-limited release of erythromycin from the SLMs consistent with earlier reports [15,18,24]. For instance, batch B1 formulation with the highest drug concentration (500 mg of erythromycin) gave a maximum release of 90%, while batch B3 with the least drug concentration (100 mg of erythromycin) gave a maximum release of 75% (Fig. 4a). Similarly results were obtained when beeswax was used. It was observed that batch C1 had maximum release (79%) as compared to other batches of the formulation with 66 and 61% for batch C2 and C3 (Fig. 4b), respectively. Regarding their sustaining potentials, all the batches of the SLM had good sustaining properties. However, batch B1 and C1 formulations produced better sustained release properties than the rest of the formulations.
Fig. 4. (a, b) (a) In vitro release profile of erythromycin from SLMs in phosphate buffer at pH 7.4; (B1, B2 and B3 were formulated with lipid matrix contain 500, 250 and 100 mg of erythromycin, respectively. MS is the commercial sample that served as a control); and (b) In vitro release profile of erythromycin from SLMs in phosphate buffer at pH 7.4; (C1, C2 and C3 were formulated with lipid matrix contain 500, 250 and 100 mg of erythromycin, respectively. MS is the commercial sample that served as a control.
250
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Fig. 5. (a–d). Time-dependent susceptibility of microorganisms to erythromycin-loaded SLMs; (a) Staphylococcus aureus (S. aureus) (b) Salmonella paratyphii (S.paratyphii) (c) Pseudomonas aeruginosa (P. aureginosa), (d) Escherichia coli (E. coli).
maintained a steady slow decreased or gradual clearance throughout the study in the treated rats. The average mean pharmacokinetic parameters (Table 3), shows that the mean Cmax values for the tested samples B1 and C1 were 3.1 and 2.9 μg/ml respectively, as compared with 2.7 μg/ml obtained in the reference sample (MS). The mean Tmax values for the B1, C1 and MS were 2.0, 1.9 and 1.0 h, respectively. However, the Cmax and Tmax values were not significantly different
3.7. Pharmacokinetics studies Data obtained from the pharmacokinetic studies are shown in Fig. 6, the SLM-loaded erythromycin showed a slow decrease in the initial peak attained in comparison to the reference drug sample as shown in Fig. 6, after oral administration. However, in contrast to the rapid exponential decrease in the reference sample, erythromycin-loaded SLMs
Fig. 6. Plasma concentration-time plots for 10 mg of erythromycin after a single oral dose of the tests and reference formulations in healthy rats (n = 5). 251
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Table 3 Pharmacokinetic parameters of B1, C1 and MS after oral administration to albino rats (mean ± SD, n = 5). Samples
AUC (μgh/ml)
Cmax (μg/ml)
Tmax (h)
T½ (h)
Ke (mg/h)
SLM-B1 SLM-C1 MS
1021.20 ± 1.21 978.10 ± 0.11 696.27 ± 0.10
3.10 ± 0.11 3.00 ± 1.02 2.70 ± 1.00
2.0 2.0 1.0
8.00 ± 0.10 8.00 ± 0.10 4.50 ± 0.10
1.20 ± 0.30 1.01 ± 0.10 5.80 ± 0.11
SLM-B1 and C1 = selected batch of erythromycin-SLMs, while MS = reference drug.
postgraduate students for their support and dedication in this research work. In addition, the principal investigator is also very appreciative of the collaborative opportunities with member of the research team that were instrumental to this achievement and many academic staff of the Department of Pharmaceutics, University of Nigeria Nsukka. The authors wish to thank Phospholipid GmbH, Köln, Germany for providing samples of Phospholipon® 90H.
(p > 0.05) among the tested formulations. However, the T1/2 value for the SLM was 8.0 h, whereas the T1/2 value for the reference drug (MS) was 4.5 h. The mean AUC values for the optimized SLMeB1, C1 and reference sample (MS) were 1021.20 ± 1.21, 978.10 ± 0.11 and 696.27 ± 0.10 μgh/ml, respectively, indicate a significant difference between the tested samples and reference drug (p < 0.05). The peak plasma level noted in the reference drug which was attained rapidly may be due to easy dissolution and release of the active content when the formulation comes in contact with gastrointestinal fluid. There was a quick fall in the plasma level within the few hours of the study. When this was compared to the SLM-loaded erythromycin, although there was a delay in attaining the plasma peak, the plasma concentration was maintained for a longer period of time than the reference sample. The obvious explanation for this alteration in both the uptake and decay or decrease of drug in the blood may be due to a 2- to 3-fold increase in the circulating t1/2 of erythromycin when administered as erythromycinloaded SLM formulation. It also should be noted that the area under the curve (AUC) for erythromycin was significantly increased by 2 to 3% when the erythromycin was formulated into SLM. The reason for this positive gain in the pharmacokinetics of the SLM formulation (batch B1 and C1) and its contribution in enhancing the in vitro activity of the formulation on the tests organisms may be attributed to the nature of the lipid used in the formulation as the P90H was able to encapsulated higher drug in its core and thereby prevented burst effect. Thus, the lipid may act as a depot and delay the release of erythromycin from the gastrointestinal lumen into the blood circulation or delay the clearance of erythromycin from the blood circulation. Consequently, the result obtained in batch B1 and C1 further confirmed the sustained release activity of this our formulation. The clinical implications of the above findings is that at therapeutic dose, the formulated SLMs would give fast and prolong effect with reduce dose frequency and subsequently enhances dosage compliance.
References [1] J.A. Baird, L.S. Taylor, Evaluation of amorphous solid dispersion properties using thermal analysis techniques, Adv. Drug Deliv. Rev. 64 (2012) 396–421. [2] Y. Kawabata, K. Wada, M. Nakatani, S. Yamada, S. Onoue, Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications, Int. J. Pharm. 420 (2011) 1–10. [3] T. Vasconcelos, B. Sarmento, P. Costa, Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs, Drug Discov. Today 12 (2007) 1068–1075. [4] G. Mooter, The use of amorphous solid dispersions: a formulation strategy to overcome poor solubility and dissolution rate, Drug Discov. Today Technol. 9 (2011) 79–85. [5] P.O. Nnamani, A.A. Attama, E.C. Ibezim, M.U. Adikwu, SRMS 142-based solid lipid microparticles: application in oral delivery of glibenclamide to diabetic rats, Eur. J. Pharm. Biopharm. 76 (2010) 68–74. [6] S. Jaspart, G. Piel, L. Delatte, B. Evrad, Solid lipid microparticles: formulation, preparation, characterization, drug release and applications, Adv. Drug Deliv. Rev. 2 (2005) 75–87. [7] M.A. Momoh, C.O. Esimone, Phospholipon 90H (P90H)-based PEGylated microscopic lipospheres delivery system for gentamicin: an antibiotic evaluation, Asian Pac. J. Trop. Biomed. 2 (2012) 889–894. [8] E.O. Uduma, O.M. Stephen, O.N. Emmanuella, T.G. Harrison, Improvement of oral efficacy of erythromycin ethyl succinate using stearic acid-Myrj-52-based SLM, J. Chem. Chem. Eng. 11 (2017) 37–44. [9] C.E. Umeyor, F.C. Kenechukwu, J.D.N. Ogbonna, Preparation of novel solid lipid microparticles loaded with gentamicin and its evaluation in vitro and in vivo, J. Microencapsul. 29 (2012) 296–307. [10] I. Friedrich, C.C. Müller-Goymann, Characterization of solidified reverse micellar solutions (SRMS) and production development of SRMS based nanosuspensions, Eur. J. Pharm. Biopharm. 56 (2003) 111–119. [11] S.A. Chime, G.C. Onunkwo, I.V. Onyishi, Kinetics and mechanisms of drug release from swellable and non swellable matrices: a review, Res. J. Pharm., Biol. Chem. Sci. 4 (2013) 97–103. [12] S.A. Wissing, O. Kayser, M.U. Muller, Solid lipid nanoparticles for parenteral drug delivery, Adv. Drug Deliv. Rev. 54 (2004) 1257–1272. [13] D.G. Fatouros, G.R. Deen, L. Arleth, B. Bergenstahl, F.S. Nielsen, J.S. Pedersen, A. Mullertz, Structural development of self nano emulsifying drug delivery systems (SNEDDS) during in vitro lipid digestion monitored by small-angle X-ray scattering, Pharm. Res. 24 (2007) 1844–1853. [14] M.U. Müller, M. Radtke, S.A. Wissing, Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Adv. Drug Deliv. Rev. 54 (2002) S131–S155. [15] M.A. Schubert, C.C. Müller-Goymann, Characterization of surface modified solid lipid nanoparticles (SLN): influence of lecithin and non-ionic emulsifier, Eur. J. Pharm. Biopharm. 61 (2005) 77–86. [16] M.A. Schubert, B.C. Schicke, C.C. Muller-Goymann, Thermal analysis of the crystallization and behaviour of lipid matrices and lipid nanoparticles containing high amounts of lecithin, Int. J. Pharm. 298 (2005) 242–254. [17] John Dike N. Ogbonna, Anthony A. Attamaa, Kenneth C. Ofokansi, Sanjay B. Patil, Ganesh D. Basarkar, Optimization of formulation processes using design expert® software for preparation of polymeric blends-artesunate-amodiaquine HCl microparticles, J. Drug Delivery Sci. Technol. 39 (2017) 36–49. [18] A.A. Attama, C.E. Okafor, P.F. Builders, O. Okorie, Formulation and in vitro evaluation of a PEGylated microscopic lipospheres delivery system for ceftriaxone sodium, Drug Deliv. 16 (2009) 448–457. [19] S.A. Chime, A.A. Attama, N.C. Obitte, In vitro and in vivo characterization of indomethacin-loaded dika fat based solid lipid microparticles, Int. J. Pharm. Sci. Rev. Res. 3 (2012) 10–16. [20] C.K. Franklin, A.A. Anthony, C.I. Emmanuel, O.N. Petra, et al., Surface-modified mucoadhesive microgels as a controlled release system for miconazole nitrate to improve localized treatment of vulvovaginal candidiasis, Eur. J. Pharm. Sci. 111 (2018) 358–375.
4. Conclusions In this study, erythromycin-loaded SLMs were formulated with SRMS based on Phospholipon® 90H and Beeswax or Softisan® 154 by melt-homogenization technique. In vitro antibacterial activities, in vivo preliminary pharmacokinetics and the physicochemical properties of SLM studies undertaken with the formulations provided promising results and demonstrated the added value of SLM, thus encouraging further development and optimization of the formulation. This formulation would be a useful alternative for enhanced oral delivery of erythromycin in the treatment of infections caused by erythromycinsusceptible micro-organisms. Conflict of interest The authors declare no conflict of interests. Acknowledgements This work received financial supports from Tertiary Education Trust Fund (TETFund) (Grant no. TETFUND/DESS/NRF/STI/13/) by Government of Nigeria. The corresponding author /principal recipient of the TETFund grant is deeply grateful to all my undergraduates and 252
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Pharm. Res. 13 (8) (2014) 1999-1205. [24] S.A. Chime, A.A. Attama, P.F. Builders, G.C. Onunkwo, Sustained release diclofenac potassium-loaded solid lipid microparticle, based on solidified reverse micellar solution (SRMS): In vitro and in vivo evaluation, J. Microencapsul. 30 (2013) 335–345. [25] S.G. Potta, S. Minemi, R.K. Nukala, C. Peinado, D.A. Lamprou, U. Urquhart, Development of solid lipid nanoparticles for enhanced solubility of poorly soluble drugs, J. Biomed. Nanotechnol. 6 (2010) 634–640.
[21] A.A. Attama, B.C. Schicke, T. Paepenmüller, C.C. Müller-Goymann, 2007. Solid lipid nanodispersions containing mixed lipid core and a polar heterolipid: characterization, Eur. J. Pharm. Biopharm. 67 (2007) 294–306. [22] M.A. Momoh, F.C. Kenechukwu, S.A. Chime, Anti-inflammatory and pharmacokinetics evaluation of PEGylated ibuprofen tablet formulation, Drug Deliv. (2013) 1–5 (DOI: 0.3109/10 717544.2013.850759). [23] F.C. Kenechukwu, C.E. Umeyor, M.A. Momoh, J.D.N. Ogbonna, S.A. Chime, P.O. Nnamani, A.A. Attama, Evaluation of gentamicin-entrapped solid lipid microparticles formulated with a biodegradable homolipid from Capra hircus trop, J.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
A new lipid-based oral delivery system of erythromycin for prolong sustain release activity
T
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Mumuni A. Momoha, , Emmanuel C. Ossaib, Omeje E. Chidoziea, Omenigbo O. Precscilaa, Franklin C. Kenechukwua, Kenneth O. Ofokansia, Anthony A. Attamaa, Kunle O. Olobayoc a
Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Nigeria c National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lipid microparticles Erythromycin stearate Prolong release Pharmacokinetics
Erythromycin-loaded solid lipid microparticles (SLM) based on solidified reverse micellar solution (SRMS) as an oral delivery formulation was studied. Hot homogenization technique was employed to prepare erythromycin stearate-loaded SLMs using blends of Softisan® 154 and Phospholipon® 90H or beeswax in the ratio of 1:2, and characterized in vitro. Antibacterial evaluation of the formulations was carried out by agar diffusion technique against some selected clinical isolates of bacterial. Preliminary pharmacokinetic study was performed after oral administration in male Albino rats. The results of matrix contain Softisan® 154 and phospholipon® 90H (1:2) showed that erythromycin-loaded SLM was smooth; particle size ranged from 10.3 ± 11.24 μm to 18.1 ± 10.11 μm and maximum encapsulation efficiency and loading capacity were 95.11 ± 0.3% and 43.22 ± 0.1 mg, respectively. While that of beeswax- containing matrix showed maximum particle size of 18.9 ± 21.10 μm, maximum encapsulation efficiency of 89.01 ± 0.11% and loading capacity of 39.02 ± 0.12 mg. All the formulations had prolonged release and antibacterial activity. Significantly (p > 0.05), prolonged plasma erythromycin concentration was obtained in the optimized formulation (> 14 h) compared with commercial sample of erythromycin tablet (10h). Erythromycin stearate-loaded SLMs formulation could serve as an alternative to conventional oral formulation of erythromycin.
1. Introduction In recent times, formulation scientists have discovered that the poor aqueous solubility and dissolution rate of active pharmaceutical ingredients (APIs) is one of the biggest challenges in pharmaceutical development. The poor solubility often results in low bioavailability of orally administered drugs, and thus results in poor therapeutic efficacy [1]. According to the Biopharmaceutics Classification System (BCS), a drug is poorly soluble if the highest dose strength is not soluble in 250 ml aqueous media over the entire pH range at 37 °C (FDA, 2000). The candidate drugs of these nature are mostly considered as Class II (IIa or IIb), which are poorly soluble and highly permeable according to the pH of the gastrointestinal fluid and tend to present solubility or dissolution rate-limited absorption [2]. Despite their high permeability, these molecules often have low oral bioavailability because of their slow and limited release of drug in gastrointestinal fluid thereby posing big challenges in clinical practice on the utilization of these molecules [3]. Therefore, one of the major tasks that the pharmaceutical industry
⁎
need to overcome is how to develop strategies that could improve the dissolution and/or apparent solubility of these drugs/compounds into orally bioavailable and therapeutically effective drugs for the benefit of patients in clinical settings [2,4]. Several approaches to overcome the poor aqueous solubility and it improve absorption after oral administration of such drugs have form the major research among the formulation scientist and their findings are well documented [5]. In comparison with other delivery system, such as liposomes, microemulsion, prodrug formation and microparticles that have been studied in the controlled release of incorporated drugs and in enhancing the solubility, improve absorption of drugs, solid lipids microparticles (SLMs) have been considered as one of the most successful strategies to maintain high degree of dispersity of drug profile of poorly soluble drugs. SLMs a new entrance in as drug delivery system and have so far been considered a promising drug carrier system, especially with a view to giving the incorporated active substance a sustained-release profile and improving the dissolution profile of poorly soluble drugs [5,6]. In contrast to polymeric drug
Corresponding author. E-mail address: [email protected] (M.A. Momoh).
https://doi.org/10.1016/j.msec.2018.12.041 Received 27 May 2018; Received in revised form 9 November 2018; Accepted 12 December 2018 Available online 13 December 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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delivery, SLMs as drug carrier has been considered as very safe and are said to be physicochemically stable as in some cases no organic chemicals is employ in its preparation [5]. Additionally, asides from its ease of preparation and scaling-up, it's have an edge over other delivery systems in the following areas; stability, low production cost and high encapsulation efficiency for lipophilic compounds [7]. The utilization of mixtures of lipids as solidified reverse micellar solutions (SRMS) in the preparation SLMs further strengthened the grip of SLMs as the last bus stop for the poorly aqueous soluble drug [8]. Formulations based on SRMS have been investigated and successfully employed to achieve controlled release and enhanced absorption of drugs [8–10]. For instance, SRMS consisting of phospholipids and solid lipids such as hydrogenated palm oil, which transform into a lamellar mesophase after melting on contact with water have widely been employed in nano-suspension [10], and in the delivery of diabetes drugs [5]. Consequently, this transformation enables controlled release of solubilized drugs, prevents drug expulsion and increase the drug encapsulation efficiency [10]. Interestingly, SRMS carriers have recently been investigated as a sustained release matrix for both hydrophilic and hydrophobic drugs; it combined several advantages of other known lipid carriers [11–13]. The recent discovery on the utilization of SLMsbased SRMS in the delivery of sensitive material against degradation and excellent in vivo tolerability is unparallel to the other system [14]. In view of these aforementioned advantages, it was the objective of this study to formulate and characterize, and in vitro erythromycinloaded – SRMS based SLMs from mixtures of lipids composed of mixture of Softisan® 154 and Phospholipon® 90H or beeswax for maintain high degree of dispersity of drugs and facilitate absorption after oral administration.
Table 1 Quantities of starting materials for the formulation of different batches of unloaded and erythromycin-loaded microparticles. Batch
Lipid matrix (g)
Sorbic acid (mg)
PVA (mg)
Sorbitol (mg)
Drug Conc. (mg)
Distilled water, q.s (% w/w)
B1 B2 B3 B4 C1 C2 C3 C4
20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0
30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0
600.0 600.0 600.0 600.0 600.0 600.0 600.0 600.0
600.0 600.0 600.0 600.0 600.0 600.0 600.0 600.0
500.0 250.0 100.0 – 500.0 250.0 100.0 –
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
Key: B1 – B3, contain 500, 250 and 100 mg of erythromycin respectively, C1 – C4 contain 500, 250 and 100 mg of erythromycin respectively, while batches B4 and C4 contains no drug.
Germany) at 8000 rpm for 4 min to produce the hot primary emulsion, which was collected in hot containers and allowed to recrystallize at room temperature. By adding decreasing concentrations of erythromycin (500, 250 and 100 mg) to the SRMS and following the above mentioned procedure, erythromycin-loaded SLMs were obtained and coded as B1, B2, and B3 respectively. Similarly SLMS based Softisan154 and Beeswax were also prepared using same drug concentration as above to obtained erythromycin loaded SLMs and coded as C1, C2, and C3 respectively. Drug free (unloaded SLMs) were similarly prepared (Table 1) and coded B4 and C4. 2.3. Characterization of SLMs
2. Materials 2.3.1. Differential scanning calorimetry (DSC) Melting transitions and changes in heat capacity as a function of the degree of crystallinity in the. physically structured lipid matrices were determined using a calorimeter (Netzsch DSC 204 F1, Germany). Approximately 5 mg of each SLMs was weighed into an aluminum pan and sealed hermetically, and the thermal behavior was determined in the range of 10 to 125 °C at a heating rate of 5 °C per min. Baselines were determined using an empty pan.
The following materials were used: Softisan® 154 (Schuppen, Condea Chemie GmbH, Germany), Phospholipon® 90H (Phospholipid GmbH, Köln, Germany), and beeswax® (Carl Roth GMbH, Germany). Erythromycin stearate was a gift from AC Drugs Ltd., Enugu State, Nigeria, and distilled water was obtained from Lion water (UNN, Nigeria). All other reagents and solvents were of analytical grade and were used as supplied. 2.1. Preparation of lipid matrix
2.3.2. Morphology and particle size analysis The particle size of the microparticles was analyzed periodically so as to know the effect of time on the formulation. Briefly, 3–5 mg from each batch of the SLMs was dispersed in distilled water and smeared on a microscope slide using a glass rod. The mixture was covered with a cover slip and viewed with a binocular microscope (Lieca, Germany) attached with a digital camera (Moticam, China) at a magnification of ×1000. The photomicrographs of the particles were also observed and taken.
The lipid matrix based on the binary mixture of 1:2 ratio of Softisan® 154 and Phospholipon® 90H (P90H) or beeswax was prepared by fusion. The ratio used in this study was based on our preliminary evaluation during the selection and screening of the lipids mixture to establish the best combination. In brief, a 10 g quantity of P90H or beeswax was carefully weighed and transferred into a crucible containing 5 g of Softisan® 154 and the mixture was heated at temperature of 60 °C on a hot plate (SR1 UM 52188, Remi Equip., India) to melt. The molten lipid mixture was then stirred thoroughly until solidification to get the solidified reverse micellar solution (SRMS).
2.3.3. Time-dependent pH stability studies The pH of the formulations, drug-loaded and unloaded (drug free) SLMs were determined in a time-dependent manner using a pH meter (Suntex TS-2, Taiwan) after 24 h, 4 weeks, and 12 weeks of storage condition in order to ascertain the stability of the formulations especially in the area of product degradation during storage.
2.2. Formulation of SLMs The following compositions for material were used in the preparation of SLM, 6.7% w/w of SRMS, 2% w/w polyvinyl alcohol (PVA), 0.1% w /w sorbic acid, 2% w/w sorbitol and enough distilled water to make 100% w/w. The hot homogenization method was adopted according to earlier work [17], with slight modifications. In each case, the lipid matrix was melted at 60 °C in a crucible, and PVA, sorbitol, and sorbic acid were dispersed in the appropriate concentration in sufficient volume of distilled water and brought to the same temperature with the lipid matrix. The hot aqueous phase was poured into the molten lipid matrix with gentle stirring with a magnetic stirrer and the mixture was further dispersed with a homogenizer (Ultra-Turrax T18, Basic, Ika,
2.3.4. Encapsulation efficiency (EE%) Approximately, 0.5% w/v dispersion of each batch of the SLMs in distilled water was prepared in a 100 mL volumetric flask, allowed to equilibrate for 48 h at room temperature, shaken and filtered. The filtrate was adequately diluted and analyzed for erythromycin content using a spectrophotometer (Unico 2102 PC UV/Vis Spectrophotometer, USA) at 214 nm. The amount of drug encapsulated in the microparticles was calculated with reference to a standard Beer's plot for erythromycin 246
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2.6. Pharmacokinetics study
to obtain the EE% using the formula below.
EE =
Actual drug content X 100 Theoretical drug content
The batches of the SLMs that show highest in recovery, loading capacity, encapsulation efficiency and release were selected and were used for the pharmacokinetic study. Following the set out criteria, batch B1 and C1 of the preparation were selected for the evaluation. Adult albino male rats weighing between the range of 255–270 g were used in the investigation. They rats were kept at the ambient temperature of 26 ± 1 °C, 12 h light/dark cycle, fed standard rat feed and had free access to drinking water. The animals were fasted for 12 h before experiment. Animal experiment in this research was in line with general standard as stipulated by our Institution's Animal Ethics Committee and in compliance with the European Community Council Directive of November 24, 1986 (86/609/EEC). Three groups (I, II and III) of five rats per group were used in this investigation. The formulations (B1 and C1) and the reference sample were orally administered to the rats. Rats in Group I received erythromycin-SLM (batch B1), rats in Group II received (batch C1), while rat in Group III received the reference sample of erythromycin (MS) tablet (10 mg/kg of body weight) in the form of suspension in 2.5 ml sterilized distilled water. Blood samples (about 0.5 ml each) were collected from the orbital plexus at 0, 1, 2, 4, 6, 8, 10, 12 h and immediately extracted in acetonitrile at blood to acetonitrite ratio of 1:3 and was subjected to low-speed centrifugation, and the supernatant collected were analyzed using a HPLC.
(1)
2.3.5. Loading capacity (LC) LC expresses the ratio between the entrapped drug and the total weight of the lipids. It was determined using Eq. (2):
LC =
Qd − Qs × 100 ql
(2)
where ql is the weight of lipid added in the formulation, Qd is the weight of erythromycin added to the formulation, and Qs is the amount of drug determined in the supernatant after separation of the lipid and aqueous phases.
2.4. In vitro release study on SLMs In vitro release was evaluated using a dialysis membrane technique as described in a previous study [9]. The polycarbonate dialysis membrane (MWCO 8000–10,000 Spectrum Labs, Netherlands) used as release barrier was pre-treated by soaking it in the dissolution medium for 24 h prior to the commencement of each release experiment. SLMs equivalent to 50 mg were placed in a dialysis membrane containing 5 mL of the dissolution media. The dialysis membrane was tied at both ends and was suspended in the basket of USP Type I dissolution apparatus (Electrolab, Mumbai, India). The baskets were immersed in 250 mL phosphate-buffered, pH 7.2, maintained at 37.0 ± 0.5 °C and was agitated at 75 rpm. At regular intervals, 5.0 mL of dissolution medium was withdrawn and was replaced with fresh buffer at the same temperature. The withdrawn samples were filtered through a 0.22 μm filter (Millipore®, USA) and analyzed for erythromycin content by HPLC method as described elsewhere [9].
2.6.1. Pharmacokinetic analysis Pharmacokinetic parameters were investigated and calculated using a computer program Calc 2002 (Korea Food & Drug Administration, Korea). The elimination rate constant (Ke) was obtained from the leastsquare fitted terminal log linear portion of the serum concentrationtime profile. The erythromycin Cmax and the corresponding Tmax were evaluated from the serum concentration-time profiles of the drug. The elimination half-life (T1/2) was calculated as 0.693/Ke. The trapezoid rule was used to investigate the area under the curve (AUC). Maximum plasma concentration (Cmax) and the time needed to reach the maximum plasma concentration (Tmax) were determined directly from the concentration-time data for the optimized SLM (B1) and the commercial sample (MS).
2.5. Antimicrobial susceptibility activity of SLMs using agar diffusion method The biological activities of erythromycin-loaded SLMs were measured by bacteria growth inhibition assay using an agar diffusion method. In another words, the antimicrobial activities of the SLM were evaluated using agar diffusion method previously reported (18). This method depends on the diffusion of antibiotics from holes on the surface of the microbial seeded agar. The test organisms (Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Salmonella paratyphii (S. paratyphii) and Staphylococcus aureus (S. aureus) as tests organisms) were standardized using 0.5 MacFarland standards which is equivalent to 1 × 10−8 colony forming unit (CFU). In brief, Mueller - Hinton agar (MHA) was prepared according to manufacturer's instruction, sterilized at 120 °C, 15 psi for 15 min, and allowed to cool to 45 °C. Thereafter, the molten MHA maintained at 45 °C was aseptically seeded with 0.1 ml of the standardized test organisms and poured into sterile plates. The set up (agar and test organism) was mixed together and was allowed to gel. Sterile cork borer was used to make holes/wells on the gel agar plate at equal distances from each other and thereafter sterile applicator was used to withdraw 0.1 ml of a batch of the SLM formulation and was added into each well (B1, B2, B3 for the loaded-SLMs), and was allowed to stand for diffusion of the antimicrobial agents. The seeded agar plates were incubated at (37.0 ± 0.5) °C for 24 h. All the tests were done in triplicates and growth was examined after incubation, with the average inhibition zone diameter (mm) of each sample determined. The same procedure was used for batch C1– C3 and a commercial sample of erythromycin (MS) was similarly evaluated and compared to the formulated erythromycin-loaded SLMs.
2.7. Statistical and data analysis Data were analyzed using SPSS Version 16.0 (SPSS Inc. Chicago, IL). All values were expressed as mean ± SD. Differences between means were assessed using one way ANOVA and student's t-test. p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of lipid matrices and SLMs The DSC results showed that the hybridization of Softisan® 154 with P90H or beeswax resulted in matrices with low enthalpies compared with the individual lipid material. The system displayed several prominent transitions upon heating which clearly showed the interaction of the lipid mixture as well as the various heat capacities of the formulation. The melting endothermic of Softisan® 154 was 61.4 °C with an enthalpy of - 41.99 mW/mg (Figure not shown). However, when the lipid was used in the formulation of SRMS matrix, the SRMS gave a melting endotherm value of 61.1 °C and an enthalpy of - 6.999 mW/mg. This value of enthalpy indicated that the matrix was less crystalline than the bulk lipid due to its lower enthalpy value and suggests that mixture of lipids can produce matrices of low crystallinity. Thus, the SRMS may have generated an imperfect matrix resulting from distortion of crystal arrangement of the bulk lipid after melting and solidification. This distortion of the lipid crystal may be an advantage in drug 247
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Fig. 1. (a, b). (a) Thermograph of unloaded SLM (4), pure erythromycin(12), batch B1 (16), B2(17) and B3(18); (b) Thermograph of unloaded SLM (4), pure erythromycin (9), C1(10), C2(11) and C3(12).
Batches B4 and C4 showed the least particle sizes throughout the period of evaluation, probably because the batch contained no drug, which could aid the growth of the particles during encapsulation. Particle size may be a function of either one or more of the following: formulation excipients, degree of homogenization, homogenization pressure, rate of particle size growth, crystal habit of the particle [11,18,21]. Alternatively, this observation may be due to sintering or Ostward ripening as postulated by the DLVO theory [23]. The particles tend to migrate toward each other and subsequently formed aggregates due to the kinetic advantage of gain by larger crystal associated with increase in the drug concentration. Similarly, larger crystals are more favoured thermodynamically and difficult to nucleate when compared with small crystals. According to the DLVO theory, there are forces that interacting between particles existing in colloidal dispersions these include electrical repulsion forces (VR) and the van der Waals attraction (VA), these parameters are said to complement each other [6]. In this context, it is very obvious that the particle size after drug incorporation was large compared with unloaded particles such that the interparticles distance was appreciably small thereby allowing the predomination of the van der Waals universal attractive forces to be predominant giving rise to larger particles on aggregation. This explanation was in agreement with earlier researcher who observed that particle in lipids formulation depend on the concentration of the incorporated drug, and it increase as concentration of the drug increases. However, in the unloaded formulation, it was observed that less particles sizes were maintained, which could be attributed to the larger inter-particulate distance such that the repulsion between the particles exists. Molecularly, the large inter-particulate distance brings about the setting of the secondary minimum. Over time, the particle tends to break-up into smaller particles thereby increasing further the inter-particulate distance. Consequently, the double-layer repulsion predominates giving a primary maximum in which case at a corresponding large thermal energy (kT) of the particles, colloidal systems thus become well dispersed and deflocculated [24].
encapsulation and entrapment [5,9,18]. However, the DSC thermogram of erythromycin shows a peak at 167.2 °C with an enthalpy of −2.661 mW/mg (Fig. 1a). When the SRMS -154 was employed to formulate SLMs, the DSC thermogram of the formulations showed different peak characteristics according to drug concentration. It was observed that B1 of the SLM formulated with 500 mg gave the minimum melting endotherm of 59.7 °C with an enthalpy of −7.623 mW/mg, whereas the unloaded (no drug) SLMs (B4) showed maximum melting endotherm (61.6 °C) together with maximum enthalpy value of −12.03 mW/mg (Fig. 1a). There was a significant difference (p < 0.05) between the SLM loaded with 500 mg of erythromycin (B1) compared to batches loaded with 250 mg (B2) and 100 mg (B3) of erythromycin. DSC thermograms of lipid carrier, beeswax, and the lipid matrix showed a sharp endothermic peak corresponding to melting transitions at 73.0, 61.4 and 66.5 °C with corresponding enthalpies of −55.7, −41.99 and − 14 mW/mg (Figure not shown), respectively. Thermograms of the erythromycin-loaded SLMs C1, C2 and C3 showed sharp endothermic peaks at 70.8, 64.8 and 66.9 °C with a corresponding transition enthalpies of –40.26, −16.03 and − 30.44 mW/mg, respectively (Fig. 1b), where the various batches are compared. However, lower enthalpy suggests less crystallinity and the possibility for retention of an entrapped drug over time, whereas high enthalpy means highly ordered crystalline arrangement (perfect crystals) which leads to drug expulsion upon crystallization of previously molten matrices [9,16,18]. 3.2. Morphology and particle size of SLMs Representative photomicrographs of batches of the SLMs containing decreasing amount of erythromycin (B1 - B3 and C1 - C3) with their corresponding unloaded batches or batches containing no erythromycin, B4 and C4 respectively, after four weeks of preparation are shown in Fig. 2. From the photomicrographs it could be seen that the SLMs were well formed, smooth, and non-porous, they were also stable and did not show sedimentation even after centrifugation at 2000 rpm for 20 min. The particle sizes within one week of formulation were small and ranged from 13.23 to 17.15 μm, and increased dose-dependently according to the concentration of entrapped erythromycin as shown in Fig. 3a. The particles sizes followed a decreasing order of magnititude: B1 > B2 > B3 > B4. Similarly, the sub-batches of C showed not appropriate increased particle size compared to the batches in B, but the particles increased as the concentration of drug increased and showed similar trend in increase in the order of C1 > C2 > C3 > C4.
3.3. Time-dependent pH stability studies on the formulations The pH of the different batches of SLMs were measured 24 h, 1 week, and 1 month after preparation to ascertain the variation of pH with time, which could be a function of degradation of the API or excipients (Fig. 3b). There was a slight decrease in the pH of the formulations within the storage period. This was also applicable to unloaded formulation (drug free) samples whose pH continued to decrease throughout the period of observation. The shifting of the pH toward 248
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Fig. 2. Photomicrographs (within 24 h) of SLMs formulated with SRMS containing (B1) 500, (B2) 250, (B3) 100; and (B4) 0 mg of erythromycin . While, C1, C2, C3, and C4 containing 500, 250, 100, and 0 mg or erythromycin, respectively.
acidity is an evidence of probably the degradation of the lipid components of the formulation and release of more free fatty acids [9,19,20]. However, the decrease in the pH of the formulation loaded with drug is not significant (p > 0.05) as compared to the decrease observed in the unloaded (drug free) formulation. This indicates that the decrease in the pH may not be enough to cause a decrease or change the expected activity of the formulation. In an earlier study, the author observed that, although, there was a tremendous improvement in the use of either synthetic or natural lipids in drug delivery, the decrease in the pH of the loaded formulation did not alter the activity of the antibiotic used in the formulation [21]. It was further suggested that such decrease or changes in the pH toward acidity could be overcome by the addition of stabilizing agent during the formulation. Similarly, previous researchers observed that Phospholipon® 90G or 90H could be used to stabilize SLM formulations and prevent product degradation [18,22]. However, since the level of decrease in pH of the loaded SLMs formulation was less than the unloaded formulation, it could be argued that the loaded formulation gave enough stabilization as compared to the unloaded; an indication that drug in the formulation did not degrade but rather improved the stability of the formulation. In other words, we can vividly argue that the changes in the pH was not associated with the drug incorporated into the lipid matrix, since the unloaded batches showed more in the decreased than the loaded sample. Scientifically, the particle surface pH in some systems may show some level of variation in the range of 2–5 units different from the pH of the bulk material because of the forces of attraction/adsorption coupled with the electrical double layer effect of other ions in the continuous phase of the same system [23].
Table 2 Result of some physical parameters of the SLMs. Formulation code
B1 B2 B3 B4 C1 C2 C3 C4
Parameters Percentage yield
EE (%)
LC (mg API/100 mg lipid)
96.10 91.12 86.23 75.50 92.10 81.12 81.23 73.50
95.11 92.23 89.85 – 89.01 79.12 78.11 –
43.22 27.42 33.26 – 39.02 29.12 31.11 –
± ± ± ± ± ± ± ±
0.11 0.31 0.01 0.12 0.41 0.31 0.01 0.12
± 0.13 ± 0.19 ± 0.01 ± 0.11 ± 0.11 ± 0.03
± 0.13 ± 0.20 ± 0.15 ± 0.12 ± 0.00 ± 0.11
Batches B1- B3 and C1-C3 contain erythromycin while batch B4 and C4 contain no drug.
3.4. Percentage yield, loading capacity and encapsulation efficiency The percentage yield or recovery rates of the formulation have a direct relationship to the methodology. As shows in Table 2, It was observed that maximum and minimum yield were observed in subbatch B1 (96.23%) and B3 (89.10%) for the loaded formulation. The sub-batch C1 and C3 showed maximum and minimum yields of 92.10 ± 0.11 and 81.23 ± 0.01% respectively. The unloaded (drug free, batch B4 and C4) showed the least recovery rate (75.50 and 73.50%, respectively) when compared to the loaded batches. In all cases, the percentage recovery across the batches increased with increase in drug loading. High values (> 70%) of the percentage of the SLMs recovered from the formulations are a strong indication that the formulation technique adopted was reliable. Fig. 3. (a, b). (a) Bar chart representing the particle size of erythromycin-loaded (B1-B3, C1-C3) and unloaded (B4 and C4) SLMs over time, were formulated with the SRMS lipid matrix. (n = 3) and (b) Timeresolved pH analysis of erythromycin-loaded (B1-B3, C1-C3) and unloaded (B4 and C4) SLMs formulated with the SRMS lipid matrix.
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3.6. Antimicrobial susceptibility activity
The role of any drug delivery system (DDS) is to deliver the incorporated drug to the target tissues intact with little or no toxic effect to other organ or system. Thus, the ability of the SLMs to accommodate active molecules is an important property. It can be expressed by the encapsulation efficiency (EE %) and loading capacity (LC). EE % defines the ratio between the weight of entrapped active pharmaceutical ingredient (API) and the total weight of API added to the dispersion, while LC expresses the ratio between the entrapped API and the total weight of the lipids [25]. As shows in Table 2, the EE% and the LC obtained for the various batches of SLMs after 4 weeks of the formulation. Both EE% and LC are dependent on factors such as the nature of the lipid, the formulation technique used and the lipophilic nature of the active pharmaceutical ingredient (API). The EE% ranged from 89.85 ± 0.4 to 95.11 ± 0.2% and 78.11 ± 0.03–89.01 ± 0.11 for batches B1 to B3 and C1 to C3, respectively. The varying EE% may be as a result of the concentration of the API used in the formulation. All the same, higher EE% values were obtained in all the batches of the formulation. However, there was no significant difference (p > 0.05) in the EE% obtained for batches B1, B2, and B3 SLMs. On the other hand, the batches in C, showed significant difference (p < 0.05) among the sub-batches in Cs. Similarly, the formulation showed high loading capacity ranging from 27. 22 ± 0.0–43.22 ± 0.1 and 31.11 ± 0.11–39.02 ± 0.12 mg for sub-batch of B and C, respectively.
The antimicrobial susceptibility testing using agar plate method was based on the diffusion of an antibiotic agent or formulation thereof through a solidified nutrient agar. The zone size around each antimicrobial disk was interpreted as size of areas of inhibition of growth (mm). The results of the antimicrobial activity of erythromycin loaded SLMs are shown in Fig. 5(a–d). From the results, the erythromycinloaded microparticles exhibited good anti-bacterial activity significantly different from the commercial sample against all the microorganisms used for the study (p < 0.05). The isolates (micro-organisms) showed sensitivity of all tested erythromycin-loaded SLM at varying degrees and are concentration dependent. The SLM-based SRMS formulations gave zones of inhibition in the following decreasing order of magnitude: B1 > B2 > B3 for SLMs for the entire test organisms. Similar results was obtained when beeswax was added to the formulation; the order of zone of inhibition in all cases was C1 > C2 > C3. The IZD was somehow low in batch B3 and C3, probably because the concentration of erythromycin contained in these batches were too low to yield concentrations equal to or above the minimum inhibitory concentration (MIC). MS, which served as the control, showed certain levels of inhibition, but somehow low when compared to the formulated SLMs at p < 0.05. Interestingly, several literatures reports the enhanced therapeutic effect, with reduced cytotoxicity, of several antimicrobial drugs when loaded in micro/nanoparticles as compared to the traditional formulation. Furthermore, when the different lipid combination (matrices) was compared, it was observed that combination of beeswax in the formulation generally produced formulation with lesser activities as compared to when P90H was used in all the batches of the formulations (B1- B3). This indicates that P90H was activity determinant in the formulation. Additionally, the formulations exhibited capacity-limited antimicrobial activity in a time-dependent, manner as observed in the IZD against the organisms tested. The antibacterial activities show timedependent increase in IZDs within 240 min implies that the formulated SLMs had potential for sustained drug release. The activity of the formulations suggests that SLMs increased the antibacterial activity of erythromycin. These results are similar to those obtained in related studies [4,5]. Earlier investigators have suggested several mechanisms for the enhanced activities of SLMs including fusion of the lipid component with the bacterial membranes [5,7,20]. Due to membrane merging, the antibiotic could easily penetrate inside the bacteria allowing the increased bactericidal efficacy observed with the drug, thus circumventing the normal pathway of penetration [21]. Generally, it's arguable that SLMs potentiate the activity of the incorporated drug either through high pay-load or inhibit drug expulsion during formulation and storage stage and the release of the drug in a controlled manner.
3.5. In vitro release The results of the in vitro release of erythromycin from the SLMs presented in Fig. 4(a, b) showed that all the erythromycin-loaded SLMs exhibited higher prolonged release properties significantly different from the reference erythromycin tablets (p > 0.05). The formulations gave a gradual and more sustained release of erythromycin over the study period. The results showed that erythromycin-loaded SLMs could be used once daily or at most twice daily as compared to the conventional formulation that must be taken thrice or more before attaining the required plasma concentration. More so, the formulations showed good encapsulation of the incorporated drug as there was no burst effect or dose dumping. The results revealed capacity-limited release of erythromycin from the SLMs consistent with earlier reports [15,18,24]. For instance, batch B1 formulation with the highest drug concentration (500 mg of erythromycin) gave a maximum release of 90%, while batch B3 with the least drug concentration (100 mg of erythromycin) gave a maximum release of 75% (Fig. 4a). Similarly results were obtained when beeswax was used. It was observed that batch C1 had maximum release (79%) as compared to other batches of the formulation with 66 and 61% for batch C2 and C3 (Fig. 4b), respectively. Regarding their sustaining potentials, all the batches of the SLM had good sustaining properties. However, batch B1 and C1 formulations produced better sustained release properties than the rest of the formulations.
Fig. 4. (a, b) (a) In vitro release profile of erythromycin from SLMs in phosphate buffer at pH 7.4; (B1, B2 and B3 were formulated with lipid matrix contain 500, 250 and 100 mg of erythromycin, respectively. MS is the commercial sample that served as a control); and (b) In vitro release profile of erythromycin from SLMs in phosphate buffer at pH 7.4; (C1, C2 and C3 were formulated with lipid matrix contain 500, 250 and 100 mg of erythromycin, respectively. MS is the commercial sample that served as a control.
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Fig. 5. (a–d). Time-dependent susceptibility of microorganisms to erythromycin-loaded SLMs; (a) Staphylococcus aureus (S. aureus) (b) Salmonella paratyphii (S.paratyphii) (c) Pseudomonas aeruginosa (P. aureginosa), (d) Escherichia coli (E. coli).
maintained a steady slow decreased or gradual clearance throughout the study in the treated rats. The average mean pharmacokinetic parameters (Table 3), shows that the mean Cmax values for the tested samples B1 and C1 were 3.1 and 2.9 μg/ml respectively, as compared with 2.7 μg/ml obtained in the reference sample (MS). The mean Tmax values for the B1, C1 and MS were 2.0, 1.9 and 1.0 h, respectively. However, the Cmax and Tmax values were not significantly different
3.7. Pharmacokinetics studies Data obtained from the pharmacokinetic studies are shown in Fig. 6, the SLM-loaded erythromycin showed a slow decrease in the initial peak attained in comparison to the reference drug sample as shown in Fig. 6, after oral administration. However, in contrast to the rapid exponential decrease in the reference sample, erythromycin-loaded SLMs
Fig. 6. Plasma concentration-time plots for 10 mg of erythromycin after a single oral dose of the tests and reference formulations in healthy rats (n = 5). 251
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Table 3 Pharmacokinetic parameters of B1, C1 and MS after oral administration to albino rats (mean ± SD, n = 5). Samples
AUC (μgh/ml)
Cmax (μg/ml)
Tmax (h)
T½ (h)
Ke (mg/h)
SLM-B1 SLM-C1 MS
1021.20 ± 1.21 978.10 ± 0.11 696.27 ± 0.10
3.10 ± 0.11 3.00 ± 1.02 2.70 ± 1.00
2.0 2.0 1.0
8.00 ± 0.10 8.00 ± 0.10 4.50 ± 0.10
1.20 ± 0.30 1.01 ± 0.10 5.80 ± 0.11
SLM-B1 and C1 = selected batch of erythromycin-SLMs, while MS = reference drug.
postgraduate students for their support and dedication in this research work. In addition, the principal investigator is also very appreciative of the collaborative opportunities with member of the research team that were instrumental to this achievement and many academic staff of the Department of Pharmaceutics, University of Nigeria Nsukka. The authors wish to thank Phospholipid GmbH, Köln, Germany for providing samples of Phospholipon® 90H.
(p > 0.05) among the tested formulations. However, the T1/2 value for the SLM was 8.0 h, whereas the T1/2 value for the reference drug (MS) was 4.5 h. The mean AUC values for the optimized SLMeB1, C1 and reference sample (MS) were 1021.20 ± 1.21, 978.10 ± 0.11 and 696.27 ± 0.10 μgh/ml, respectively, indicate a significant difference between the tested samples and reference drug (p < 0.05). The peak plasma level noted in the reference drug which was attained rapidly may be due to easy dissolution and release of the active content when the formulation comes in contact with gastrointestinal fluid. There was a quick fall in the plasma level within the few hours of the study. When this was compared to the SLM-loaded erythromycin, although there was a delay in attaining the plasma peak, the plasma concentration was maintained for a longer period of time than the reference sample. The obvious explanation for this alteration in both the uptake and decay or decrease of drug in the blood may be due to a 2- to 3-fold increase in the circulating t1/2 of erythromycin when administered as erythromycinloaded SLM formulation. It also should be noted that the area under the curve (AUC) for erythromycin was significantly increased by 2 to 3% when the erythromycin was formulated into SLM. The reason for this positive gain in the pharmacokinetics of the SLM formulation (batch B1 and C1) and its contribution in enhancing the in vitro activity of the formulation on the tests organisms may be attributed to the nature of the lipid used in the formulation as the P90H was able to encapsulated higher drug in its core and thereby prevented burst effect. Thus, the lipid may act as a depot and delay the release of erythromycin from the gastrointestinal lumen into the blood circulation or delay the clearance of erythromycin from the blood circulation. Consequently, the result obtained in batch B1 and C1 further confirmed the sustained release activity of this our formulation. The clinical implications of the above findings is that at therapeutic dose, the formulated SLMs would give fast and prolong effect with reduce dose frequency and subsequently enhances dosage compliance.
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4. Conclusions In this study, erythromycin-loaded SLMs were formulated with SRMS based on Phospholipon® 90H and Beeswax or Softisan® 154 by melt-homogenization technique. In vitro antibacterial activities, in vivo preliminary pharmacokinetics and the physicochemical properties of SLM studies undertaken with the formulations provided promising results and demonstrated the added value of SLM, thus encouraging further development and optimization of the formulation. This formulation would be a useful alternative for enhanced oral delivery of erythromycin in the treatment of infections caused by erythromycinsusceptible micro-organisms. Conflict of interest The authors declare no conflict of interests. Acknowledgements This work received financial supports from Tertiary Education Trust Fund (TETFund) (Grant no. TETFUND/DESS/NRF/STI/13/) by Government of Nigeria. The corresponding author /principal recipient of the TETFund grant is deeply grateful to all my undergraduates and 252
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