Non-Absorbable Oral Gentamicin Sulphate: Biopharmaceutical and Dosage Form Evaluation

Gentamicin sulphate is an antibiotic belonging to the aminoglycosides and to class III of the Biopharmaceutical Classification System (BCS) (Pignatello et al., 2016; Baasov et al., 2015; Ito et al., 2005). Gentamicin sulphate is highly water soluble, but has very low intestinal permeability. The drug has been used extensively through parenteral administration in the treatment of wide range of susceptible bacterial infections. The wide use is because of its broad spectrum of activity. Conventionally, gentamicin sulphate is administered parentally (Imamura and Adams, 2003; Labiris et al., 1999). The drug has also been presented for dermal topical applications (Imamura and Adams, 2003; Ipsen et al., 1991; Nnamani et al., 2013; Picket al., 1997; Ravis et al., 2013) and as inhalations (Labiris et al., 1999; Aquino et al., 2012; Goldman et al., 1990; Lim et al., 2002). It has been evaluated for rectal local activity (Fix et al., 1983) as well as implants (Krasko et al., 2007; Soriano et al., 2000).

As noted earlier, the low intestinal permeability makes oral administration, hence formulation, very difficult. However, in veterinary practice, oral gentamicin sulphate has been recommended in weaning swine for the control and treatment of colibacillosis caused by strains of E. coli sensitive to gentamicin, and for the control and treatment of swine dysentery associated with Treponema hyodysenteriae (Legal Information Institute, 2016). It has also been evaluated in dogs in combination with labrasol to enhance its intestinal mucosa permeability (Rama Prasad et al, 2003).

In humans, oral administration of gentamicin sulphate has been included in the treatment trial of necrotizing enterocolitis, a condition of death of tissue in the intestine, often occurring mostly in premature or sick babies (Gephart et al, 2012; Shah and Sinn, 2012). This is a form of localised drug action in which the gentamicin sulphate acts on the diseased tissues. The widespread use of gentamicin sulphate through the oral route in humans to achieve systemic therapeutic concentration has been limited by low intestinal permeability. Early researches have centred on the incorporation of permeation enhancers to achieve intestinal absorption and systemic circulation. Verma et al (2014) in a review enumerated some of these permeation enhancers. Attempts have been made to formulate the drug for oral administration by incorporating some of these intestinal mucosa penetration/permeation enhancers to achieve systemic circulation (Anilkumar et al., 2011; Rama Prasad et al., 2003; Shaikh et al., 2012). Umeyor et al. (2016) have achieved improved systemic circulation of orally administered gentamicin sulphate by surface modified self-nano-emulsifying formulations (SNEFs). However, the drug has not been formulated into dosage form(s) for local activity in the gastrointestinal tract (GIT). An unaided formulation of the drug administered orally will invariably accumulate in the gut, and the drugs’ broad spectrum antibacterial activity could be used for local action against bacteria pathogens inhabiting the GIT. Representative enterobacteria, including, but not limited to, E. coli, S. typhi, K. pneumonia, S. aureus etc., have been known to cause diseases in the GIT (Walker et al., 2014).

Gentamicin sulphate powder is well known for its hygroscopic nature. Presenting the drug powder into granules is hindered by atmospheric moisture absorption. Due to the drug powder's hygroscopic nature, wet granulation and direct compression into tablet/capsule dosage forms become impossible. Preliminary investigation had shown that it is possible to use polyethylene glycol 4000 (PEG 4000) as a granulating aid to hold the powder particles together.

In this research, therefore, gentamicin sulphate was evaluated for suitability in the treatment of GIT localised susceptible bacterial infections. The gentamicin sulphate powder was granulated using PEG 4000 as the granulating aid. The granules were formed into different batches of capsules of 250 mg and the release profile of the formed capsules ascertained.

For a possible GIT local active dosage form, there is need to ensure that the drug is not absorbed appreciably from the GIT into the systemic circulation even when administered with potential permeation enhancers. To show that gentamicin sulphate does not get into the systemic circulation when administered PO, a sensitive bacterium was selected and used as a biomarker for the drug permeation study. Various bacteria species were incubated with 4.0 μg/ml of gentamicin sulphate. This represents the highest serum drug level after parenteral administration (CPACPS, 2002). The most sensitive bacterium being Staphylococcus aureus, as shown in Table 2, was therefore selected for the permeation studies.

It can be seen from Table 2 that all the bacteria were sensitive to gentamicin sulphate from 30 min after administration to 2 h after administration. Only B. subtilis and S. aureus were sensitive at the third hour. The S. Aureus had the highest sensitivity for all the period under review with IZD of 20, 14, 15.7 and 4 mm corresponded to the periods of 0.5, 1, 2 and 3 h, respectively.

It has been shown that gentamicin sulphate given to rats orally could appear in the blood of the rats if the administered dose is up to 10,000 mg/kg (EMEA, 2001). Hence, to ascertain the minimum concentration of gentamicin sulphate that can appear in the blood of rat after oral administration, gentamicin sulphate was administered orally to the rats at doses from 40 mg/kg and up to 1,000 mg/kg, as shown in Table 3. The oral administration was started at 40 mg/kg because this dose was preliminarily given parenterally (im) and the serum concentration after 1 h inhibited the test bacterium (S aureus). The 1,000 mg/kg corresponds to one-tenth of the orally administered dose elsewhere (EMEA, 2001). The serum collected at specified time interval after oral administration of various doses of the drug, as shown in Table 3, was used to inoculate S. aureus-seeded Mueller–Hinton agar plates. From Table 3, it can be seen that doses of 400 mg/kg gentamicin sulphate and below did not get into the blood at concentrations that inhibit bacteria growth in vitro. However, from gentamicin sulphate concentrations of 500 mg/kg and above, the drug was absorbed into the blood because the sera have concentrations that inhibited bacteria growth in vitro (Table 3).

To rule out the possibility of some permeation enhancers, at their pharmaceutically recommended usage levels aiding the drug at 400 mg/kg (and below) to cross the mucosal barriers, the drug was administered to the rats at the dose level of 400 mg/kg (using 500mg/kg as control) and in the presence of the permeation enhancers (Table 4) (Kibbe, 2000). It is quite evident from Table 4 that gentamicin sulphate concentrations of 400 mg/kg and below given orally could not permeate into the systemic circulation even in the presence of the permeation enhancers. However, at the dose of 500 mg/kg and above, the sera from rats inhibited the bacteria growth in vitro. This cannot be attributed to the permeation enhancers because similar values of IZDs were obtained in their absence (Table 4).

However formulating the drug at this dose level of 500 mg/kg, which permeates into the blood system, will correspond to giving a 70 kg body weight adult 35 g/dose as tablet of the drug; an outrageous dose. This of course indicates the suitability of the drug being evaluated as a potential drug for GIT local activity. Such evaluation was subsequently done by determining the dose of the drug. The doses of the drug were determined by administering gentamicin sulphate, as shown in Table 5. The filtrate collected from a 10% suspension of rat's droppings inhibited all the test bacteria at the following doses:14.28 mg/kg (1,000 mg/70 kg), 10.71 mg/kg (750 mg/70 kg) and 7.14 mg/kg (500 mg/70 kg) body weights. There was no activity on any of the bacteria when gentamicin sulphate was given at 3.57mg/kg (250 mg/70 kg) body weight. These doses are very much acceptable and realistic in dosage form design.

The duration of activity of gentamicin sulphate administered orally was thereafter determined by monitoring the administered drug for 18 h, as shown in Table 6. The rats were given the drug at 7.14 mg/kg (500 mg/70 kg), the lowest dose that produces activity in vitro. The filtrate from a 10% suspension of rat droppings inhibited the growth of all the bacteria up to the 8^th hour (Table 6). For all the bacteria tested, inhibition peaked at the 8^th hour. This implies that the drug should be administered orally on 8-hourly intervals. This frequency of administration will maintain sufficient drug concentration level in the GIT to limit the proliferation of bacteria. However, doses of up to 14.28 mg/kg (1,000 mg/70 kg) and 10.71 mg/kg (750 mg/70 kg), as obtained in Table 5, could be administered. It is very important to note that gentamicin sulphate given orally does not cause lesion on the GIT mucosa at these acceptable doses of 14.28 mg/kg (1,000 mg/70 kg), 10.71 mg/kg (750 mg/70 kg) and 7.14 mg/kg (500 mg/70 kg). Such mucosal damage could not be possible because higher doses of 10,000 mg/kg body weight have been administered without such damage (EMEA, 2001). Hence, important fundamental biopharmaceutical and formulation issues that will enable ushering gentamicin sulphate into usage as oral tablets and solutions for the treatment of GIT localised infections that ravage the greater population of the world are possibly addressed.

Pure gentamicin sulphate powder is known for its hygroscopic character. The pure powder absorbs moisture from the environment and becomes sticky, making the powder difficult to granulate. This presents serious formulation challenges. To overcome this challenge requires the incorporation of excipients that will retard or discourage the absorption of moisture from the atmosphere (Table 1). To this end, N-modified maize starch possesses such attribute judging from its hydrophobic nature (Nwakile, 2017). This attribute of the N-modified maize starch is expected to help limit the atmospheric moisture-absorbing ability of gentamicin sulphate granules formulated with it. To limit the atmospheric moisture-absorption potential of the gentamicin sulphate powders, some other additive like silicone dioxide (Carb-o-sil^®) was also included. Carb-o-sil^® is a well-known desiccant approved for pharmaceutical use. Other ingredients included in the formulation were Ac-di-sol^®. PEG 4000 was employed as the granulating aid because it performs better than PVP and gelatine in preliminary studies. Based on the administrable doses discussed earlier, 250 mg gentamicin sulphate granules were formed and manually packaged into capsules to a total weight of 350 mg of the gentamicin sulphate granules. Dissolution sink was tested for the presence of gentamicin sulphate by incubating the sink with S. aureus-seeded molten Mueller–Hinton agar. The inhibition zone diameter was used to deduce the concentration using the calibration curve established for the purpose. The calibration curve was established using S. aureus and pure gentamicin sulphate powder, as shown in the Figure 1.

Figure 1

As can be seen from Figure 2, the formulated gentamicin sulphate capsule released enough concentration of the drug that inhibits the test microorganism within 2 min of dissolution (Fig 2).

Figure 2

Gentamicin sulphate at dose level of 400 mg/kg and below given orally could not get into the systemic circulation even in the presence of commonly available permeation enhancers that include PEG, Tween^® 80, kolliphor^® 188, glycerine, sodium lauryl sulphate, citric acid and oleic acid. The drug at 14.28 mg/kg (1,000 mg/70 kg), 10.71 mg/kg (750 mg/70kg) and 7.14 mg/kg (500 mg/70 kg) orally administered inhibited some frequently encountered pathogenic enterobacteria. Orally administered gentamicin sulphate was found to maintain effective inhibition concentration in the GIT for 8 h pointing to an 8-hourly administration. The above-stated doses are acceptable in the dosage form design; it is possible to formulate a non-absorbable oral gentamicin sulphate dosage form for local activity in the GIT using existing conventional solid dosage formulating equipment. Such oral dosage(s) will be very important in controlling, in a very simple way, the ever debilitating GIT pathogens without recourse to drastic changes in conventional formulation pattern.

To that effect, gentamicin sulphate 250 mg granules was formulated. The gentamicin sulphate granules, which were quick drying, were packaged into capsules, which released enough concentration of the drug that inhibits the test organism within 2 min of dissolution. Oral dosage forms of gentamicin sulphate will expand the usage of gentamicin sulphate, thereby exploiting the drug's wide spectrum of activity.