Management of diabetic foot ulcers: a 25% lidocaine topical cream formulation design, physicochemical and microbiological assessments

Abstract

Background

Given the importance of surgical debridement in healing of diabetic foot ulcers, effective local anaesthesia is required to manage the related extreme pain. The pharmaceutical proprietary products currently available have low concentrations and do not exceed 5% w/w local anaesthetic.

Objective

Formulation design of a lidocaine cream of 25% and assessment of the intrinsic stability.

Methods

A cream pharmaceutical form was chosen for its ability to cross the skin barrier and effectively anaesthetise the skin. The choice of cream formula is based on changes in the size of the emulsions and resistance to physical stress. Stability tests were assessed over a 6-month period in terms of physical (evaluation of oil droplets), microbiological (germ count and identification, and preservative antimicrobial efficacy) and chemical parameters (content and pH).

Results

Under the study conditions, the drug product displayed good physicochemical and microbiological stability for 6 months at 20°C and 40°C, and no degradation product was detected. Due to the systemic adverse effects of lidocaine, the pH stability guarantee the drug product tolerance along with very weak systemic passage.

Conclusions

Given the good physicochemical and microbiological stability of the drug product over 6-month period, it has been made available to the clinical unit. An average of 250 patients per year benefit from the treatment with an excellent efficacy/tolerability ratio.

Introduction

Underlying diabetic neuropathy and arteriopathy¹ cause deformities and hyperkeratosis, potentially culminating in amputation. A significant reduction in plantar pressure among other things is carried out in anticipation of disease evolution and thus its seriousness. This is achieved by removing plantar hyperkeratosis efficiently and regularly at the earliest opportunity.^{2 3} This treatment calls for effective anaesthesia on the peripheral nerves to reduce locally the sensation of pain. Lidocaine (2-(diethylamino)-N-(2,6-dimethyl phenyl acetamide) is used as a local anaesthetic that acts by reversibly inhibiting the transmission of nerve impulses. It binds to sodium channel receptors by reducing their activity and acts as a cell membrane stabiliser.⁴ It is therefore used to alleviate pain during the surgical debridement of diabetic foot ulcers. The pharmaceutical proprietary products currently available have low concentrations and do not exceed 5% w/w local anaesthetic. A new formula with a lidocaine concentration of 25% (w/w) has been considered in this context. A cream pharmaceutical form was chosen due to its ability to cross the skin barrier and effectively anaesthetise the skin.⁵

Hence, the aims of the present study are the formulation design and the determination of the physicochemical and microbiological stability of lidocaine cream. For that purpose, a stability-indicating high-performance liquid chromatography (HPLC) method was developed to assay the active substance in the presence of preservative ingredients or any degradation products. Specific emphasis was laid on the drug product tolerability.

Materials and methods

Material

Reagents and raw materials

HPLC grade acetonitrile and methanol were obtained from VWR (Fontenay Sous-Bois, France) and purified water by the Aguettant Laboratory (Lyon, France). The chemical substances, namely lidocaine hydrochloride, methyl-4-benzoate (MB), propyl-4-benzoate (PB) and diazolidinyl urea (DU) were supplied by Sigma-Aldrich (Steinheim, Germany). The pharmaceutical raw materials (Emulsan, guar gum and lidocaine hydrochloride) were purchased from Cooper (France). The culture media were supplied by Biomerieux (France), and the reference bacterial and fungal strains were provided by the BioReference Laboratory, Eurofins (Lille, France) and the American Type Culture Collection (ATCC).

Instrumentation

HPLC–UV analysis

A Dionex Ultimate 3000 LC system (DIONEX, Les Ulis, France) was used to separate lidocaine active pharmaceutical ingredient (API) in the presence of preservative ingredients or degradation products. A C18 analytical column was selected (Waters, Ireland), 250 mm × 4 mm × 5 µm, maintained at 25°C±1°C. The flow rate and injection volume were set at 1.5 mL/min and 20 µL, respectively. The detection wavelengths were defined for lidocaine and the ingredients of the preservative (MB, PB and DU) at 210, 214 and 254 nm, respectively. The nature of the mobile phases was chosen according to the compounds physicochemical properties, and mobile phases were composed of acetonitrile: phosphate buffer 0.1M (40/60) for lidocaine, MB and PB assays, and methanol: water (20/80) for DU analysis. The run was set under isocratic elution mode.

The Microscope Olympus BX51 system was used to determine the size of the emulsions, and the Heraeus Biofuge Primo R centrifuge machine was used to study the stress of the cream.

Methodology

Preformulation tests

As the purpose of this study was to develop a cream formula containing 25% lidocaine with satisfactory physicochemical and microbiological stability, several proportions of Emulsan excipient were tested. The latter is an oil/water (o/w) emulsion containing an oily phase (Vaseline oil, octyl palmitate, polyacrylamide, C13-C14 isoparaffin), an aqueous phase (demineralised water), an anionic surfactant (laureth 7), an emulsifier (propylene glycol isoceteth-3-acetate) and an antimicrobial preservative (Microcare DMP: mixture comprising DU, MB and PB). Since Emulsan is miscible with water, numerous formulations (F1–F5) were prepared by varying the hydrophilic-lipophilic balance (HLB) of the surfactant (table 1). An emulsion-stabilising agent, namely guar gum (thickening agent), was also used for the formulation tests.

Table 1.

Formulae evaluated from F1 to F5

	F1	F2	F3	F4	F5
Emulsan (g)	73	63	43	23	3
Gomme guar (g)	2	2	2	2	2
Lidocaïne (g)	25	25	25	25	25
Water (g)	0	10	30	50	70

The preliminary evaluation leading to the choice of formula was performed over 15 days and focused solely on physical stability (size of the emulsions and resistance to centrifugation).

After selecting the formula, the stability tests were carried out on three pilot batches produced in accordance with good preparation practices. The cream was packaged in 20 g impermeable, opaque tubes (30 units per batch). Each batch was divided in half and stored in the oven at 20°C±0.3°C and at 40°C±0.3°C. The new formula underwent physicochemical and microbiological tests on days 0, 7, 15, 30, 90 and 180. Each test was performed on three tubes corresponding to one tube per batch and per storage condition.

Physical stability

Appearance and resistance of the cream to centrifugation

The appearance (colour and odour) of the cream complied with the European Pharmacopoeia (2.3.4).⁶ The preparation was then subjected to physical stress: an amount of 5 g of cream was transferred in a glass phial, and then centrifuged at 3500 rpm (Heraeus Biofuge Primo R) for 10 min at 20°C (±0.2°C).

Changes in the size of the emulsions

Changes in the size of the oil droplets in the emulsion were monitored using the Olympus BX51 Microscope System (Tokyo, Japan). The glass microscope slides were prepared with approximately 10 mg of cream stained with Sudan red III, then delicately covered with a cover slip followed by the application of a drop of immersion oil. For each batch, the size of the cream emulsions was measured in triplicate on 60 droplets and then compared with that of the control on day 0.

Chemical stability

Validation protocol

Reverse phase HPLC method was developed to quantify the active substance and preservative ingredients in accordance with guide ICH Q2 (R1).⁷ Specificity was established on the basis of separations and peak purity analysed using the photodiode array detector. Linearity and accuracy were studied over 3 days at 60%–140% of the target concentration of 25 µg/mL, 10 µg/mL and 100 µg/mL for lidocaine (reconstituted form (crème) and chemical reference substance), benzoate derivatives (MB and PB) and DU, respectively. Reproducibility and repeatability were evaluated by injecting six solutions at the same target concentrations of the four products over 3 days. The limits of quantification and limits of detection were defined by considering the signal–background noise ratio of 10:1 and 3:1, respectively.

pH measurement

The pH is the critical quality attribute of the drug product ensuring the stability and the tolerance of the cream. The pH of the preparation was measured using a Mettler Toledo SevenMulti pH-metre, suitable for semisolid pH measurements. A rate of two measurements per batch and per storage condition was applied.

Quantification of the active substance and preservative ingredients

The cream (200 mg) was dissolved in 20 mL of acetonitrile (assay of lidocaine and benzoate derivatives) or taken up in 20 mL of water (DU assay), then subjected to ultrasound at 40°C (25 min) and centrifugation (3500 rpm for 10 min) procedures in succession. The supernatant was filtered through a 0.22 µm Millipore filter and then analysed by HPLC. The lidocaine assay required additional dilution to 1/100. Each batch was analysed in triplicate.

Microbiological stability

The microbiological parameters were evaluated in accordance with European Pharmacopoeia recommendations (2.6.12 and 2.6.13).^{8 9}

Media fertility and cream properties

The fertility of the culture media was assessed using two reference strains: Bacillus subtilis (ATCC 6633) and Candidas albicans (ATCC 90028) seeded on selected solid culture media (Trypticase Soy and Sabouraud). The test for specified germs and the total aerobic micro-organism count were determined by inoculating 0.1 mL of a 10 g cream suspension on Trypticase Soy and Sabouraud agars incubated at 30°C and 25°C (±0.3°C), respectively, for 7 days.

Antifungal efficacy of the preservative

The antimicrobial efficacy of the preservative was assessed using the Candidas albicans strain (ATCC 90028) and the method described in chapter 5.1.3 of the European Pharmacopoeia.¹⁰

A 10⁶/mL inoculum of Candida albicans (ATCC 90028) was prepared to evaluate the efficacy of the preservative. The cream was introduced into three sterile tubes (Falcon, BD) at the rate of 20 mL per tube. Two hundred microlitres of Candida suspension were added to two tubes and 200 µL of sterile water to the control tube. The viability of the inoculum was checked by inoculation on CHROMagar medium. The tubes were then stored in darkness at ambient temperature. One millilitre of cream was collected at different time period (2, 7, 14 and 28 days), and mixed with sterile water to obtain various dilutions of the starting suspension, that is, 1/10, 1/100, 1/1000 and 1/10000. Five hundred microlitres of each dilution was then diluted in 9 mL of water. One hundred microlitres of this final dilution was then inoculated on CHROMagar medium and incubated in an oven at 37°C for 72 hours.

This step was completed by evaluating the chemical stability of the preservative ingredients (MB, PB and DU).

Results and discussion

Formulation tests

HLB changes alter the stability of the cream. As shown in figure 1, subjected to physical stress by centrifugation, this variation manifests as instability of the cream characterised by creaming followed by sedimentation due to the variation in density between the lipophilic and hydrophilic phases. This phenomenon is described by Stokes’ law in a diluted emulsion according to the following equation:

Figure 1.

Appearance of formulae F1–F5 after centrifugation.

$${\displaystyle V_c=\frac{\mathrm{\Delta }\rho d^{2}g}{18\eta }}$$

where ${\displaystyle V_c}$ is the creaming/sedimentation rate (m/s), d is the diameter of the drop (w), g is the acceleration of gravity (m/s²), ${\displaystyle \mathrm{\Delta }\rho }$ is the difference in density (kg/m³) and ${\displaystyle \eta }$ is viscosity (Pa·s).^{11 12}

This premature destabilisation is more marked with formulae F2, F3 and F4. Furthermore, under normal storage conditions (20°C–25°C), the increased hydrophilia of the cream generates microscopic instability characterised by a difference to varying degree in the time taken to reach the emulsion sizes. The average size of the droplets was 9.4 µm, 11.7 µm, 15.3 µm, 14.0 µm and 7.3 µm for F1, F2, F3, F4 and F5, respectively. Apart from formula F1, a coalescence phenomenon appears to occur with the other formulae and is essentially related to droplet fusion (figure 2). This phenomenon is illustrated by figure 2 showing changes in the direct micelles from a spheroidal form at day zero towards an ellipsoidal form on day 7 for formulae F2, F3, F4 and F5.¹³

Figure 2.

Optical microscopic pictures of the emulsions of formulae F1–F5 over 15 days at 20°C.

Based on the above, the formula that presents good surfactant affinity for the continuous hydrophilic phase is formula F1 comprising lidocaine hydrochloride (25% w/w), guar gum as the emulsion-stabilising agent (2% w/w) and emulsan (73% w/w).^{14 15}

Optimisation of chromatographic parameters

Given the elevated octanol–water partition coefficients (LogP) of the active substance and two preservative benzoate derivatives (1.96 and 3.04, respectively, for MB and PB), a reverse phase C18 column (5 µm, 250 mm × 4 mm) was chosen for the tests.^{16 17} Separation was initially tested with variable proportions of a mobile phase comprising acetonitrile and a (0.1 M) phosphate buffer at a fixed wavelength of 210 nm. The low preservative content of the cream led to preservative quantification at wavelengths where lidocaine showed selectively an absence of absorbance. Thus, the assay of the two benzoate derivatives (MB and PB) was coupled with the lidocaine assay but detected at 254 nm. As for DU, its very low solubility in acetonitrile and methanol led us to assay it in its undiluted format only, as described by Williams et al ¹⁸ and Doi et al.¹⁹

Validation of the chromatographic method

Lidocaine (active substance alone (AS) and in the reconstitued cream (CR))

The chromatograms obtained from the analysis of samples subjected to stress conditions displayed excellent peak separation, confirmed by peak purity analysis using the photodiode array detector (figure 3). Lidocaine, as an AS and in the CR drug product responded linearly with linear correlation coefficients of 0.985 and 0.983, respectively (figure 4). Linear adjustment was validated by virtue of Fischer’s statistical test carried out with analysis of variance. Comparison of the linear regression slopes between the CR and AS confirmed the absence of a matrix effect associated with formula excipients (t_exp=0.26<t_{5% 26ddl}=2.056). The y-intercepts (a) are comparable (aAS=aCR≠0), indicating the possibility of using the active substance in the routine compilation of calibration ranges. The limits of detection and quantification, based on measuring the signal/background noise ratio, were determined at 3.10 µg/mL and 9.60 µg/mL, respectively. The method proved to be precise (repeatability CV=0.22% and reproducibility CV=4.44%) and exact, with excellent recovery (figure 4) and relative error at 5.41%.

Figure 3.

Cream component chromatograms: (A) lidocaine; (B) methyl-4-benzoate and propyl-4-benzoate; and (C) diazolidinyl urea.

Figure 4.

(A) Linearity: example of the calibration curve of lidocaine in the cream (reconstituted form) and (B) accuracy profile of lidocaine validation (acceptance criterion ±2%).

Preservatives: MB, PB and DU

In the range of the analytical procedure, linear relations were established between the areas obtained and the concentrations of three preservatives with correlation coefficients r²≥0.98. Excellent peak resolution was obtained, and validation results demonstrated suitable level of precision and accuracy to quantify these preservatives in the cream (data not shown).

Chemical stability

Content uniformity

The results of the stability tests carried out on 3 batches over 6 months highlighted slight variations in lidocaine content compared with the theoretical value (<±5%) at both 20°C and 40°C (table 2C). No degradation product was detected under such conditions.

Table 2.

Physicochemical and microbiological parameters of the cream over 6 months: (A) Physical; (B) microbiological; (C) chemical stability of lidocaine and pH variation; (D) chemical stability of preservative components

(A)	N	D0		D7		D15		D30		D90		D180
		25°C	40°C	25°C	40°C	25°C	40°C	25°C	40°C	25°C	40°C	25°C	40°C
Colour	3	White	White	White	White	White	White	White	White	White	White	White	White
Odour	3	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil
Phases separation after centrifugation	3	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil
Average size of the emulsions±SD (µm)	3	14.23± 5.5	15.23±6.2	16.03±5.6	14.85±4.9	16.37±6.3	15.14±4.9	14.08±7.1	16.17±6.2	15.49±5.4	17.13±4.4	15.42±6.8	16.36±5.9

(B)	N	D0		D7		D15		D30		D90		D180
		25°C	30°C	25°C	30°C	25°C	30°C	25°C	30°C	25°C	30°C	25°C	30°C
Total aerobic micro-organism count (colony-forming units/10 mL)	3	<5	<5	<5	<5	<5	<5	<5	<5	<5	<5	<5	<5
Specified germs (Escherichia coli, Staphyloccocus aureus and Salmonella)	3	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil	Nil

(C)	N	D0		D7		D15		D30		D90		D180
		20°C	40°C	20°C	40°C	20°C	40°C	20°C	40°C	20°C	40°C	20°C	40°C
Lidocaine (CR) (m/m±SD) (%)	3	25.5±1.6	25.5±1.6	22.8±0.8	25.5±0.6	23.9±1.8	24.7±1.9	23.1±1.4	24.3±1.1	23.8±3.4	23.3±1.7	24.9±1.9	24.2±2.1
pH variation	3	4.79	4.79	4.86	4.87	4.84	4.9	4.87	5.02	5.1	5.2	5.5	5.5

(D)	N	D0		D7		D90				D180
		20°C	40°C	20°C	40°C	20°C		40 °C		20 °C		40°C
MB (Av±SD) (µg/mL)	3	7.76±0.18	7.76±0.18	–	–	–		–		7.46 ± 1.05		7.71±0.65
PB (Av±SD) (µg/mL)	3	1.59; 5.6	1.59; 5.6	–	–	–		–		1.43 ± 0.72		1.45±1.3
DU (Av±SD) (µg/mL)	3	15.33±2.8	15.33±2.8	–	–	–		–		14.55 ± 2.33		12.69±4.67

Av, Average; CR, cream drug product; D, day,

pH variation

The pH is a crucial parameter for monitoring the stability of the cream. In fact, variations in pH are indicative of chemical reactions and provide information on the quality of the final preparation. The pH of the preparations varied slightly over 6 months at 20°C and 40°C, ranging from 4.79 to 5.50 (table 2C). These pH values are compatible with those recorded in healthy skin, namely 4.50–6.0.^{20 21} Since it is used in topical treatment, the pH of the CR (4.7–5.5) is lower than the pKa of lidocaine (7.16).²² Lidocaine is therefore quite totally present in its ionised form, since at approximatively 2 unit pH below the pKa (pH of the cream) the percentage of unionised form for lidocaine is less than 0.5%, guaranteeing weak systemic passage. In fact, only the non-ionised form is likely to cross the transmembrane barrier. In addition, the ischaemic and very limited surface treated with the cream does not favour the systemic passage of the active pharmaceutical ingredient. Moreover, Natella et al have demonstrated that the passage from the dermal to the systemic circulation of 25% lidocaine ointment is virtually undetectable.²³ However, the possibility of systemic passage of lidocaine has been reported when used concomitantly with a topical preparation with a high dose of lidocaine (30%) and using phototherapy in laser surgery.²⁴ Moreover, particular caution must also be taken in a context of hypoproteinaemia resulting in a marked decrease in plasma protein binding and in cases of renal and hepatic impairment likely to decrease elimination clearance.^25–27

Chemical stability of the preservative

The quantitative analysis of the preservative ingredients showed reproducible results at 20°C and 40°C, with residual deviations ≤±10% (table 2D).

Physical stability

As shown in table 2A, formulation F1 displayed good physical stability at 20°C and 40°C for 6 months. Indeed, no change in colour or odour was observed. Similarly, no phase separation was detected after centrifugation. These results are corroborated by the microscopic study that confirmed the repeatability and reproducibility of the size of the emulsions, disregarding any phenomenon of cream coalescence.

Microbiological stability

Media fertility and cream properties

After seeding the inocula on solid medium, the formation of a homogeneous layer was used to check the fertility of the culture media. Subsequent monitoring of cream inocula over 6 months confirmed the absence of specified germs (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella), and the total aerobic micro-organism count was below 5 CFU/10 mL of cream (table 2B).

Antifungal efficacy of the preservative

The preservative triggered logarithmic reduction of more than 4 of the fungal population for 28 days, which demonstrates its efficacy to protect the cream from any external contamination.

In addition, lidocaine possesses intrinsic antibacterial and antifungal properties that confer additional protection to the cream.^{28 29}

Conclusion

The article establishes the main quality attributes of 25% lidocaine cream for diabetic foot surgical debridement. According to the medical staff, the new formulation is much more efficient in pain control than the 5% lidocaine commercially available creams. On the whole, the proper choice of the excipients ensured the drug intrinsic stability. The cream displays a good physicochemical and microbiological stability over 6 months. The properly control of the cream pH below the pKa value of lidocaine, guaranteeing weak systemic passage and thus the tolerance of the drug product. Given the large number of patients benefiting from this cream (250 per year), the stability data have led us to apply to the regulatory agency (National Agency for Medicines and Health Products) the authorisation to produce the cream as an hospital preparation. Finally, the development of methods to assay and identify the active substance and preservative ingredients (DU, MB and PB) as well as determination of the physicochemical and microbiological stability of the cream clearly open the opportunity to anticipate the manufacturing process and ensure the drug product availability to clinical departments in a hospital context.

What this paper adds.

What is already known on this subject

• Surgical debridement of diabetic foot ulcers is mandatory.

• Effective local anaesthesia is required to manage the related extreme pain.

• Absence of appropriate drug products on the market.

What this study adds

• Design of a new formulation of a lidocaine cream of 25% for the management of diabetic foot ulcers.

• Assessment of the intrinsic stability:

  • Stability-indicating assay method was developed for API and impurities.

  • Physicochemical and microbiological stability studies were performed.

• The drug product displayed an excellent efficacy/tolerability ratio.