In an attempt to reach better treatment of skin infections, gel formulations containing Cefotaxime (CTX) were prepared. The gel was formulated using Carbopol 934 (C934), Hydroxypropyl Methylcellulose 4000 (HPMC 4000), Carboxymethylcellulose Sodium (Na CMC), Pectin (PEC), Xanthan Gum (XG), or Guar Gum (GG). Thirteen different formulas were prepared and characterized physically in terms of color, syneresis, spreadability, pH, drug content, and rheological properties. Drug-excipients compatibility studies were confirmed by FTIR and then
Several antimicrobial agents are available in the market in different pharmaceutical dosage forms for the treatment of skin and soft tissue infections (SSTIs). These preparations are designed to be administered either orally or parenterally or topically. Topical preparations have several benefits compared with systemic therapy [
Controlled release of antibiotics at site of infection is a new strategy being employed to treat chronic infections [
Hydrogels, swollen three-dimensional networks of hydrophilic polymers held together by association bonds or cohesive forces, are of special interest in controlled release applications, because of their soft tissue biocompatibility, the ease with which drugs are dispersed in matrix, and the high degree of control achieved by selecting the physical and chemical properties of polymer network. Hydrogels have been investigated extensively for application as carriers in diffusion-controlled release devices [
Cefotaxime (CTX) sodium as an antimicrobial agent is a semisynthetic broad spectrum third-generation cephalosporin against Gram positive and Gram negative bacteria and some pseudomonal species as well as some anaerobic bacteria [
To our knowledge, there is neither marketed topical formulation of this essential group nor research study performed to investigate topical cephalosporin preparations. Based on this knowledge, it was of interest to formulate CTX as topical preparation to treat bacterial skin and soft tissue infections more efficiently.
The present study was conducted to formulate topical hydrogel formulations of CTX. Natural polymers such as Pectin (PEC), Xanthan Gum (XG), Guar Gum (GG), synthetic polymer, namely, Carbopol 934 (C934), and semisynthetic polymers such as Hydroxypropyl Methylcellulose (HPMC) and Carboxymethylcellulose Sodium (Na CMC) were used as gelling agents. Effect of propylene glycol (PG) used as a penetration enhancer on the release had been studied. Hydrogels were evaluated for their physical appearance, rheological behavior, spreadability, drug release, and antimicrobial activity. Since different microorganisms can exist on the margins of chronic wounds [
Cefotaxime (CTX) sodium and Hydroxypropyl Methylcellulose 4000 (HPMC 4000) were purchased from Sigma, USA. Xanthan Gum was purchased from Ultrafine (India). Guar Gum was purchased from Premcem Gums Ltd. (India). Sodium carboxymethyl cellulose (Na CMC) and Pectin (Pec) were obtained from Shinestu Chemical Co., France. Carbopol 934 (C934) and Triethanolamine (TEA) were purchased from Loba Chemie, Mumbai, India. Propylene glycol (PG) and sodium benzoate are obtained from Merck, Germany. Fucidin is a product of LEO Pharmaceutical products, Denmark.
FTIR spectra were performed to find out any possible drug-polymer interactions. Samples of CTX and of each polymer used (XG, GG, PEC, HPMC, Na CMC, and C934) as well as samples of their physical mixtures in the ratio of 1 : 1 were grounded separately and mixed thoroughly with potassium bromide. A total of 1 mg of each sample and 100 mg of potassium bromide (KBr) were ground uniformly and compressed to form a KBr film disc. Potassium bromide discs were prepared by compressing the powders at a pressure of 5 tones for 5 min in a hydraulic press. Data were scanned over a spectral region from 400 to 4000 cm−1 and the obtained spectra were analyzed [
Drug-loaded hydrogels were prepared according to previously demonstrated procedure [
Composition of CTX topical hydrogels (% w/w).
Ingredients | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 | F10 | F11 | F12 | F13 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTX | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | — | — |
XG | 2 | 3 | — | — | — | — | — | — | — | — | — | — | — |
GG | — | — | 2 | 3 | 4 | 5 | — | — | — | — | — | — | — |
PEC | — | — | — | — | — | — | 6 | 7 | — | — | — | — | — |
Na CMC | — | — | — | — | — | — | — | — | 4 | 7 | — | — | — |
HPMC | — | — | — | — | — | — | — | — | — | — | 3 | 4 | — |
C934 | — | — | — | — | — | — | — | — | — | — | — | — | 2 |
PG | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Sodium benzoate | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
Purified water to | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Physical properties of CTX topical gel formulations (F1–F13).
Formulations | Color | Homogeneity | Grittiness | pH | % drug content |
---|---|---|---|---|---|
F1 | Opaque, yellowish | +++ | — | 6.6 | 96 ± 0.3 |
F2 | Opaque, yellowish | +++ | — | 6.5 | 98.8 ± 0.1 |
F3 | Translucent yellowish | +++ | — | 5.9 | 95.4 ± 0.2 |
F4 | Translucent yellowish | +++ | — | 5.6 | 98.9 ± 0.3 |
F5 | Translucent yellowish | +++ | — | 5.7 | 97.5 ± 0.4 |
F6 | Translucent yellowish | +++ | — | 5.6 | 97.8 ± 0.1 |
F7 | Opaque, buff | +++ | — | 4.0 | 98 ± 0.2 |
F8 | Opaque, buff | +++ | — | 4.0 | 98.3 ± 0.1 |
F9 | Shiny, transparent, yellowish | +++ | — | 6.2 | 97.5 ± 0.2 |
F10 | Shiny, transparent, yellowish | Clumpy | — | 6.3 | 95 ± 0.1 |
F11 | Transparent | +++ | — | 5.7 | 98 ± 0.3 |
F12 | Transparent | +++ | — | 5.9 | 97.8 ± 0.4 |
F13 | Transparent | +++ | — | 6.3 | 96 ± 0.2 |
++: good; +++: very good; —: no grittiness.
To find out the mechanism of drug release, the release data was fitted in Korsmeyer-Peppas model as follows:
Kinetic study of the
Formulation code | Zero-order | First-order | Higuchi model | Korsmeyer-Peppas model |
---|---|---|---|---|
Correlation coefficient ( | ||||
F1 | 0.848 | 0.652 | 0.976 | 1.330 |
F2 | 0.913 | 0.663 | 0.990 | 0.834 |
F3 | 0.689 | 0.644 | 0.843 | 2.510 |
F4 | 0.785 | 0.603 | 0.922 | 1.597 |
F5 | 0.875 | 0.647 | 0.975 | 1.256 |
F6 | 0.881 | 0.648 | 0.978 | 1.330 |
F7 | 0.805 | 0.241 | 0.936 | 8.730 |
F8 | 0.812 | 0.179 | 0.940 | 11.15 |
F9 | 0.896 | 0.589 | 0.980 | 0.629 |
F10 | 0.927 | 0.661 | 0.993 | 0.844 |
F11 | 0.820 | 0.579 | 0.943 | 1.069 |
F12 | 0.857 | 0.655 | 0.967 | 1.421 |
F13 | 0.969 | 0.693 | 0.995 | 0.860 |
MICs of CTX and the different gel formulations were performed in cation-adjusted Müller Hinton broth (Oxoid, Hampshire, UK) (MHB) by means of microdilution broth method in accordance to National Committee for Clinical laboratory Standards [
Sensitivity of the standard strains and the clinical isolates under test to various CTX formulations was determined by Kirby-Bauer well diffusion susceptibility test described in CLSI [
Susceptibility of the bacterial strains in planktonic form was tested against different gel formulations using the modified quantitative method originally introduced by Hammond et al. [
The final result recorded represented
To evaluate the pharmaceutical potential of C934-CTX hydrogel (F13) as a novel local treatment for SSTIs, antimicrobial effect of this hydrogel was investigated in a murine surgical site infection model.
Rats of each group were intraperitoneally anesthetized with xylazine (8 mg/kg body weight) and ketamine (30 mg/kg body weight), the hair on the lower back was shaved and the skin was cleansed with antiseptic solution (10% povidone-iodine solution). A 1-cm-long, full thickness incision wound was created on the dorsal side of the rat. Approximately 1 cm of silk suture infected with either
Antibiotic treatments were started 24 h after incision and suturing. After 24 h, the animals were sacrificed and 1 cm2 tissue was cut from the wound area in order to examine the effectiveness of the formulated gel F13 for infection treatment. Equal sections of the isolated skin tissues were used for reading bacterial bioburden. Viable counts of bacteria per section were analyzed in tissue homogenate.
Quantification of viable bacteria in the homogenate was done by culturing serial 10-fold dilutions of the bacterial suspension onto nutrient agar plates. The plates were incubated at 37°C for 24 h and the organisms were quantified by counting the number of CFU/section.
Other tissue sections were further used for histological study. They were fixed with formalin and embedded in paraffin. Consequently, the sections were stained using Hematoxylin and Eosin (H&E), and then they were dried overnight. The stained tissues were microscopically examined and the most detailed and clear tissue slides were chosen [
FTIR spectrum of free CTX (Figure
FTIR spectra of CTX (a), C934 (b), and physical mixture of CTX and C934 (c).
Figures
Results of the visual inspection of the prepared CTX hydrogels are presented in Table
pH values of the prepared formulas were in the range of 5-6 which is considered acceptable to avoid skin irritation upon application [
The data presented in Table
Good spreadability is one of the criteria for gel to meet ideal qualities. It is the term expressed to denote the extent of area to which gel readily spreads on application. Therapeutic efficacy of a gel formulation also depends on its spreading value [
Spreading coefficient of different hydrogel formulations F1–F13.
It can be concluded that the prepared hydrogels fulfilled the requirement of gel-based formulations for dermatological use which should have several favorable properties such as greaseless and ease of spreadability [
It was reported that viscosity is an important physical property of topical formulations, which affects rate of drug release [
Viscosity in centipoises of different hydrogel formulations F1–F13.
The release profiles of CTX from different hydrogel formulations are shown in Figures
Drug release profiles of hydrogel formulations F1 and F2.
Drug release profiles of hydrogel formulations F3–F6.
Drug release profiles of hydrogel formulations F7–F10.
Drug release profiles of hydrogel formulations F11–F13.
The release of the drug from the prepared hydrogels was ranked in case of hydrogels containing XG in the order F1
Same results were obtained for the other prepared hydrogels. Drug release from PEC-based hydrogels, namely, F7 and F8, was very fast as more than 95% of the drug content was released within 45 min. Hydrogel F7 showed faster and higher drug release than formulation F8. In case of hydrogels containing Na CMC, the release was ranked in the order F9
It was also observed that the drug release did not depend only on hydrogels’ viscosity but it was also influenced by the type and network structure of each polymer used. For example, the viscosities of F2 (XG-based hydrogel) and F11 (HPMC-based hydrogel) are nearly in the same range (9537 and 9817 centipoises) but F2 released about 93% of its drug content within 6 h, whereas F11 released its drug content completely after 4 h. On the other hand, hydrogels F5 (GG-based hydrogel) and F12 (HPMC-based hydrogel) with a viscosity of 10788 and 10981 centipoises, respectively, both released about 95% of their drug content after 4 h. Increasing viscosity to 59042 centipoises as in case of hydrogel F10 (Na CMC-based formulation) released the same amount of the drug at the same time (4 h).
Among all hydrogel formulations, C934 (F13) showed superior sustained drug release followed by HPMC 4000 (F11) and XG (F1). C934 is a hydrophilic polyacrylic acid polymer and its carboxyl groups become highly ionized after neutralization with TEA, forming a gel due to electrostatic repulsion among charged polymer chains. Increasing pH of the prepared hydrogel to be suitable for skin application resulted in uncoiling of the polymer chains due to ionization of its carboxyl groups and subsequently forming a rigid gel [
Results of
The progressive decrease in the amount of drug released from hydrogels, as the release test proceeded, was attributed to gradual increase in concentration of the eroded polymer which increased the viscosity of the system. Consequently, the diffusion of the drug through the membrane decreased.
Different mathematical models were used to describe the kinetics of CTX hydrogels. The values of the release exponent (
The Higuchi kinetic plots were found to be fairly linear as indicated by their highest regression values. The correlation coefficients values (
The release exponents (
CTX hydrogels (F1, F11, and F13) which showed promising results as sustained release formulations were subjected to stability studies at refrigerator and ambient room conditions for 3 months. After storage for 3 months, hydrogels did not show any change in color, odor, pH, drug content, and rheological properties. Additionally, no phase separation occurred. This indicated that the drug was stable in gels even after 3 months of short term storage and the gel formulations were physically and chemically stable.
It is clear from above discussion that F1, F11, and F13 are the best formulations among all the prepared formulations; therefore, they were subjected for further investigation for microbiological activities.
SSTIs are considered as one of the serious problems worldwide, especially due to bacterial resistance. Shortage in marketed topical dosage forms containing different groups of antibiotics aroused the need of oral and parenteral applications with their accompanied side effects.
Cefotaxime (CTX) sodium as one of cephalosporin broad spectrum antibiotics has a high safety margin and was reported to be effective against different multidrug resistant pathogen as by the FDA.
In the present study, effect of the prepared CTX topical gel formulations was investigated through different microbiological techniques.
MIC (
Bacterial Strains | CTX | F13 | F11 | F1 |
---|---|---|---|---|
| 0.06 | 0.06 | 1 | 2 |
| 1 | 1 | >2 | >2 |
| 31.25 | 31.25 | 62.5 | 31.25 |
| 62.5 | 125 | 250 | 62.5 |
| 0.25 | 0.25 | 0.5 | 1 |
MRSA | 7.8 | 15.6 | 31.25 | 31.25 |
Agar well diffusion assay for antibacterial activities of different CTX gel formulations against selected pathogens.
Tested formulas showed significant results
Although the tested formulations demonstrated better effect against
Figure
Generally,
Same results were found by Anacona and Da Silva as they found that the zone of inhibition of CTX on
The action of the three topical gel formulations containing 1% CTX was evaluated against the previously selected pathogens. Total killing was noticed after 24 h by using any of the selected formulas against
Killing pattern of different CTX gel formulations against
Against isolates used, different results were obtained. An inhibition of about 4-log difference than control was obtained by F1 against
C934-CTX gel formulation (F13) showed the most bactericidal effect against both
On the other side, MRSA was almost not greatly affected by application of F11 and F1 since the difference in log number of survivors was less than 2 log. However, upon application of F13, the reduction in CFU was significant compared to control reading (>5 log).
Observed variation in formulas’ effect on the isolates may be related to the difference in nature of the three polymers used in the formulas concerning their chemical structure and viscosity. Furthermore, good spreadability of F13 formula compared to F1 and F11 can be a probable cause for better killing activity since spreadability directly affects therapeutic efficacy of the drug. The present results were consistent with previously reported work done by Dua et al. [
Killing curve for the prepared topical gel F13 (GIII) against (a)
Microscopic appearance of skin tissues sections after H&E staining, 500x. (a) Untreated skin tissues of rat after 24 h microbial inoculation with MRSA isolates. (b) Skin tissue treated with F13 gel formulation after 24 h microbial inoculation with MRSA isolates. (c) Untreated skin tissues of rat after 24 h microbial inoculation with
Untreated tissues (Figures
SSTIs are considered a problem in the field of medicine for a long time. Third-generation antibiotic, Cefotaxime (CTX), proved to be very effective against most strains of bacterial pathogens.
In the present investigation, CTX was successfully incorporated into different gel formulations. Among all gel formulations, CTX gel (F13) prepared from C934 (2%) as gel reservoir proved to be the formula of choice, showing good characteristics and controlling the drug release for long period of time. Furthermore, it showed promising antibacterial activity
CTX gel formulation F13 could be very promising and innovative topical alternative for treatment of skin infections caused by Cefotaxime-susceptible bacteria and play a vital role in drug efficiency. These findings may open new avenues for the treatment of dermal infections by local application of tailored antibiotic gel. However, further preclinical and clinical studies are recommended to support its efficiency claims in humans.
The authors declare that there are no competing interests regarding the publication of this paper.
This research project was supported by a grant from the Research Center of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University.