Aim: To evaluate in vitro the effectiveness of several anti-infective agents alone and in combination against Leishmania donovani . Method: A convenient stratified sampling method was used to obtain selected anti-infective agents. For individual drug samples, Half Maximal Inhibitory Concentrations (IC 50 ) were obtained using the broth dilution method. The IC 50’s of the drugs which were active against L. donovani were used as reference values to prepare drug combinations for the modified microdilution checkerboard method. Results: Five (5) out of the fifty-six (56) drugs used showed activity (inhibition of cell growth) against L. donovani cells. They include Quinine sulphate (IC50 = 0.089 μg/ml), gentamicin (IC50 = 8.1 μg/ml), amodiaquine (IC50 = 138 μg/ml) and the two standard drugs: Amphotericin B (IC50 = 6.3 μg/ml) and Pentamidine (IC50 = 25 μg/ml). The remaining fifty-one (51) drugs did not show any inhibition within the range of concentrations used (1.25 - 160 μg/ml). The drug combinations of Pentamidine/Amodiaquine, Pentamidine/ Quinine sulphate, Pentamidine/Gentamicin, Amphotericin B/Quinine Sulphate, Amphotericin B/ Gentamicin, Amodiaquine/Quinine sulphate and Amodiaquine/Gentamicin showed synergistic effects against L. donovani whereas the Amphotericin B/Amodiaquine combination was antagonistic. Notable in the results obtained was the high effectiveness of quinine sulphate in inhibiting the growth of L. donovani. Quinine sulphate, though not indicated for leishmania treatment, was more effective than the two standard drugs and has a potential of playing a significant role in the treatment of leishmaniasis. Conclusion: This study has revealed five (5) anti-infective agents that by themselves or in combinations show activity against L. donovani. Some of the drug combinations which showed synergism should further be investigated. These results have to be confirmed by in vivo studies to define their roles in leishmaniasis treatment.
Leishmaniasis is a major vector borne disease caused by the obligate intramacrophage protozoa of the genus Leishmania [
Historically, the treatment of leishmaniasis has been based on the use of pentavalent antimonial drugs. Recently, an increased incidence of emergence of antimony resistant parasite strains has demanded a shift in focus from antimony to other anti-leishmanial agents including Miltefosine, Amphotericin B, Pentamidine, Paromomycin and Sitamaquine among others [
Amphotericin B and Pentamidine are the second line alternative drugs [
Although the last century has been characterized by a drastic dropping in the mortality caused by infectious diseases, leishmaniasis still remains a dreadful menace to human health and therefore there is the need for efficient control, which requires the steady development of new, more powerful, less toxic and inexpensive drugs for alternative treatment. The search for efficacious and, at the same time, safer anti-leishmania compounds is a continual process that needs convenient, reproducible and scalable drug screening assays.
The goal of this project is to evaluate the in vitro activity of individual and combinations of anti-infective agents against Leishmania donovani. The objectives are to determine from susceptibility studies, the single anti- infective agents that show activity against L. donovani, and to obtain their fifty percent Inhibitory Concentrations (IC50’s). The information obtained will be used to prepare possible combinations of the anti-infective agents at various concentrations and the susceptibility of L. donovani to these drug combinations determined. Finally the combination indices will be calculated and used and to characterize the activity of the drug combinations as synergistic, additive or antagonistic at the various concentrations.
The test organism, Leishmania donovani (WHO strain DD8) was a gift from Dr. Neelo Singh of the Leishmania Research Society, India. Culture media, M199, Alamar blue and all other reagents used for experiments were purchased from VWR, USA.
Drug standards were obtained as gifts from the Centers for Disease Control, Atlanta, GA, USA. All other antimicrobials were purchased as tablets, capsules, or injectables from various pharmacies in Ghana and the United States of America. A total of fifty-six (56) drugs, including the reference standards were used (
ANTI-INFECTIVE AGENTS | IC50 (mg/ml) | IC50 (µg/ml) |
---|---|---|
AMPHOTERICIN B (REFERENCE) | 0.0063 | 6.3 |
PENTAMIDINE (REFERENCE) | 0.025 | 25 |
QUININE SULFATE | 0.000089 | 0.089 |
AMODIAQUINE | 0.138 | 138 |
GENTAMICIN | 0.0081 | 8.1 |
HYDROXYCHLOROQUINE | NA | NA |
ARTEMETHER | NA | NA |
LUMEFANTRINE | NA | NA |
PRIMAQUINE | NA | NA |
CEFAZOLIN | NA | NA |
CEFOTETAN | NA | NA |
CEFEPIME | NA | NA |
AMPICILLIN | NA | NA |
NAFCILLIN | NA | NA |
PENICILLIN G SODIUM | NA | NA |
FLUCLOXACILLIN | NA | NA |
AMPICILLIN/SULBACTAM | NA | NA |
POLYMIXIN B | NA | NA |
TETRACYCLINE | NA | NA |
SULFAMETHOXAZOLE/TRIMETHOPRIM | NA | NA |
SULFADOXINE/PYRIMETHAMINE | NA | NA |
CHLOROQUINE | NA | NA |
CEFOXITIN | NA | NA |
TIGECYCLINE | NA | NA |
CEFTAZIDIME | NA | NA |
RIFAMPICIN | NA | NA |
CEFUROXIME | NA | NA |
MEROPENEM | NA | NA |
DOREPENEM | NA | NA |
IMIPENEM | NA | NA |
PIPERACILLIN/TAZOBACTAM | NA | NA |
AMIKACIN | NA | NA |
TOBRAMYCIN | NA | NA |
CEFACLOR | NA | NA |
CIPROFLOXACIN | NA | NA |
LEVOFLOXACIN | NA | NA |
DOXYCYCLINE | NA | NA |
DICLOXACILLIN | NA | NA |
TETRACYCLINE | NA | NA |
AZITHROMYCIN | NA | NA |
---|---|---|
CLARITHROMYCIN | NA | NA |
ERYTHROMYCIN | NA | NA |
CLINDAMYCIN | NA | NA |
CHLORAMPHENICOL | NA | NA |
AMOXICILLIN | NA | NA |
AMOXICILLIN/CLAVULANATE | NA | NA |
CEFTRIAXONE | NA | NA |
FLUCLOXACILLIN | NA | NA |
PENICILLIN G SODIUM | NA | NA |
NITROFURANTOIN | NA | NA |
AZTREONAM | NA | NA |
OSELTAMIVIR | NA | NA |
VANCOMYCIN | NA | NA |
ISONIAZID | NA | NA |
ETHAMBUTOL | NA | NA |
PYRAZINAMIDE | NA | NA |
Working drug solutions of concentrations between 1.25 and 160 µg/ml were prepared from each stock solution of 4 mg/ml. Aliquots (100 µl) of each drug concentration were pipetted in triplicates into 96-well plates. To each of the aliquot was added 50 µl of M199 media followed by the inoculation of the wells, under aseptic conditions. 50 µl inoculum-equivalent to a McFarland turbidity standard absorbance of 0.02 and containing approximately 1.88 × 106 cells/ml of L. donovani was used. Negative control wells containing 200 µl of the media were included in the same plate. Each plate had positive control wells, which contained 50 µl of inoculum and 150 µl of media. The plates were covered and incubated at 26˚C for 24 hours, after which 10 µl alamarBlue® was added to each well and incubation continued for another 24 hours. The plates were read (λex =540nm; λem = 590 nm) after the 48-hour incubation period using a fluorescent microplate reader. Percentage inhibitions were calculated for each concentration, and a graph of percentage inhibition against log of drug concentrations was plotted to obtain the IC50 for each anti-infective agent.
Using IC50’s obtained for individual drug susceptibility tests as reference values, combinations of anti-infective agents at various concentrations were prepared and used to determine the susceptibility of L. donovani. Briefly, 50 µl of the first anti-infective of the combination was put, in increasing order of the seven (7) concentrations, into wells along the ordinate, while 50 µl of the second drug was put into wells along the abscissa (
antagonism is present if there is a reduced effect of a combination of antimicrobials observed in comparison with the effect of the most effective individual substance [
For Pentamidine/Quinine combination, the combination index (CI) was calculated as:
CI = (Conc. of Pentamidine in the well)/(IC50 of Pentamidine) + (Conc. of Quinine in the well)/(IC50 of Quinine) + {(Conc. of Pentamidine × Conc. Of Quinine)/(IC50 of Pentamidine × IC50 of Quinine)}.
This was repeated for each combination and used to characterize the activity of the drug combinations as synergistic, additive or antagonistic at the various concentrations.
Five (5) out of the fifty-six (56) drugs used showed activity (inhibition of cell growth) against L. donovani cells. This includes the two standard drugs; Amphotericin B and Pentamidine. The remaining fifty-one (51) drugs did not show any inhibition within the range of concentrations used (1.25 - 160 µg/ml) (
IC50’s obtained were 0.089, 6.3, 8.1, 25, and 138 µg/ml for Quinine Sulphate, Amphotericin B, Gentamicin, Pentamidine, and Amodiaquine respectively.
Other than testing the susceptibility of micro-organisms to individual anti-microbial agents, their susceptibility to combined anti-infectives too can be tested for. Multi-drug susceptibility tests do not only give results for susceptibility, but also qualifies the activity of the combined anti-infective agents [
The minimum inhibitory concentration (MIC) is used to measure the potency or effectiveness of an antimicrobial agent, and not necessarily the IC50 [
of inhibition of microbial growth, the agent can be said to be effective against the micro-organism. It could be inferred from the results that most of the combined anti-infectives were effective against L. donovani at the various concentrations used. Comparing the number of wells that showed activity against L. donovani to the number of wells that showed no inhibitory effect, it could be inferred that Amodiaquine/Quinine was 61.22% effective, Pentamidine/Quinine was 89.80% effective, Pentamidine/Amodiaquine was 83.67% effective and Gentamicin/Amodiaquine was 100% effective (
The results from the microdilution method also indicated that only the combination Amphotericin B/Gentam- icin showed a 100% inhibitory activity against L. donovani. The drug combination Quinine Sulphate/Amphot- ericin B gave a 98% inhibitory activity against L. donovani. The combination of Amphotericin B/Amodiaquine
Drug Combination | % Inhibition | % Synergy | % Additivity | % Antagonism | Interpretation |
---|---|---|---|---|---|
Pent/Quin | 89.80 | 89.80 | 0 | 0 | Synergy |
Pent/Amod | 83.67 | 36.73 | 14.29 | 32.65 | Synergy |
Amod/Quin | 61.22 | 46.94 | 0 | 14.28 | Synergy |
Gent/Amod | 100 | 48.98 | 10.20 | 40.82 | Synergy |
AmphoB/Quin | 98 | 83.67 | 12.25 | 2.04 | Synergy |
AmphoB/Amod | 69.94 | 25 | 2.04 | 42.9 | Antagonism |
AmphoB/Gent | 100 | 81.6 | 10.2 | 8.16 | Synergy |
Gent/Quin | 85.7 | 85.7 | 0 | 0 | Synergy |
also gave a 69.4% inhibitory activity against L. donovani while that of Gentamicin/Quinine Sulphate showed an inhibitory activity of 85.7% against L. donovani. Although the combination of Amphotericin B/Gentamicin sho- wed an activity of 100% against L. donovani, the two drugs showed a synergistic potential of 81.6% when used in their respective concentrations against L. donovani. Also 10.2% of the total number of microtiter wells sho- wed additivity whiles 8.16% of the wells showed antagonistic effect. Moreover, all of the wells with this combination showed percentage inhibitions greater than 50% (IC50) (data not shown) with the lowest percentage inhibition in this plate being 58.62% and the highest percentage inhibition being 74.31% (
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorgamism) by half. For any two agents that inhibit the same biological process, the agent with the lower IC50 value is more effective at inhibiting that biological process. IC50 was measured for drugs that showed activity against L. donovani and then compared to that obtained from standard (second line) drugs used in the treatment of the disease, Pentamidine and Amphotericin B. The lower IC50 of Quinine Sulphate than either Amphotericin B or Pentamidine is an indication that it is more effective than either drug. Gentamicin, on the other hand is less effective than Amphotericin B, but more effective than Pentamidine as indicated by the IC50’s. Finally, the IC50 of Amodiaquine shows that it was the least effective drug. A higher concentration of Amodiaquine is needed to inhibit L. donovani cell growth to the same extent as the standard drugs.
Notably, each combination of anti-infective agent had effectiveness against L. donovani. However, the aim of drug combinations is to achieve susceptibility with synergy. Drug synergy, the combined boost of drug efficacy, is a highly pursued goal of combinational drug development [
From the characterization by the modified micro-dilution checkerboard method, combinations of Amodiaquine/Quinine showed 46.94% synergy and 14.28% antagonism out of the total 61.22% effectiveness. Pentamidine/Quinine showed 89.80% synergy which was the same as the percentage of its effectiveness. Pentamidine/Amodiaquine showed 36.73% synergy, 14.29% additivity and 32.65% antagonism out of the 83.67% effectiveness. Gentamicin/Amodiaquine showed 48.98% synergy, 10.20% Additivity and 40.82% antagonism (
For a drug combination to be considered good to be developed for clinical use, it is desired that it shows synergism as well as good percentage inhibition. From the results, Amodiaquine/Quinine had percentage inhibitions ranging from 31.77% to 69.13%. Most of the inhibitions were more than 50% with just three (3) wells showing less than 50% inhibition. Pentamidine/Quinine showed percentage inhibitions ranging from 22.71% to 61.55%, with thirteen (13) wells showing less than 50% inhibition. Pentamidine/Amodiaquine showed percentage inhibitions ranging from 35.60% to 67.27%, with just one (1) well showing less than 50% inhibition. Gentamicin/ Amodiaquine showed percentage inhibitions ranging from 63.87% to 71.34%, with none of the wells showing less than 50% inhibition.
The mechanisms of action of two drugs have an effect on their overall effect in combination. Therefore for the use of the CI in characterizing the effect of two drug combinations, there is the assumption that the two agents being combined have different mechanisms of action and exhibit a dose-response relationship [
Amodiaquine is a 4-aminoquinoline similar in structure and activity to chloroquine. It has been used as both an antimalarial and an anti-inflammatory agent for more than 40 years. The mode of action of amodiaquine has not yet been determined. However, in general, 4-aminoquinoline derivatives appear to bind to nucleoproteins and inhibit DNA and RNA polymerase. High drug concentrations are found in the malaria parasite’s digestive vacuoles [
Quinine is a quinolone-containing antimalarial. These drugs are thought to act by interfering with the digestion of haemoglobin in the blood stages of the malaria life cycle. The drug diffuses down the pH gradient to accumulate in the acidic vacuole of the parasite. The high intravacuolar concentration of quinine is proposed to inhibit the polymerisation of haem. As a result, the haem which is released during haemoglobin breakdown builds up to poisonous levels, thereby killing the parasite with its own toxic waste [
Assuming that these two drugs employ these same mechanisms in L. donovani, the extreme difference in their mechanism of action could account for the greater synergy they exhibited.
Pentamidine is active against a variety of protozoal infections, including many trypanosomes. Although its mechanism of action has not yet been defined, evidence exists that the drug is concentrated in the organism by an energy-dependent high uptake system. The drug then binds to the parasite’s DNA and interferes with its synthesis of RNA, DNA, phospholipids and proteins [
Pentamidine/Amodiaquine had percentage synergism of 36.73% which was quite low. Again, assuming that the drugs employ these same mechanisms in Leishmania, it could be observed that both drugs could have some similarity in their mechanisms of action. Thus, both drugs tend to be accumulated in the parasite and eventually inhibit DNA synthesis. When two drugs have a similar effect in action but act by different mechanisms, they are expected to exhibit more of summation than synergism when combined [
Gentamicin/Amodiaquine could also have exhibited low percentage synergy, 48.98% for the same reason as Pentamidine/Amodiaquine. This is because Gentamicin is an aminoglycoside. It interferes with protein synthesis by binding to the 30S ribosomal subunits. Gentamicin has been reported to be effective in combination with Paromomycin topically for the treatment of cutaneous leishmaniasis [
From the susceptibility screening test of individual anti-microbials, Quinine Sulphate, Amodiaquine and Gentamicin showed considerable activity against L. donovani as well as to the standard drug therapies; Amphotericin B and Pentamidine. Seven of the eight drug combinations showed synergistic activity against L. donovani. This would have to be confirmed by further in vitro and in vivo studies and possibly affect treatment options for leishmaniasis.
We wish to acknowledge the immeasurable help provided by Dr. Neelo Singh for providing us with all the L. donovni parasites we used. The entire research was funded by the Office of Research, Innovation and Development (ORID) of the University of Ghana.
HenryNettey,Grace LoviaAllotey-Babington,Benoit BangaNguessan,BarimaAfrane,MustafaTagoe,AnokyeAbabio,PatienceBotchway,YvonneDarko,ClementSasu,AlexanderNyarko, (2016) Screening of Anti-Infectives against Leishmania donovani. Advances in Microbiology,06,13-22. doi: 10.4236/aim.2016.61002