Antiretroviral activity of protease inhibitors against Toxoplasma gondii

Terapia antiretroviral de inibidores da protease contra Toxoplasma gondii

TECHNICAL REPORT

Antiretroviral activity of protease inhibitors against Toxoplasma gondii

Terapia antiretroviral de inibidores da protease contra Toxoplasma gondii

Lianet Monzote; Marta Rodríguez; Yenisey Alfonso; Raymundo Cox

Parasitology Department, Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba

^{Correspondence to}

SUMMARY

The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE). These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API) on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC₅₀ value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

Keywords: Toxoplasma gondii; Antiretroviral protease inhibitors; Toxoplasmic encephalitis.

RESUMEN

La introducción de la terapia antirretroviral de alta efectividad ha causada una marcada reducción en la ocurrencia y curso clínico de las infecciones parasitarias, incluyendo la toxoplasmosis encefálica (TE). Estos cambios han sido atribuidos a la restauración celular. Este estudio fue desarrollado para examinar la actividad de seis inhibidores de proteasas antirretrovirales (IPA) sobre taquizoitos de Toxoplasma gondii. Los seis IPA mostraron actividad anti-Toxoplasma, con valores de CI₅₀ entre 1.4 y 6.6 µg/mL. Futuros estudios a nivel molecular deben ser realizados, los cuales podrán delucidar si el uso de IPA pudiera beneficiar o no a los pacientes que sufren de TE.

Toxoplasma gondii is a widely distributed apicomplexan parasite of great medical importance. Most primary infections are asymptomatic, and may affect up to one-third of the human population worldwide⁷. In immunocompromised patients, especially those with acquired immunodeficiency syndrome (AIDS), reactivation of latent infection causes necrotic lesions producing life threatening encephalitis⁹. The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and clinical course of different parasitic infections, including toxoplasmic encephalitis (TE). Although these changes have been attributed to the restoration of cell-mediated immunity induced by either non-nucleoside reverse transcriptase inhibitors or protease inhibitors, in combination with at least two nucleoside reverse transcriptase inhibitors included in HAART, there are evidences that antiretroviral protease inhibitors (APIs) have a direct inhibitory effect on the proteases of some parasites⁴. Previously, experimental studies demonstrated the antiparasitic activity of APIs against Plasmodium falciparum¹⁰ and Leishmania spp¹³. Against T. gondii, the in vitro inhibition has been observed for two protease inhibitors (indinavir and nelfinavir)⁶. The aim of this study was to investigate the in vitro effects against T. gondii of six available APIs, which are used in the treatment of Cuban patients with AIDS.

Tachyzoites of the virulent T. gondii RH strain maintained through serial intraperitoneal passages in Swiss mice were used. Tachyzoites were harvested from mouse peritoneal fluids 72 h post-infection and purified by centrifugation and needle extraction in the moment of use.

Female Swiss Webster mice, with a body weight of approximately 22 to 26 g, were obtained from The National Centre of Laboratory Animals Production (CENPALAB), Cuba. The animals were kept according to "Guidelines on the Care and Use of Laboratory Animals".

Atazanavir (Bristol-Myers Squibb, USA), fosamprenavir (GlaxoSmithKline, United Kingdom), indinavir (Novatec, Havana City, Cuba), nelfinavir (CIPLA, India), ritonavir (CIPLA, India) and saquinavir (Hoffmann-La Roche SA Basilea, Switzerland) were dissolved in dimethylsulfoxide (DMSO) at final concentration of 10 mg/mL. Sulfadiazine and pyrimethamine (Ipca Laboratories Ltd, India) was used as reference drug dissolved in DMSO. The products were stored at 4 ºC.

Biological evaluation: Resident macrophages were collected from the peritoneal cavities of normal Swiss Webster mice in ice-cold RPMI 1640 medium (SIGMA, St. Louis, Mo, USA) supplemented with antibiotics, and seeded at 3 x 10⁵ cell/well in a 24 well plate and incubated for two hours at 37 ºC in 5% CO₂. Non-adherent cells were removed by washing with PBS, and then, T. gondii tachyzoites were added at a 1:3 parasite/macrophage ratio, and the cultures were incubated for one hour. Free parasites were removed by washing and the drugs were added at final concentrations between 1.25 to 10 µg/mL. The cultures were incubated for 48 h in same conditions. The cells and parasites were then fixed in absolute methanol, stained with Giemsa, and examined under light microscopy. The percentage of infected macrophage was determined by the counting of 1000 cells. The IC₅₀ value was determined from the lineal equation obtained from concentration-response curves⁸. Three replicates were carried out and the results are presented as average ± standard deviation.

Cytotoxicity assay: Resident macrophages were collected and seeded as previously described in plates of 96 wells. Dilutions of the APIs were added and the cytotoxicity was determined after three days of incubation using a colorimetric assay with 15 mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (SIGMA, St. Louis, MO, USA) and an additional incubation for four hours. The formazan crystals were dissolved by the addition of 100 mL of DMSO and the optical density was determined using an EMS Reader MF Version 2.4-0, at a test wavelength of 560 nm and a reference wavelength of 630 nm¹⁵. The 50% cytotoxic concentration (CC₅₀) was obtained from dose-response curves fit to data by means of the equation for the sigmoidal E_max model². Three replicates were carried out and the results are presented as average ± standard deviation. Selectivity indices (SI) were then calculated by dividing the CC₅₀ for peritoneal macrophage of mice by the IC₅₀ for T. gondii³.

Statistical analysis: The three values of IC₅₀ obtained from each experiment were used and compared by Mann-Whitney test (Statistical for Windows Program, Release 4.5, StatSoft, Inc. 1993). Statistical differences were when p < 0.05.

The APIs were evaluated against intracellular tachyzoites resident in peritoneal macrophage from OF-1 mice. All tested APIs demonstrated anti-Toxoplasma activity at micromolar concentrations after 48 hours (Table I). The best activity was shown by atazanavir, which had a statistically different IC₅₀ value (p < 0.05) compared with the other APIs. The compounds were toxic to mammalian host, with selectivity index from 6 to 15 and IC₅₀ values from 15.9 to 76.8 µg/mL. Sulfadiazine and pyrimethamine were active in a similar concentrations reported^3,5. Nevertheless, both compounds showed higher cytotoxicity compared with APIs, with selectivity index of 4 and 1, respectively.

Several studies concerning Toxoplasma/HIV co-infections have been published; particularly focused on diagnosis, pathogenesis and treatment. The treatment of these patients with APIs has been considered as one of the more promising studies. Currently, APIs have been also reported as potential drugs for the treatment of intracellular protozoa. All compounds studied have been used to treat HIV patients due to their potent in vitro antiviral activity. Specifically for T. gondii, all evaluated APIs exhibited in vitro activity and were selectivity better than other clinically used drugs such as sulfadiazine and pyrimethamine. Calculated IC₅₀ were similar to those previously reported by DEROUIN & SANTILLANA-HAYAT which reported that nelfinavir and ritonavir showed an IC₅₀ of 4.0 and 5.4 µg/mL, respectively⁶. The activity showed by PI, together with their cellular permeability properties could justify the further exploration of APIs as anti-Toxoplasma drugs in TE and ocular infections. In addition, available APIs can gain roles as treatment for toxoplasmic complicated infections due to availability of the drug and potential activity on resistant strains⁶.

The mode of action of APIs on parasites, including T. gondii, still needs to be clarified. Nevertheless, in protozoan parasites the aspartyl protease enzymes seem to play important roles in the life cycle and display diverse functions, including the invasion of host cells and tissues, the degradation of mediators of the immune response and the hydrolysis of host proteins for nutritional purposes^11,12. For T. gondii, seven genes coding for putative aspartic proteases have been identified with known human and apicomplexan proteases¹³. In this sense, SANTOS et al. demonstrated that nelfinavir powerfully inhibited the hydrolysis of HIV peptidase substrate by L. amazonensis extract¹²; while ANDREWS et al. reported that in vitro enzyme assays with plasmepsins II and IV from P. falciparum are both inhibited by saquinavir and ritonavir¹. Further experiments with toxoplasma enzyme are required to back up this hypothesis.

In conclusion, our results demonstrated for the first time the potential activity of APIs (atazanavir fosamprenavir, indinavir and saquinavir) against T. gondii, which have not been reported previously. In this sense, in vitro data suggest the possibility that HIV patients receiving PI therapy might also benefit from an effect against Toxoplasma due to inhibition of parasite growth, and consequently limiting the development of the TE disease. Further studies about the method of the action of APIs using T. gondii enzymes, as well as the interaction with the co-infection HIV-parasite can help to clarify the influence of API in the development of TE in AIDS patients. In addition, new strategies can be useful in designing new PI with specific activity on protozoa parasites, as well as the exploration of their potential in patients infected only with T. gondii.

Received: 16 January 2012

Accepted: 27 July 2012

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Publication Dates

• Publication in this collection
  14 Jan 2013
• Date of issue
  Feb 2013

History

• Received
  16 Jan 2012
• Accepted
  27 July 2012