Su RB et al / Acta Pharmacol Sin 2003 Sep; 24 (9): 918-922

Antimalarial effect of agmatine on Plasmodium berghei K173 strain

SU Rui-Bin¹, WEI Xiao-Li², LIU Yin, LI Jin

Beijing Institute of Pharmacology and Toxicology, Beijing 100850;

²Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China

¹ Correspondences to SU Rui-Bin, PhD. Phn 86-10-6693-1621. Fax 86-10-6821-1656. E-mail Ruibinsu@yahoo.com

Received 2002-08-06 Accepted 2003-01-21

KEY WORDS agmatine; difluoromethylornithine; antimalarials; spermidine; Plasmodium berghei

ABSTRACT

AIM: To study the antimalarial effect of agmatine (Agm) on chloroquine-susceptible Plasmodium berghei K173 strain (S strain) and the P berghei K173 resistant strain (R strain). METHODS: The antimalarial effects of Agm on P berghei K173 S strain and R strain were evaluated by Peters 4-d suppression test in mice. RESULTS: Agm (12.5-200 mg/kg, ig, daily) decreased the parasitemia for both P berghei K173 S strain (IC₅₀=139 mg/kg) and R strain (IC₅₀=126 mg/kg) in mice. Subcutaneous injection (sc) of Agm (5-40 mg/kg, tid) showed relatively stronger antimalarial effect than intragastric gavage (IC₅₀=30 mg/kg ) in P berghei K173 S strain. Spermidine antagonized the antimalarial effect of Agm for P berghei K173 S strain and R strain. Agm did not reverse the chloroquine resistance of P berghei K173 S strain. dl--Difluoromethylornithine (DFMO, sc) decreased the parasitemia of P Berghei K173 S strain and this effect was antagonized by spermidine. CONCLUSION: Agm has an antimalarial effect and the mechanism is related to its inhibition of polyamine synthesis.

INTRODUCTION

Spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, and interference with polyamine biosynthesis by inhibition of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infection^[1]. Blocking spermidine and spermine synthesis in P falciparum-infected erythrocytes with irreversible inhibitors of AdoMetDC prevents the growth of the parasite in vitro. D,L--Difluoromethyl-ornithine (DFMO), an inhibitor of ODC, which can inhibit the synthesis of spermine and spermidine and influence the content of the polyamines, is able to decrease the growth rate and proliferation of Leishmania mexicana promastigote, and parasite multiplication could be resumed by addition of exogenous polyamines^[2]. Furthermore, the addition of DFMO to human P falciparum-infected red cells in continuous culture decreased parasite growth and intracellular polyamine concentrations^[3]. DFMO also blocked exoerythrocytic schizogony of Plasmodium berghei in mice and in cultured human hepatoma cells and these effects were also reversed by administration of exogenous spermidine^[4]. 1,7-Diaminoheptane, an inhibitor of deoxyhypusine synthase, which could decrease the intracellular spermidine content, inhibits the proliferation of malaria parasites of P falciparum NF54 strain in vitro; this effect could be reversed by spermidine^[5]. All these results extend previous observations that the polyamines, especially spermidine, are crucial for differentiation and proliferation of malarial parasites, thus interference with polyamine biosynthesis may be a viable chemotherapeutic target of malaria parasites.

Agmatine (Agm) is a primary amine formed by decarboxylation of L-arginine by an enzyme, L-arginine decarboxylase. Agm was a constituent of bacteria, plants and many invertebrates, and was believed to be a metabolic intermediate in the formation of spermine and spermidine^[6]. Agm is proved to inhibit NOS^[7] and ODC activity and hold back cell proliferation^[8]. Agm is also a moderate inhibitor of homospermidine synthase and suppresses the parasitemia of the chloroquine-susceptible NF54 strain and the less chloroquine-susceptible R strain in vitro^[5]. In addition, Agm is able to deplete polyamine in hepatocytes by inducing spermidine/spermine acetyltransferase^[9]. All these results inferred that Agm might antagonize the effect of spermidine on cell proliferation. In the present study we investigated the effect of Agm on P Berghei K173 strain and the Plasmodium berghei K173 resistant strain (RCQ/K173) in mice and analyzed whether this effect was related to the inhibition of polyamine synthesis.

MATERIALS AND METHODS

Animals and malarial parasites Male and female (1:1) Kunming mice [20 g±2 g, Grade II, Certificate No 01-3023, provided by the Experimental Animal Center of Academy of Military Medical Sciences (AMMS)] were used in all experiments. Animals were randomly assigned to treatment groups and maintained on a 12-h light/dark cycle and given ad libitum access to food and water, strictly in compliance with the guidelines established for the use of experimental animals by the European Community. Ten mice were used in each group, and each animal was used only for one treatment.

P berghei K173 S strain was introduced in 1983 from the Peters Laboratary of London Academy of Tropical Medicine and Hygiene. P berghei K173 R strain was cultured to be highly resistant to chloroquine by using high concentration chloroquine on P berghei K173 S strain continuously for 200 generation, index of resistance (RI) was 110.

Mice were injected with 1×10⁷ malarial parasite intraperitoneally (ip). One hour later, drugs were injected alternately for a 4-d suppression test. Four days later, tail blood was used to make thin blood slice and parasitemia was counted by microscopy. The inhibitory rate of drugs was expressed as [(Parasitemia in control group _ Parasitemia in drug treated group)/ (Parasitemia in control group)×100 %].

Drugs and administration Agm sulphate and spermidine were obtained from Sigma (St Louis, MO, USA). DFMO hydrochloride was purchased from Merck KgaA (Darmstadt, Germany). All drugs were dissolved in normal saline and freshly prepared on the experimental day. Agm was injected ig or sc. Spermidine and DFMO were given sc.

Antimalarial effect of Agm on P berghei K173 S strain Mice were ip injected with red blood cells infected by P berghei K173 strain. One hour later, the mice were continuously treated with different doses of Agm by ig (12.5-200 mg/kg, daily) or sc (5-40 mg/kg, tid) injection for 4 d. To study the influence of spermidine on the antimalarial effect of Agm, spermidine (30 mg/kg, sc, daily) was coadministered with Agm (40 mg/kg, ig).

Antimalarial effect of Agm on P berghei K173 R strain Mice were intraperitoneally injected with red blood cells infected with P berghei K173 R strain. One hour later, mice were continuously treated with Agm (12.5-200 mg/kg, ig, daily) for 4 d. To investigate whether Agm could reverse the chloroquine resistance of malarial parasites, mice were co-pretreated with Agm (100 mg/kg, ig) and chloroquine (0.22-3.5 mg/kg, ig). In another experiment, Agm (12.5-200 mg/kg, ig) was coadministered with chloroquine (1.75 mg/kg, ig). To study the influence of spermidine on the antimalarial effect of Agm, spermidine (5-60 mg/kg, sc, daily) was coadministered with Agm (100 mg/kg, ig).

Antimalarial effect of DFMO on P berghei K173 S strain Mice were ip injected with red blood cells infected by P berghei K173 S strain. One hour later, the mice were continuously treated with DFMO (10-50 mg/kg, sc, daily) for 4 d. To study the influence of spermidine on the antimalarial effect of DFMO, spermidine (30 mg/kg, sc, daily) was coadministered with DFMO (10-50 mg/kg, sc, daily). Tail blood was used to make thin blood slice and parasitemia was counted by microscopy in all antimalarial experiments.

Statistics The parasitemia in red blood cells were expressed as mean±SD. ANOVA was used to analyze the statistical significance of differences between groups. Difference was significant at P<0.05.

RESULTS

Antimalarial effect of Agm on P berghei K173 S strain In Peters 4-d suppression test, the average parasitemia of P berghei K173 S strain was 222 per 1000 red blood cells. Agm (12.5-200 mg/kg, ig, daily) decreased the parasitemia dose-dependently, the parasitemia reduced to 7 % at 200 mg/kg (n=6, P<0.01 vs control), the inhibitory rate reached 68.5 % at the dose of 200 mg/kg, and IC₅₀ was about 139 mg/kg (Tab 1). The antimalarial effect of Agm (10-40 mg/kg, sc, tid) was increased, the inhibitory rate reached 68.9 % at the dose of 40 mg/kg and the IC₅₀ was 30 mg/kg (Tab 2). These results first demonstrate that Agm had an antimalarial effect in vivo. Spermidine (30 mg/kg, sc) itself did not exhibit any antimalarial effect, however, the effect of Agm on parasitemia was inhibited significantly by coadministration of spermidine (30 mg/kg), Indicating that the effect of Agm is related to its influence on synthesis of polyamines.

Tab 1. Antimalarial effect of Agm (ig, daily) on P berghei K173 S strain in mice. n=6. Mean±SD. ^bP<0.05, ^cP<0.01 vs NS.

NS: normal saline; Agm: agmatine.

Tab 2. Antimalarial effect of Agm (sc, tid) on P berghei K173 S strain in mice and the influence of spermidine (sc, qd) on the effect of Agm. n=6. Mean±SD. ^bP<0.05 vs NS, ^eP<0.05 vs Agm 40 mg/kg.

NS: normal saline; Agm: agmatine; Spd: spermidine.

Tab 3. Antimalarial effect of Agm (ig, daily) on P berghei K173 R strain in mice. n=6. Mean±SD. ^bP<0.05 vs NS.

NS: normal saline; Agm: agmatine.

Antimalarial effect of Agm on P berghei K173 R strain Agm (12.5-200 mg/kg, ig, daily) showed a dose-dependent inhibitory effect on the proliferation of P berghei K173 R strain, the inhibitory rate reached 70.2 % at the dose of 200 mg/kg, and the IC₅₀ was 126 mg/kg (Tab 3). This effect of Agm was the same as that on P berghei K173 S strain. Chloroquine at 1.75 mg/kg had no inhibitory effect on P berghei K173 R strain. Coadministration of different doses of chloroquine (0.22-3.5 mg/kg) with Agm (100 mg/kg) did not increase the antimalarial effect of Agm (Tab 4). Agm (12.5-200 mg/kg) did not enhance the antimalarial effect of chloroquine (1.75 mg/kg) too (Tab 5). These results indicated that Agm could not reverse chloroquine resistance of malaria parasites. Spermidine (5-60 mg/kg) significantly inhibited the antimalarial effect of Agm (100 mg/kg) on P berghei K173 R strain in a dose-dependent manner (n=6, P<0.05 vs Agm 100 mg/kg; Tab 6), the inhibitory rate of Agm decreased from 44.0 % (Agm 100 mg/kg) to 8.9 % in the presence of spermidine 60 mg/kg. This result further proved that the effect of Agm was exerted through the inhibition of spermidine synthesis.

Tab 4. Influence of Agm (100 mg/kg, ig, daily) on the antimalarial effect of chloroquine (0.22-3.5 mg/kg, ig, daily) on P berghei K173 R strain in mice. n=6. Mean±SD. ^bP<0.05 vs Agm 100 mg/kg.

NS: normal saline; Agm: agmatine; CQ: chloroquine.

Tab 5. Influence of Agm (12.5-200 mg/kg, ig, daily) on the antimalarial effect of chloroquine (1.75 mg/kg, ig, daily) on P berghei K173 R strain in mice. n=6. Mean±SD. ^bP<0.05 vs CQ 1.75 mg/kg.

NS: normal saline; Agm: agmatine; CQ:chloroquine.

Tab 6. Influence of spermidine (5-60 mg/kg, sc, daily) on the antimalarial effect of Agm (100 mg/kg, ig, daily) on P berghei K173 R strain in mice. n=6. Mean±SD. ^bP<0.05 vs NS, ^eP<0.05 vs Agm 100 mg/kg.

NS: normal saline; Agm: agmatine; Spd: spermidine.

Antimalarial effect of DFMO on P berghei K173 strain DFMO (10-50 mg/kg, sc, daily) inhibited the proliferation of P berghei K173 S strain significantly, the inhibitory rate reached 73.1 % at the dose of 50 mg/kg, IC₅₀ was 19 mg/kg (n=6, P<0.05 vs control, Tab 7). Spermidine (30 mg/kg, sc) itself had no influence on parasitemia of P berghei K173 S strain, however, coadministration of spermidine with DFMO antagonized the effect of DFMO. In the presence of spermidine (30 mg/kg, sc), the inhibitory rate of DFMO was decreased from 73.1 % to 53.8 % (n=6, P<0.05 vs DFMO 50 mg/kg), indicating that the antimalarial effect of DFMO was also exerted through the inhibition of spermidine synthesis.

Tab 7. Antimalarial effect of DFMO (sc, daily) on P berghei K173 strain in mice and the influence of spermidine (sc, daily) on the effect of DFMO. n=6. Mean±SD. ^bP<0.05 vs NS; ^eP<0.05 vs DFMO 50 mg/kg group.

NS: normal saline; DFMO: D,L-a-difluoromethylornithine; Spd: spermidine.

DISCUSSION

Agm, a product of arginine decarboxylation in mammalian cells, can be metabolized by agmatinase to putrescine, and the latter can be synthesized to spermidine and spermine. Spermidine and spermine were reported to increase the activity of malarial DNA polymerase^[9] and play important roles in the translation of the DNA polymerase mRNA and regulating the cell cycle of the malarial parasite. And DFMO, an inhibitor of ODC, which can inhibit the synthesis of spermine and spermidine, inhibited proliferation of P falciparum. Therefore, spermine and spermidine are essential for the differentiation and proliferation of cells and malarial parasites, and the use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials. As far as the important role of spermidine in cell proliferation, it is possible that Agm may be important in promoting the proliferation of malarial parasites too. However, Agm is believed to govern cell polyamines by inducing antizyme which in turn suppresses ODC activity and polyamine uptake in vitro and in vivo^[10] and in addition, Agm was able to deplete polyamine in hepatocytes and inhibit cell proliferation^[11]. Thus it is also possible that Agm may have an opposite effect like spermidine, which is to inhibit the differentiation and proliferation of cells and malarial parasites. Agm was reported to inhibit the proliferation of malarial parasites of P falciparum NF54 strain in vitro^[5]. Consistent with this study, the result in our present experiment demonstrated that Agm had an antimalarial effect in mice in vivo, and spermidine antagonized the effect of Agm. This result further proved the negative effect of Agm on spermidine content.

DFMO, an irreversible inhibitor of mammalian ODC activity, caused polyamine depletion in the infected cells and inhibited synthesis of DNA in the malarial parasites grown in cultured human erythrocytes, at the same time, the growth rate of the parasites was inhibited as well^[12]. In our experiment sc injection of DFMO inhibited proliferation of malarial parasites, and this effect was also reversed by spermidine. These results inferred that both Agm and DFMO might exert their antimalarial effect through inhibition of ODC activity and spermidine synthesis.

The resistance of P berghei to many drugs is thought to correlate with the alteration of polyamine metabolism in the malarial parasites. In the mice infected with drug resistant malarial parasites, the contents of spermidine and spermine in the infected erythrocytes increased significantly, and spermidine was thought to be a very important factor that led to drug resistance^[13]. Both CQS and CQR infections produced similar changes in polyamine profiles of various tissues. An increase in spermidine level was more prominent as compared to putrescine and spermine, leading to an overall increase in spermidine/spermine ratio. This ratio is an important index of cellular proliferation^[14]. Normal erythrocytes contained traces of polyamine while the erythrocytes loaded with malarial parasites of P berghei exhibited appreciably higher polyamine levels. Again, CQS as well as CQR P berghei, exhibited qualitatively and quantitatively similar polyamine profiles thus ruling out a role of polyamines in CQ-resistance in malaria. Although spermidine was proved to inhibit the uptake of chloroquine in malarial parasites, spermidine combined with chloroquine did not influence the antimalarial effect of chloroquine in chloroquine resistant malarial parasites, thus chloroquine resistance is not merely attributed to the insufficient quantity of chloroquine in the drug resistant parasites^[15]. In the present study, Agm inhibited the proliferation of malarial parasites of P berghei K173 R strain as well as the S strain, and spermidine antagonized these effects, these results further supported that spermidine was related to the proliferation of malarial parasites. However, Agm did not reverse the effect of chloroquine on the R strain, thus spermidine is not a factor that is related to chloroquine resistance of the R strain.

In conclusion, for the first time we found that Agm had an antimalarial effect on both P berghei K173 S strain and R strain, the mechanism is through inhibition of the synthesis of polyamine especially spermidine. Spermidine is not related to chloroquine resistance of P berghei K173 R strain and Agm could not reverse chloroquine resistance of malarial parasites.

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