Plasmogine

ABSTRACT

Plasmogine,  a  combined  extract  of  Dichroa febrifuga,  Artemisia annua and Coptis teeta   was tested for antimalarial activity against Plasmodium berghei-mouse in vivo and in vitro sensitivity against two isolates of Plasmodium falciparum in culture using standard methods. It was observed that in vivo ED₉₀ was 100 mg/kg/day and minumum schizont inhibition concentration (MIC) was found to be 50 ΅g/ml in vitro.

INTRODUCTION

Malaria remains the most important of the tropical diseases. It is widespread throughout the tropical, sub-tropical and many temperate regions of the world. Malaria is the communicable disease which infects 270 million people worldwide (WHO, 1990) and claims 2.5 million lives annually. Malaria in humans is caused by four species of protozoa parasites of genus Plasmodium, namely P.falciparum, P.vivax, P.malariae and P.ovale.

Of the four species, P.falciparum accounts for the most of the malarial infections in Africa and Southeast Asia (including Myanmar). In Myanmar, 11% of the population (~ 4.98 millions) lives in no risk area but 89 %( ~ 40.29 millions in 1994 ) are living in malaria endemic areas (VBDC  Annual  Report , 1994). According to the National  Health  Programmes of Myanmar, malaria is the first priority disease in terms of morbidity and mortality (National Health Plan, 1996 ).

The existence of many genetic variants in humans with innate resistance against malaria infection is in example of evolutionary adaptation in the host brought about by an endemic disease. While the human host is gradually adapting to the malarial parasites over an evolutionary time scale of perhaps many thousands of years, the parasites are also adapting to pressure put upon them by the host over far shorter periods through the use of antimalarial drugs. The relatively fast response of parasites  to the drug pressure is largely not understood, but probably relates to their short life cycles and particular genomic structures which allow them to undergo rapid changes.

The earliest description of drug resistance dates to 1910 when resistance of P.falciparum to quinine was reported from Brazil. Resistance to dihydrofolate reductase (DHFR) inhibitors such as pyrimethamine was noted soon after their introduction at the end of the second world war. Drug resistance become a major problem with the emergency of resistance in P. falciparum to the most potent and widely used synthetic antimalarial drug, Chloroquine. The first evidence came almost simultaneously from South American and Southeast Aisa in the late 1950’s. The story is similar for other antimalarials. For example, resistance to Mefloquine, a derivative of 4 quinoline methnol, appeared only a few years after commercial release.

At increased prevalence of multidrug resistance strains of P. falciparum continue to reduce to effectiveness of currently available antimalarial drugs. Nonetheless, in the absence of effectiveness and practical preventive measures, chemotherapy and chemoprophylaxis remain the only options for reducing morbidity and mortality in many of the malaria-endemic countries. It is obvious that P. falciparum in Myanmar has developed resistance to all kinds of blood schizonticides; the longer the half life in the circulation of the drug the sooner the parasite develops resistance to the drug. Thus t^1/2  of chloroquine, mefloquine, sulfadoxine and pyrimethamine are about a month, 3 weeks, 200 hrs and 80 hrs respectively; the shorter the t^1/2 of the drug the longer time the parasite takes to develop resistance. It is note worthy there is still only occasional reports of resistance to quinine to whose  t^1/2   is 11-16 hours. 

Therefore many scientists concentrate their works on compounds of plant origin. One of the most poten antimalarial isolated from Quinghaosu plant is artemisinine. The therapeutic effectiveness of Quinghaosu and its derivatives (sesquiterpene lactone peroxides )and their possible development in accordance with international standards have continued to be the subject of intense study. These naturally occurring peroxide  compounds have been extensively studied in China since 1970. Research on the hemisuccinyl derivative, artesunate, and the methyl ether derivative, artemether (focusing on the production methods), the  standardization of formulations and the  development of methods for pharmacokinetic and  metabolic studies is being carried  out.  

As the antimalarial activity of the Quinghaosu series of compounds appears todepend upon the 1,2,4 trixane ring structure of the basic molecule a number of  simpler compounds containing this ring have been synthesized and are now being   evaluated for efficacy against P.falciparum. They do appear to have blood schizontocidal activity.   

Another promising and locally available plant is Dichroa febrifuga ( Chang shan in Chinese, and Yin Pyar in Myanmar ). This plant is cultivated and widely grown in Pyin Oo Lwin area, Upper Myanmar. Some alkaloids have been isolated from the root of this plant and proved to be the active principle for antimalarial drug.

The third antiprotozoal herbal medicine was observed in rhizome of  Coptis teeta ( Golden thread, Khan tauk myit in Myanmar ). Coptis teeta growsin icy mountains of  Kachin State, Myanmar and India and is used in Ayurvedic medicine. The roots of the commercially important plant Coptis teeta has been examined chemically, and found to contain the alkaloid, berberine (identified by elemental analysis, U.V and I.R spectroscopy, preparation of derivatives and by conversion to the tetrahydro-base) and an unstable new alkaloid, m.p.181-182˚ in yields of 11.0 and 3.6 percent respectively. Chinese herbalist also use preparations made from Coptis teeta to relieve high fever and delirium. Modern scientific research supports many of the traditional uses of coptis including anti-protozoal effects.

Our study was conducted to test the efficacy of FAME’s  “Plasmogine”  on in vivo  rodent malaria P.berghei system and in vitro sensitivity assays with P. falciparum in the process of formulation of new antimalarial drug with higher potency and lower resistancy.

MATERIALS AND METHOD

In vivo Mouse Model

Plasmodium berghei - mouse model system as described by Peters(1970 )⁹ was used to screen the effect of the drugs. Both therapeutic and suppressive tests were done to determine the efficacy of Plasmogine.

In vitro sensitivity assay

In vitro sensitivity assay of Plasmogine was carried out with two isolates of Plasmodium falciparum in microtitre plate containing RPMI1640 (LPLF) medium using the methods described by WHO. The concentrations tested were 25, 50, 100, and 200 ΅g/ml. The desired concentrations of drug were obtained by  dissolving in the culture medium.

Extraction and purification of Plasmogine :

The extraction and purification of Plasmogine was done according to the standardized methods.

A capsule of Plasmogine contains the total alkaloids of Dichroa febrifuga, Artemisia annua, and Coptis teeta in the ratio of 1.20 gm, 0.06 gm, and 0.01 gm respectively. 

Examination of acute and sub-acute toxicity :

Acute and sub-acute toxicity tests were carried out according to the methods described by Walter Reed Army Research Institute, USA with some minor modification by Pharmacology Research Division of Department of Medical Research (Lower Myanmar).

Three  groups of  ddy mice ( 10 mice/each group ) were given oral Plasmogine 100, 200, 400, 800, 1,600 mg/kg/day  respectively for four days. No sign of acute and sub-acute toxicity were observed after eight weeks. Pathological examinations of blood and organ tissues revealed no changes compared to untreated controls. 

Sources of ingredients of Plasmogine:

Artemisia annua (Quinghaosu), Dichroa febrifuga (Chan Shan , Yin-pyar) were collected from FAME’s plantation site, Pyin Oo Lwin. Coptis teeta (Khan tauk  myit ) was locally purchased from Kachin state.

RESULTS

The effect of Plasmogine  on mice infected with Plasmodium berghei is summarized in Table (1).

Table 1.The effects of oral Plasmogine100mg/kgon the mice infectedwith P. berghei   (At day 6 after infection).

A  - Control (Infected but untreated)
B- Plasmogine 100 mg/kg/day
C- Chloroquine 20 mg/kg/day orally for 4 days
n-  Number of mice survived at day 6          
n-  Number of mice died at day 6            
N- Total number of mice inoculated with Plasmodium berghei  

The highest parasitaemia rates ( 44.24 ± 3.26% ) were recorded in control mice (infected  but  untreated) with a mortality recorded in control mice(infected but untreated) with a mortality rate of 100% on 6 ^th  day after infection. The oral Plasmogine (100 mg/kg/day  for 4 days)  was found to be effective for rodent malaria. There was no mortality in the group of mice receiving oral chloroquine (20mg/kg/day for 4 days ). 

Both the test ( suppressive and therapeutic ) showed the effectiveness of Plasmogine in ddy mice infected with Plasmodium berghei. The effective dosage was observed to be 100mg/kg/day for 4 days. 

The in vitro sensitivity assay with two isolates of Plasmodium falciparum was shown in Table 1. The in vitro schizont inhibition was recorded after 24-hr exposure to Plasmogine at the concentrations of 25, 50, 100, and 200 ΅g/ml. The In vitro schizont inhibition was marked at the concentration of 50 ΅g/ml with the average inhibition rate (mean of three experiments with two isolates of Plasmodium falciparum) of 95%.

Table 2. Mean schizont inhibition rates of Plasmodium falciparum after 24-hr exposure to Plasmogine  in vitro.

DISCUSSION

Sodium artesunate displayed potent blood schizontocidal activity against the asexual forms of P.falciparum at erythrocytic stage in cultures as well as against P. berghei in  mice and P .knowlesi in monkeys. The use of subcutaneous or intravenous route of administration will not alter the effect of artesunate on the parasites. The parasite clearance time was shorter than all other antimalarials currenty in use. Comparative  treatment with sodium artesunate and quinine dihydrochloride, both given intravenously to  P. knowlesi infected rhesus monkeys, indicated that the former was significantly better even at a dosage equivalent to one-tenth of the later. Thus, time required  for   90% parasite clearance by sodium artesunate at 3.16mg/kg was only 13.2 hours,  whilst by quinine dihydrochloride at 31.6 mg/kg was 50.5hours. In our study it was observed that the potency of the drug as evaluated against P. falciparum in cultures was also higher than that of chloroquine and quinine. 

In the early 1940s, Jang and his colleagues identified the structure of dichroine and found it to be effective against chicken malaria. Clinical trial using the water-based extract of Yin-pyar to treat patients suffering from P. falciparum or P. vivax showed a significant effective rate. The total alkaloids of Yin-pyar are 26-50 times more potent than quinine in antimalarial  activity. Among them, gamma--dichroine is the most effective, being about 100 times more potent than quinine, - beta-dichroine is the next most potent. The LD₅₀ of the total alkaloids in mice is 7.8 mg/kg (oral); the LD₅₀ of ,  - beta-dichroine is 6.6 mg/kg. 

Berberine (an alkaloid of Coptis teeta) and 50% water-soluble alcoholic extract of rhizome of Coptis teeta have been reported to be amoebicidal and trichomonadocidal by some workers from Myanmar.

According to the WHO Guidelines on combination therapy,FAME Pharmaceuticals Enterprise has initiated to produce an antimalarial herbal drug using the total alkaloids extracted from three reputed medicinal plants which process antimalarial activity as well as antiprotozoal activity. Therefore,  FAME has designated the combined extracts of total alkaloids from three reputed medicinal plants Dichroa febrifuga, Artemisia annua and Coptis teeta and registered as Plasmogine. 

In our study with Plasmogine, the effective dose was observed to be 100mg/kg /day for four days. No sign of toxicity were observed with Plasmogine even at the dosage of 1600mg/kg/day for four days. The in vitro drug sensitivity assays with P.falciparum  also revealed the effectiveness of Plasmogine with the minimum schizont inhibition of 50 ΅g/ml.

CONCLUSION

The efficacy of Plasmogine on human malaria parasites (with special emphasis on
 P.falciparum  and P.vivax infected subjects) will be carried out in near future. The Myanmar National Ethical Committee on testing of new drug is now undertaking all the necessary measures to approve the clinical trials of Plasmogine on human subjects.

Web Developer: Myanmars.NET, Yangon, Myanmar. © Copyright 2004-2006 by Fame Pharmaceuticals Co., Ltd.