ANTIMALARIAL COMPOSITION COMPRISING ARJUNETIN AND ARTEMISININ

Abstract

The present invention provides an antimalarial composition comprising arjunetin and artemisinin. The composition exhibits synergistic antimalarial effect against malaria causing Plasmodium species including Plamsodium falciparum, P berghe species. The composition comprising Arjunetin and Artemisinin demonstrated potent antimalarial activity in vivo and in vitro with better survival and low parasite burden in the blood.

Specification

Description:FIELD OF THE INVENTION
The present invention is related to an antimalarial composition comprising arjunetin and artemisinin. The antimalarial composition is effective against malaria causing species of Plasmodium, specifically P. falciparum and P. berghei.
BACKGROUND OF THE INVENTION
Malaria, a significant parasitic disease affecting humans globally, infects approximately 270 million people worldwide. Tragically, around 2 million lives are lost to malaria each year. The parasite's ability to express variant antigens on the surface of infected red blood cells allows it to evade the host's immune response, contributing to its survival. Despite a substantial scientific progress over the past two decades, malaria continues to be a worldwide burden. Ever growing resistance towards the currently available antimalarial drugs is a challenge to combat malaria. Drug resistance exists in almost all antimalarial drugs currently in use, leading to an urgent need to identify new antimalarial drugs. Plasmodium falciparum malaria still remains one of the deadliest diseases in the world. Efficient treatment is jeopardized by parasite resistance to artemisinin and its derivatives, and by poor access to treatment in endemic regions. Traditional anti-malarial remedies still offer new tracks for identifying promising antiplasmodial molecules, and a way to ensure that all people have access to care.
Medicinal plants are a promising source of new drugs to tackle this problem. Terminalia Arjuna (TA) (Family: Combretaceae), a tree native to the Indian subcontinent, is extensively used in many traditional forms of medicine for the treatment of hypertension and coronary heart diseases (Dwivedi S et al., 2014), earning it the epitaph of "Guardian of the heart". It is known to confer cardioprotective properties such as strengthening heart muscles and improving the functioning of cardiac muscles, thereby alleviating heart failure, angina and hypercholesterolemia. Stem and bark of this plant exhibit various other therapeutic properties such as expectorant, anti-dysenteric, purgative, laxative and have also been used to treat leucoderma, anemia, hyperhidrosis, asthma, and tumors (Jain, S, 2009). In addition, it has also been reported that the bark of T. arjuna also possesses good anticancer, antiviral, and antimicrobial activities. It has been hypothesized that the cardioprotective activity of TA bark extracts is likely by the activation of endogenous antioxidant molecules by triterpenes, namely arjunolic acid. plant-derived phytochemicals can offer a new direction in developing methods for disease control. The major advantage of these traditional systems of medicine such as Ayurveda, Siddha, or Unani is that, they depend on natively available flora and has been in use for treatment of almost all kinds of maladies for centuries. Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant.
Another compound, Artemisinin is derived from the (Artemisia annua), sweet wormwood plant, and is used in the treatment of malaria caused by Plasmodium falciparum. Artemisinin and its derivatives are now standard treatment worldwide for P. falciparum malaria.
Another aspect in malaria treatment is the development of resistance to known drugs such as chloroquine, mefloquine, quinine, sulfadoxine , posing a threat to malaria control and thereby leading to increased malaria morbidity and mortality. Resistance to currently available antimalarial drugs has been confirmed in some Plasmodium species, Plasmodium falciparum and P. vivax.
P. falciparum has developed resistance to nearly all currently available antimalarial drugs, including sulfadoxine/pyrimethamine, mefloquine, and quinine. While resistance to these drugs is less widespread in some regions, multi-drug resistant malaria can still have a significant impact. Recently, partial artemisinin resistance has emerged independently in parts of Southeast Asia, South America, and East Africa, affecting the effectiveness of artemisinin-based combination therapy-the primary class of antimalarials used worldwide.
The present invention provides an antimalarial composition to effective against Plasmodium species that is safe and non-toxic even at high doses. The antimalarial composition of the invention could be helpful for inhibiting resistant strains of malaria causing Plasmodium species.
SUMMARY
One aspect of the current invention is an antimalarial composition comprising arjunetin and artemisinin.
In one aspect, arjunetin is derived from Terminalia arjuna.
In one aspect, the amount of arjunetin in the composition is in the range of 0.0005-0.005% (wt/wt).
In one aspect, the amount of arjunetin in the composition is 0.001% (wt/wt). In one aspect, the amount of artemisinin in the composition is in the range of 0.005-0.015% (wt/wt). In one aspect, the amount of artemisinin in the composition is 0.010% (wt/wt). In one aspect, the composition has synergistic antimalarial activity against Plasmodium species selected from P. falciparum or P. berghei.
In one aspect, the composition comprises Dihydroartemisinin.
In one aspect, the composition is stable at room temperature for several days.
In one aspect, the antimalarial composition comprises arjunetin in the range of 0.7 to 4 µM and artemisinin in the range of 1-4 nM.
One aspect of the invention is a method of making the antimalarial composition comprising arjunetin and artemisinin wherein arjunetin is derived from the bark of Terminalia arjuna, the method comprising the step of mixing artemisinin with arjunetin wherein the amount of arjunetin is in the range of 0.7 to 4 µM and amount of artemisinin in the range of 1-4 nM.
BRIEF DESCRIPTION OF FIGURES:
Fig 1. Shows structure of Arjunetin.
Fig. 2. Shows Growth Inhibition of P. falciparum by Arjunetin (IC50: 13.4 uM).
Fig. 3. Shows In vivo efficacy of Arjunetin on P. berghei parasitemia and survival of infected mice.
Fig 4. Shows In vivo efficacy of the Formulation of Arjunetin with Artemisinin on P. berghei parasitemia in blood and survival of infected mice.
Fig 5. Shows Isobologram showing interaction between Artemisinin and Arjunetin against Plasmodium falciparum 3D7 strain.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
The current invention encompasses an antimicrobial composition comprising arjunetin and artemisinin.
Previous studies have shown that, ethanolic extract of T. arjuna bark exhibits radical scavenging anti-oxidant activity and also effectively inhibited catalase activity. Arjunetin was isolated as a bio-active compound from ethanolic bark extract apart from various other compounds such as oleanane, triterpenoids type compounds viz., oleanolic acid, arjunolic acid, arjunolitin, Of the other compounds, Arjunetin showed significant inhibition of catalase activity as compared to the other compounds. In vitro and preclinical experimental results of arjunetin against Plasmodium berghei infected mice showed arjunetin act as potential drug candidates against malarial disease and showed activity even at sub-micromolar concentration with no cytotoxicity.
ADVANTAGES OF THE INVENTION
• Demonstration of arjunetin for antimalarial activity
• Arjunetin with artemisinin showed synergistic effect on antimalarial activity
• Therapeutic effectiveness, and safety (against cell cultures studies)
• Minimal side effects.
• Absence of obvious toxicity in cell cultures studies even at high dosing
• Non-problematic synthesis with potential for scale-up
• Molecular docking and simulations studies revealed that arjunetin showed equivalent binding affinity of FDA approved antiviral drug Remdesivir.
• Antiviral properties of the composition.
DEFINITIONS:
As used herewith, the term "arjunetin" a oleanane triterpenoid compound (CAS No. 31297-79-7) found in various plants including in the bark and roots of the Terminalia arjuna tree (Family: Combretaceae), Arjunetin appears as yellow crystals and is soluble in alcohols and organic solvents, with slight solubility in water.
As used herewith, the term "artemisinin" refers to a a sesquiterpene lactone first isolated from Artemisia annua L. It can penetrate the blood-brain barrier and has no obvious toxic or side effects on the central nervous system. Artemisinin has a peroxide bridge on its chemical structure. This peroxide bridge is found to have a significant role in its antimalarial activity based on several proposed mechanisms of artemisinin efficacy
As used herewith, the term "dihydroartemisinin or dihydroqinghaosu or artenimol or DHA (CAS No. 71939-50-9) is a drug used to treat malaria.

As used herewith the term "antimalarial composition" or "antimalarial formulation" or "formulation" herewith refers to a composition effective against malaria causing Plasmodium species Plasmodium falciparum, Plasmodium berghei, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi.
As used herewith the term "IC50" the (half maximal inhibitory concentration), refers to the potency of a substance or a composition/formulation in inhibiting a specific biological or biochemical function. It quantifies how much of an inhibitory substance is needed to inhibit a given biological process or component by 50%.
As used herewith, the term "parasitemia" refers to the quantitative content of the parasite in the blood sample. Parasitemia can be computed by following formula;
% parasitemia = (parasitized RBCs/total RBCs) × 100
Parasite counts are generally performed on thick blood films. If there is a no thick film or it is damaged, a thin film count is performed. A thin film count is also performed when there are > 100 parasites in each field of the thick film, which corresponds to > 80,000 parasites/μL.
As used herewith, the term "trophozoite" refers to an activated, growing or vegetative stage in the life cycle of the parasites such as malaria caysing Plasmodium species such as Plasmodium falciparum,
EMBODIMENTS
The current invention encompasses an antimalarial composition comprising arjunetin and artemisinin.
The foregoing description of the specific embodiments willfully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such as specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications. However, all such modifications are deemed to be within the scope of the claims.
The scope of the embodiments will be ascertained by the claims to be submitted at the time of filing a complete specification.
One embodiment of the invention is an antimalarial composition comprising arjunetin and artemisinin.
In one embodiment of the invention, arjunetin is derived from Terminalia arjuna. In one embodiment of the invention, arjunetin has a purity greater than 99% .In one embodiment of the invention, arjunetin has a purity of 99.67%. In one embodiment of the invention, arjunetin is derived from the bark of Terminalia arjuna.
In one embodiment of the invention, the amount of arjunetin in the antimalarial composition is in the range of 0.0005-0.005% (wt/wt). In one embodiment of the invention,the amount of arjunetin in the composition is in the range of 0.001-0.005% (wt/wt).In one embodiment, the antimalarial composition comprises arjunetin in the range of 0.7 to 4 µM and artemisinin in the range of 1-4 nM.
In one embodiment, the antimalarial composition comprises arjunetin in the range of 0.7 to 3.3 µM and artemisinin in the range of 1-4 nM. In one embodiment, the antimalarial composition comprises arjunetin in the range 1 to 3.2 µM and artemisinin in the range of 1-2 nM. In one embodiment, the antimalarial composition comprises arjunetin in the range of 0.7 to 3.1 µM and artemisinin in the range of 1-4 nM
In one embodiment, the amount of arjunetin in the antimalarial composition is 0.001% (wt/wt).
In one embodiment of the invention, the amount of artemisinin in the antimalarial composition is in the range of 0.005-0.015% (wt/wt). In one embodiment of the invention, the amount of artemisinin in the composition is in the range of 0.002-0.015% (wt/wt). In one embodiment, the amount of artemisinin in the antimalarial composition is 0.010% (wt/wt).
In one embodiment, the antimalarial composition has synergistic antimalarial activity against plasmodium species selected from P. falciparum, P. berghei, P. vivax, P. knowlesi or P. ovale. In one embodiment, the antimalarial composition has synergistic antimalarial activity against plasmodium species selected from P. falciparum, or P. berghei.
In one embodiment, the antimalarial composition further comprises Dihydroartemisinin. In one embodiment, the amount of Dihydroartemisinin used is 2mg/kg in combination with arjunetin (10mg/kg).
In one embodiment, the antimalarial composition is stable at room temperature for several days. In one embodiment, the antimalarial composition is stable at room temperature for up to 180 days. In one embodiment, the antimalarial composition is stable at room temperature for up to 90 days.
In one embodiment, the antimalarial composition comprising arjunetin and artemisinin can be administered by orally, parenterally, rectally, intramuscular or intravenous routes.
In one embodiment, the antimalarial composition comprising arjunetin and artemisinin inhibits catalase enzyme of malaria causing Plasmodium species.
One embodiment of the invention is a method of making the antimalarial composition comprising arjunetin and artemisinin wherein arjunetin is derived from the bark of Terminalia arjuna, the method comprising the step of mixing artemisinin with arjunetin wherein the amount of arjunetin is in the range of 0.7 to 4 µM and amount of artemisinin in the range of 1-4 nM.
In one embodiment, arjunetin is mixed with artemisinin in powdered form. In one embodiment, artemisinin is mixed with arjunetin in a powdered form.
In one embodiment, the mixing of arjunetin and artemisinin is done at a temperature of 25-35°C.
In one embodiment, the antimalarial composition comprising arjunetin and artemisinin has a shelf life up to 12 months.
In one embodiment, the antimalarial composition comprising arjunetin and artemisinin has a shelf life of 12 months
In one embodiment of the invention, the method of making the antimalarial composition comprising arjunetin and artemisinin wherein arjunetin is derived from the bark of Terminalia arjuna, the method comprises the steps of;
a. Mixing artemisinin with arjunetin wherein the amount of arjunetin is in the range of 0.7 to 4 µM and amount of artemisinin in the range of 1-4 nM; and
b. Dissolving the mixture in physiological buffer.
In one embodiment, the physiological buffer is a bicarbonate or a phosphate buffer.
In one embodiment, the mixture in step a. is mixed with saline for administering the antimalarial composition,
In one embodiment, the ratio of mixture in step a to volume of saline is determined based upon body weight of a subject.
In one embodiment, the amount of mixture in step a. dissolved in saline is 0.20-0.30 mg per kg. In one embodiment, the amount of mixture in step a. dissolved in saline is 0.24 mg per kg.
In one embodiment, the antimalarial composition is administered orally, intravenously, intramuscularly, inhalably. In one embodiment, the antimalarial composition is administered orally. In one embodiment, the antimalarial composition is injected intravenously. In one embodiment, the antimalarial composition is injected in a subject infected with malaria causing Plasmodium species.
EXAMPLES
Example 1 :Growth inhibition Assay of Arjunetin against Plasmodium falciparum
Ring stage parasites of Plasmodium falciparum were treated with different concentrations of Arjunetin (0.78-100 M) for 72 hours and number of trophozoites formed were counted under the microscope following Giemsa staining. Percentage of growth inhibition was calculated with respect to untreated control. Figure 2. shows Growth Inhibition of P. falciparum by Arjunetin. The IC50 observed was 13.4uM.
Example 2: In vivo antimalarial efficacy of Arjunetin against P. berghei
P. berghei ANKA strain (ATCC, India) parasitized mouse erythrocytes (1x105) were injected intraperitoneally in Swiss albino mice. One group of mice was injected with Arjunetin (10 mg/Kg, intraperitoneal) and the other group of mice was left as untreated. Parasitemia was determined daily from Giemsa stained tail blood smears.
Figure 3 shows In vivo efficacy of Arjunetin on P. berghei parasitemia and survival of infected mice
Example 3: Formulation of Arjunetin with Artemisinin demonstrates potent in vivo antimalarial efficacy against P. berghei
P. berghei ANKA strain (ATCC, India) parasitized mouse erythrocytes (1x105) were injected intraperitoneally in five groups of Swiss albino mice (Figure 4). Parasitemia of P. berghei parasites was monitored from Giemsa-stained tail blood smears. The mice treated with the Formulation of Arjunetin with Artemisinin demonstrates better survival and low parasite burden in the blood.
Example 4: Isobologram demonstrating synergism between artemisinin and arjunetin
Isobologram demonstrating synergism between artemisinin and arjunetin For combination studies, double-fold serial dilutions up to six concentrations, were mixed in checkerboard combination to assess all possible combinations. Artemisinin and arjunetin demonstrated synergistic effect at concentrations much lower than their respective IC50s. This data suggests that arjunetin enhances the antimalarial effect of artemisinin and can be proposed for its use in combination regimens.
Table 1: Concentrations of Arjunetin and Artemisinin used in the checkerboard assay
Artemisinin (nM) Arjunetin (µM)
32 100
16 50
8 25
4 12.5
6 6.25
3 3.125
2 1.5625
1 0.78125
Table 2: List of concentrations of Arjunetin and Artemisinin combination that exhibited antimalarial synergistic effect
Arjunetin 3.1 µM + Artemisinin 2nM
Arjunetin 3.1 µM + Artemisinin 1nM
Arjunetin 1.5 µM + Artemisinin 2nM
Arjunetin 1.5 µM + Artemisinin 4nM
Arjunetin 1.5 µM + Artemisinin 1nM
Arjunetin 0.7 µM + Artemisinin 2nM
Arjunetin 0.7µM + Artemisinin 1nM
Figure 5 shows Isobologram showing interaction between artemisinin and arjunetin against Plasmodium falciparum 3D7 strain.
, Claims:1. An antimalarial composition comprising arjunetin and artemisinin.
2. The composition of claim 1 wherein arjunetin is derived from Terminalia arjuna.
3. The composition of claim 1 wherein the amount of arjunetin in the composition is in the range of 0.0005-0.005% (wt/wt).
4. The composition of claim 2 wherein the amount of arjunetin in the composition is 0.001% (wt/wt).
5. The composition of claim 1 wherein the amount of artemisinin in the composition is in the range of 0.005-0.015% (wt/wt).
6. The composition of claim 1 wherein the amount of artemisinin in the composition is 0.010% (wt/wt).
7. The composition of claim 1, wherein the composition has synergistic antimalarial activity against plasmodium species selected from P.falciparumor or P. berghei.
8. The composition of claim 1, wherein the composition comprises Dihydroartemisinin.
9. The composition of claim 1, wherein the composition is stable at room temperature for several days.
10. The composition of claim 1 the antimalarial composition comprises arjunetin in the range of 0.7 to 4 µM and artemisinin in the range of 1-4nM.
11. A method of making the antimalarial composition comprising arjunetin and artemisinin wherein arjunetin is derived from the bark of Terminalia arjuna, the method comprising the step of mixing artemisinin with arjunetin wherein the amount of arjunetin is in the range of 0.7 to 4 µM and amount of artemisinin in the range of 1-4 nM.

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