Novel combination Kit for Treatment of Malaria
Field of the Invention
The present invention relates to a novel combination kit for the treatment of malaria kit
contains formulation which is useful for the prevention and/or treatment of various medical indications
associated with malaria in humans and animals. Particularly the present invention further relates to the
process for the preparation of anoral formulation containing sulfadoxine-pyrimethamine (20:1) [SPJand
a,P-arteether [ART], its pharmaceutical compositions, for the management of malaria. The present
invention also relates to a method of treatment of malaria comprising of administeringorally three
antimalarial agents sulfadoxin-pyrimethamine and a,p-arteether in a predetermined doses and in a
predetermined sequence for a period of two to five days.
Background of the Invention:
Malaria is a major health problem in tropical and subtropical countries almost 106 countries has a major
effect of it. Malaria has foremost affect on morbidity and mortality even in the twenty first century.
Approximately more than a million people die from it every year, which includes the maximum number
of children [D.R. Gwatkin, M. Guillot, P. Heuveline, The burden of disease among the global poor.
Lancet, 354 (1999) 586-589]. Malaria is caused by Plasmodium species in which most life threatening is
Plasmodium falciparum. Malaria prevalence is on the rise due to the development of multiple drug
resistant (MDR) parasites, a poor rate of discovery in antiparasitic segments and high cost of antimalarial
drugs. This has led to rising numbers of malaria-related deaths per year [R.W. Snow, C.A. Guerra, A.M.
Noor, H.Y. Myint, S.I. Hay, The global distribution of clinical episodes of Plasmodium falciparum
malaria. Nature, 434 (2005) 214-217] and highlighted the requirement of new antimalarial compounds.
Artemisinin, a sesquiterpene lactone isolated from the plant Artemesiaannuah. has come out as a ray of
hope to cure MDR parasites to cure malaria and now its derivatives are being broadly used as antimalarial
drugs. It is particularly effective against P. falciparum malaria parasites that are resistant to traditional
antimalarial drugs [V. Dhingra, K. Vishweshwar Rao, M. Lakshmi Narasu, Current status of artemisinin
and its derivatives as antimalarial drugs. Life Sci, 66 (2000) 279-300, S.R. Meshnick, Artemisinin:
mechanisms of action, resistance and toxicity, International Journal for Parasitology, 32 (2002) 1655-
1660, P.M. O'Neill, Medicinal chemistry: A worthy adversary for malaria. Nature, 430 (2004) 838-839].
The treatment by administration of artemisinin-type compounds usually are associated with other
traditional drugs [R.T. Eastman, D.A. Fidock, Artemisinin-based combination therapies: a vital tool in
efforts to eliminate malaria, Nat Rev Microbiol, 7 (2009) 864-874]. Artemisinin derivatives including
dihydroartemisinin, artesunate, artemether and a,p-Arteether [S.R. Meshnick, Artemisinin: mechanisms
of action, resistance and toxicity. International Journal for Parasitology, 32 (2002) 1655-1660] believed to
be the keystones of the treatment for P. falciparum malaria due to their high potency and rapid action.
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a,P-Arteether (ART) is an ethyl ether derivative of dihydroartemisinine and is one of the most promising
candidates for the cure of malaria. ART contain stable endoperoxide bridge, which is cleaved by the intraparasiticheme
and ultimately becomes a carbon centered free radical, which then functions as an
alkylating agent, reacting with both heme and parasitic proteins (but not DNA), resulting in antimalarial
activity. ART also possesgametocytocidal properties by inhibiting parasitetransmission which possibly
decrease the maturity of antimalarialresistance [F. Nosten, N.J. White, Artemisinin-based combination
treatment of falciparum malaria, Am J Trop Med Hyg, 77 (2007) 181-192, U. Eckstein-Ludwig, R.J.
Webb, I.D. Van Goethem, J.M. East, A.G. Lee, M. Kimura, P.M. O'Neill, P.G. Bray, S.A. Ward, S.
Krishna, Artemisinins target the SERCA of Plasmodium falciparum. Nature, 424 (2003) 957-961]. ART
has been found to reduce haemozoin development as well as haemoglobin degradation. ART has now
become the milestone in the treatment of malaria and protection of ART against the development of
resistance is a key factor in the fight against malaria. One strategy that may achieve this objectiveis the
use of ART in combinations with antimalarials having independent modes of action. The concept of
combination drug therapy is based on the synergistic or additive potential of two or more drugs to
improve therapeutic efficacy and also delay the development of resistance to the individual components
of combination. This concept that resistance could be delayed or prevented by combining drugs with
different targets was first developed in the treatment of tuberculosis [J. Grosset, Bacteriologic basis of
short-course chemotherapy for tuberculosis, Clin Chest Med, 1 (1980) 231-241] and has been adopted
widely for the treatment of HIV, leprosy and cancer. Artemisinin-based drug combinations have been
proposed as an option for treatment of MDR malaria [N.J. White, P.L. Olliaro, Strategies for the
prevention of antimalarial drug resistance: Rationale for combination chemotherapy for malaria.
Parasitology today (Personal ed.), 12 (1996) 399-401]. The artemisinin derivatives are currently the most
potent antimalarials and to date no clinical resistance has been documented but for few reduced efficacy
reports of artesunate has been noticed in Pallin region. The basis for adopting artemisinin-based drug
combinations is the ability of the artemisinin to reduce significantly the initial parasite biomass, leaving
only a small residual number of parasites to be eliminated by high concentrations of the companion drug.
This minimizes the risk of parasite exposure to suboptimal antimalarial drug levels thus reducing the risk
of selection of resistant strains [N. White, Antimalarial drug resistance and combination chemotherapy,
Philos Trans R Soc Lond B Biol Sci, 354 (1999) 739-749]. Furthermore, because the artemisinin
derivatives reduce gametocyte carriage, its combination might have a lead role in the reduction of malaria
transmission [R.N. Price, F. Nosten, C. Luxemburger, F.O. ter Kuile, L. Paiphun, T.
Chongsuphajaisiddhi, N.J. White, Effects of artemisinin derivatives on malaria transmissibility. Lancet,
347(1996)1654-1658].
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Reference may be made to South African Patent No. 2007/06835 dated 29.05.2007 and US patent
application no 2006/0141024 (Synergistic combination kits of a,P-arteether, sulfadoxin and
pyrimethamine for the treatment of severe/multi-drug resistant cerebral malaria. R. Tripathi, S. K.Puri, J.
S. Srivastav, S. Singh, O. P.Asthana and A. K. Dwivedi) wherein it was reported earlier by inventor's
group that a combination kit containing injectable preparation of a,P-arteether and oral preparation of
sulfadoxine-pyrimethamine (20:1) is effective for treatment of chloroquine resistant P. falciparum
malaria.
Fansidar, a synergistic combination of sulfadoxine-pyrimethamine (20:1) (SP) is currently
advocated as the first line drug for treatment of chloroquine resistant P. falciparum malaria. However,
recent reports on the clinical failure with SP are likely to compromise its large scale application,
particularly in the Asian and African continent [B.R. Ogutu, B.L. Smoak, R.W. Nduati, D.A. Mbori-
Ngacha, F. Mwathe, G.D. Shanks, The efficacy of pyrimethamine-sulfadoxine (Fansidar) in the treatment
of uncomplicated Plasmodium falciparum malaria in Kenyan children, Trans R Soc Trop Med Hyg, 94
(2000) 83-84;A.M. Ronn, H.A. Msangeni, J. Mhina, W.H. Wernsdorfer, I.C. Bygbjerg, High level of
resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania, Trans R Soc
Trop Med Hyg, 90 (1996) 179-181]. It has also been highlighted as emerging public health disaster facing
sub-Saharan Africa if P. falciparum develops resistance to SP as quickly as it did in south-east Asia and
South America [C.H. Sibley, J.E. Hyde, P.F.G. Sims, C.V. Plowe, J.G. Kublin, E.K. Mberu, A.F.
Cowman, P.A. Winstanley, W.M. Watkins, A.M. Nzila, Pyrimethamine-sulfadoxine resistance in
Plasmodium falciparum: what next?. Trends in Parasitology, 17 (2001) 570-571]. Although alternate antifolate
combinations like chlorproguanil-dapsone are being explored, there is an urgent need to add a third
drug to SP combination to prolong its useful therapeutic life. In areas where there is currently little SP
resistance, early use of combination regimens will increase the chance of preventing the spread of
resistance. In such areas, it is therefore important to establish combination therapy.
The combination of an artemisinin derivative with SP will delay or prevent the emergence of
resistance to SP. Artemisinin derivatives achieve substantial and rapid decreases in parasite load but have
a short half-life; combination with a longer-acting drug, such as SP, which acts on a different target,
protects against the emergence of artemisinin-resistant parasites[N.J. White, F. Nosten, S. Looareesuwan,
W.M. Watkins, K. Marsh, R.W. Snow, G. Kokwaro, J. Ouma, T.T. Hien, M.E. Molyneux, T.E. Taylor,
C.I. Newboid, T.K. Ruebush, 2nd, M. Danis, B.M. Greenwood, R.M. Anderson, P. Olliaro, Averting a
malaria disaster. Lancet, 353 (1999) 1965-1967]. However, some important shortcomings of artemisinins,
including short half-life usually between 3 h and 5 h, poor aqueous solubility, and thus low oral
bioavailability (~40%) is the limiting step for it [F.I.D. Konotey-Ahulu, Averting a malaria disaster. The
Lancet, 354 (1999) 258] but fortunately arteether (ART) has the half life of 23-30h. Therefore this is the
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artemisinin derivative with longest half life.ARTis only offered as oily formulations for intramuscular
(i.m.) injection, which demands sterile dosage forms and a qualified health workforce. In addition,
intramuscular administration is not suitable to deliver the antimalarial drug for the treatment of cerebral
malaria, or when quickly suppression of the parasite is required [Q.-G. Li, J.O. Peggins, L.L.
Fleckenstein, K. Masonic, M.H. Heiffer, T.G. Brewer, The Pharmacokinetics and Bioavailability of
Dihydroartemisinin, Arteether, Artemether, Artesunic Acid and Artelinic Acid in Rats, Journal of
Pharmacy and Pharmacology, 50 (1998) 173-182]. Developing novel approaches to administer these
derivatives by oral route would be precious in overcoming their therapeutic limitations.
SMEDDS(self-microemuisifying drug delivery system)
SMEDDS are lipid based formulations which offer the potential for enhancing the absorption of poorly
soluble drugs including ART by self-emulsifying themselves when they come in contact with the aqueous
phase. The superior performance of SMEDDS may be accredited to the formation of the fine emulsion
droplets and subsequent lipolysis and formation of mixed micelles providing larger surface area for the
absorption of the drug. SMEDDS increases the mucosal permeability due to the presence of surfactants
and improves the lymphatic absorption of the drug due to the long chain oil present in it. These SMEDDS
also provide the stability to the drug due to the absence of the aqueous phase related degradation.
SMEDDS of some of the active pharmaceutical ingredients are also available commercially, which
include cyclosporine, ritonavir,saquinavir,amprenavir and valsartan[T.R. Kommuru, B. Gurley, M.A.
Khan, I.K. Reddy, Self-emulsifying drug delivery systems (SEDDS) of coenzyme QIO: formulation
development and bioavailability assessment, Int J Pharm, 212 (2001) 233-246; Preparation and
bioavailability assessment of smedds containing valsartan, Adhvait R. Dixit, Sadhana J. Rajput, Samir G.
Patel: AAPS PharmSciTech, 2010, 11, 314-321]. Mandawgadeet.al. developed a self-microemulsifying
drug delivery systems using natural lipophile (N-LCT) as an oily phase and commercially available
modified oil (Capryol 90).[Application to P-artemether delivery, S. D. Mandawgade; S. Sharma; S.
Pathak; V. B. Patravale, International Journal of Pharmaceutics 2008, 362 (1-2), 179-183].
Objects of the present invention
The main object of this invention is to provide a novel combination kit for treatment of malaria for a
period of one to five days schedule.
Further object of this invention is to provide a SMEDDS formulation containing a/p arteether sulfadoxine
and pyrimethamine.
Yet another objective of the present invention is to provide a process of preparation of above mentioned
combination kit for the management of various medical indications associated with malaria in humans
and animal such as P. falciparum, P. vivax, MDR P. falciparum, XDR P. falciparum, MDR- P. vivax,
XDR P. vivax.
ifc
Yet another object of this invention is to provide a combined dosage form for sulfadoxine-pyrimethamine
and a,p arteether in single formulation.
Further object of this invention is to provide a formulation with easy route of administration, where the
dose will be given orally.
Further object of this invention is to provide a formulation of a,P-Arteether, sulfadoxin and
pyrimethamine with reduced toxicity.
Summary of the invention:
Accordingly, the present invention provides a combination kit for the treatment of multidrug resistant
malaria for a period of one to five days schedule wherein the kit comprising;
a. a synergistic single oral formulation of anti-malarial agents containing a/p arteether,
sulfadoxine and pyrimethamine in the form of SMEDDS;
b. an oral formulation containing a/p arteether in the form of SMEDDS or containing a/p
arteether with one tenth dose of sulfadoxine and pyrimethamine as mentioned in step a in the
form of SMEDDS;
c. instruction manual for the administration of the three antimalarial drugs.
In an embodiment of the present invention wherein single oral formulation in the form of SMEDDS
comprising a/p arteether in the ratio ranging between 15 to 250, sulfadoxine in the ratio ranging
between 47.6 to 500 and pyrimethamine in the ratio ranging between 2.4 to 25.along with the
pharmaceutically acceptable diluents, surfactants, co-surfactants.
In an embodiment of the present invention wherein oral formulation containsa/p arteether in the form
of SMEDDS comprising a/p arteether along with the pharmaceutically acceptable diluents, surfactants,
co-surfactants wherein the ratio of a and P-arteether is 30±5:70±5.
In yet another embodiment of the present invention wherein the instruction manual for administering
anti-malarial agents for one to five days treatment comprising:
i) instructions for administration of a synergistic oral SMEDDS formulation of anti-malarial
agents containing a,P arteether, sulfadoxine and pyrimethamine on day one;
ii) instructions for administration of oral SMEDDS formulation containing a,P arteether or
containing a/p arteether with one tenth dose of sulfadoxine and pyrimethamine (as
mentioned above in step i) per day in predetermined doses and predetermined dose
schedule for next two to five day therapy.
In one more embodiment of the invention wherein the process for preparation of a synergistic single
oral SMEDDS formulation comprising the steps:
(^
1) a,P-arteether, sulfadoxine and pyrimethamine are dispersed in alcohol followed by the
addition of natural glycerides or semi-synthetic glycerides and surfactant under agitation at
200-1000 rpm for a 2-60 minutes at 10-40° C until homogenization,
ii) the final mixture is vortexed vigorously for 2-60 min to achieve complete mixing and
homogenized the product until a clear solution of SMEDDS is obtained,
Hi) examining the SMEDDS for any signs of turbidity or phase separation prior to selfemulsification
and globule size studies, and equilibrating the SMEDDS to ambient
temperature for 2-50 hr and then stored at room temperature.
In one more embodiment of the invention wherein the process for preparation of an oral SMEDDS
formulation of a,P arteether comprising the steps:
I. a,P-arteether is dispersed in alcohol followed by the addition of natural glycerides or semisynthetic
glycerides and surfactant under agitation at 200-1000 rpm for 2-60 minutes at 10-
40° C until homogenization,
II. the final mixture is vortexed vigorously for 2-60 min to achieve complete mixing and
homogenized the product until a clear solution of SMEDDS is obtained,
III. examining the SMEDDS for any signs of turbidity or phase separation prior to selfemulsification
and globule size studies, and equilibrating the SMEDDS to ambient
temperature for 2-50hr and then stored at room temperature.
In an embodiment of the present invention wherein the solvent used in the preparation of the
formulation is selected from a group of primary or secondary aliphatic alcohols such as ethanol, 2-
propanol.
In an embodiment of the present invention wherein the diluent used is anatural glycerides or semisynthetic
glycerides and is selected from a group consisting of naturally occurring oil, synthetic source or
combination thereof such as Coconut oil. Corn oil. Cottonseed oil, Olive oil. Palm oil. Peanut
oil (Ground nut oil), Rapeseed oil, Safflower oil, Sesame oil, Soybean oil, Captex 355 (Glyceryl
Tricaorylate/Caprate), Captex 200 (Propylene Dicaprylate/Dicaprate Glycol), Captex 8000 (Glyceryl
Tricaprylate(Tricaprylin)), Witepsol (90:10 % w/w cl2 Glyceride tri: diesters), Myritol 318 (c8/clO
triglycerides) Isopropyl myristate (Myristic acid isopropyl ester).
In an embodiment of the present invention wherein the surfactants used in the preparation is the
generally regarded as safe (GRAS) is selected from a group consisting of Tween 20, Tween 40, Tween
60, Tween 65, Tween 80, Tween 85, Span 40, Span 60, Span65, Span 80, PEG 200, PEG 300, PEG 400,
PEG 600, PEG 1500, PEG 6000, Pluronic F-68, Pluronic F-127, PLUROL, PVA, BRIJ 35, BRIJ 56,
Carbopol 934, Carbopol 940, Cremophor EL, Labrafac, Labrosol, Lauroglycol 90, Sodium Dodecyl
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Sulphate, Sodium Deoxycholate, POE 40 Stearate, POE 25 Propylene Glycol Stearate, POE 20 Oleyl
Ether, Transcutol, Gelucire 44/14, Gelucire 34.
In an embodiment of the present invention wherein the co-surfactants used in the preparation is
generally regarded as safe (GRAS) and is selected from a group consisting of Transcutol, glycerin,
ethylene glycol, propylene glycol, ethanol, propanol.
A combination kit is useful for the treatment of malaria including malaria caused by P. falciparum or
P. Vivax administration of a SMEDDS formulation to a subject in need containing 15 to 250 mg of a/p
arteether 47.6 to 500 mg of sulfadoxine and 2.4 to 25 mg of pyrimethamine on day one and SMEDDS
formulation containing 15 to 250 mg of a/p arteether per day in predetermined doses and predetermined
dose schedule for next two to five day therapy.
Detailed description of the invention:
The novel combination therapy of the present invention comprising the use of a,P-arteether in
combination with sulfadoxine and pyrimethamine has distinct advantages in terms of improved
effectiveness in the case of multi-drug resistant P. falciparum malaria because of the synergistic
antimalarial activity of both the drugs. Thus the use of the present invention, containing a,P-arteether,
sulfadoxine and pyrimethamine becomes a very useful treatment from the stand point of its effectiveness
in the case of multi-drug resistant P. falciparum malaria/cerebral malaria.
In the present investigation, we have developed SMEDDS formulation of combination of three drugs
sulfadoxine-pyrimethamine (20:1) (SP) and a,P arteether(ART) by using different polymers and
surfactant. ART in combination with SP can be given orally for effective treatment of malaria and can
reduce the effective dose of individual drug for the treatment of MDR malaria by increasing its
bioavailability without causing any toxicity or reducing the toxicity associated with arteether and SP if
there any. We aimed to design ART-SP loaded SMEDDS that could self-emulsify spontaneously when
came in contact with a physiological medium. ART has been reported for poor absorption when given as
in aqueous solution with a base line in mind that when ART is given with the fat rich diet, it has an
improved absorption. Hence, the lipid-based SMEDDS were designed based on the literature to enhance
the solubility and thus bioavailability of ART and to obtain self-emulsifying properties with the selected
excipients. The choice of the ratio between the various excipients required (oil, surfactant, co-surfactant
and co-solvent) was guided mainly by the "Lipid Formulation Classification System" [C.H. Sibley, J.E.
Hyde, P.F.G. Sims, C.V. Plowe, J.G. Kublin, E.K. Mberu, A.F. Cowman, P.A. Winstanley, W.M.
Watkins, A.M. Nzila, Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?.
Trends in Parasitology, 17 (2001) 570-571, N.J. White, F. Nosten, S. Looareesuwan, W.M. Watkins, K.
Marsh, R.W. Snow, G. Kokwaro, J. Ouma, T.T. Hien, M.E. Molyneux, T.E. Taylor, C.I. Newbold, T.K.
Ruebush, 2nd, M. Danis, B.M. Greenwood, R.M. Anderson, P. Olliaro, Averting a malaria disaster,
8
^
Lancet, 353 (1999) 1965-1967], which shows better characteristics and properties for formulations of type
111 A. Hence, the lipid-based formulations were designed based on the literature to enhance the solubility
of ART and SP to obtain self-emulsifying properties with the selected excipients. Four subgroups of
excipients were included in the composition of our lipid-based formulations that contained 27.5% natural
glycerides (sesame oil, groundnut oil or soybean oil), 27.5% semi-synthetic glycerides (Maisine 35-1 or
Labrafils), 35% surfactant (Cremophor EL or Tween 80) and 10% co-solvent (ethanol), or 30% natural
glycerides, 30%) semi-synthetic glycerides, 30% surfactant and 10%) co-solvent. Thirty-six formulations
were identified as clear oil-surfactant-co-solvent mixtures in the absence and the presence of ART.
Following aqueous dilution at a formulation/water ratio of 1:250 (w/v), (almost) clear and fine (micro)
emulsions were spontaneously formed by gentle agitation. The solubility of ART in the lipid-based
formulations was evaluated. ART was solubilized up to 200±10 mg/g for all lipid formulations tested.
The solubility of ART in the ethanol-free lipid-based formulations was reduced to 130 ± 10 mg/g, which
is consistent with results previously reported by Pouton [C.W. Pouton, C.J. Porter, Formulation of lipidbased
delivery systems for oral administration: materials, methods and strategies, Adv Drug Deliv Rev,
60 (2008) 625-637, C.W. Pouton, Formulation of poorly water-soluble drugs for oral administration:
physicochemical and physiological issues and the lipid formulation classification system, Eur J Pharm
Sci, 29(2006)278-287].
Several formulations were identified as clear oil-surfactant-co-solvent mixtures in the absence and the
presence of ART and SP. Following aqueous dilution at a formulation/water ratio of 1:100 (w/v), (almost)
clear and fine (micro) emulsions were spontaneously formed by gentle agitation.
We aimed to design a combination formulation of sulfadoxine, pyrimethamine and ART that can selfemulsify
spontaneously when it comesin contact with the gastro-intestinal fluids. For the preparation of
ART-sulfadoxine and pyrimethamine lipid-based combination formulations, ART, sulfadoxine and
pyrimethamine were dispersed in ethanol followed by the addition of natural glycerides (sesame oil,
groundnut oil or soybean oil), semi-synthetic glycerides (Maisine 35-1 or Labrafils, Labrafac) and the
surfactants (Cremophor EL and span 80) under agitation at 500 rpm for a few minutes at room
temperature until homogenization. A series of SMEDDS were prepared as a mixture at ambient
temperature. The final mixture was vortexed vigorously for 20 min to achieve complete mixing and
homogenized the product until a clear solution was obtained. The SMEDDS were examined for any signs
of turbidity or phase separation prior to self-emulsification and globule size studies. These SMEDDS
were equilibrated to ambient temperature for 24 h and then stored at room temperature.
To determine the feasibility of the self-(micro) emulsification, each mixture (1 g) was then slowly
titrated with distilled water (250 ml, 37''C), gently stirred and visually examined for transparency [S.-M.
Khoo, A.J. Humberstone, C.J.H. Porter, G.A. Edwards, W.N. Charman, Formulation design and
bioavailability assessment of lipidic self-emulsifying formulations of halofantrine, International Journal
of Pharmaceutics, 167 (1998) 155-164, L. Ould-Ouali, A. Arien, J. Rosenblatt, A. Nathan, P. Twaddle, T.
Matalenas, M. Borgia, S. Arnold, D. Leroy, M. Dinguizli, L. Rouxhet, M. Brewster, V. Preat,
Biodegradable self-assembling PEG-copolymer as vehicle for poorly water-soluble drugs, Pharm Res, 21
(2004) 1581-1590].
The dosage of the drugs depends on the need of an individual and the dosages described herein are adult
doses. However, this invention is not limited to the dosage of the combination regimen described herein
and may be varied according to medical advice. Accordingly the specific dosage described in the typical
embodiment is only illustrative and non-limiting formulations for other dosage forms are also included in
the scope of this invention.
Characterization of the SMEDDS
Globule size and zeta potential
SMEDDS was added to a volumetric flask containing 25 ml of TDW and agitated to form a fine
emulsion. The globule size has been determined by ZetasizerNano ZS model (Malvern Instruments) and
the zeta potential was determined by laser Doppler anemometry using a Malvern Zetasizer. All the
SMEDDS has been diluted with TDW to an appropriate concentration before determining the zeta
potential. The measurements were carried out in the fully automatic mode. Each sample was analyzed
thrice.
SMEDDS has been characterized by diluting them with the aqueous phase in a volumetric flask
containing TDW and agitated to form a fine emulsion. The visual parameters revealed the formation of
spontaneous clear emulsion and the globule size in a range of 55-160 nm. All the SMEDDS has been
diluted with TDW to an appropriate concentration before determining the zeta potential. The
measurements were carried out in the fully automatic mode. Each sample was analyzed thrice.
The SMEDDS consisted of oil, surfactants and ART should be a clear and monophasic liquid at ambient
temperature when introduced to aqueous phase and should have good solvent properties to allow
presentation of the ART in solution.
Self-emulsification of SMEDDS in simulated gastro-intestinal fluids
The size and solubility of SMEDDS were measured in TDW, in simulated gastric fluid (prepared by
dissolving 2.0 g of sodium chloride and 3.2 g of purified pepsin, in 7.0 mL of hydrochloric acid and water
up to 1000 mL) and in simulated intestinal fluid (prepared by dissolving 6.8 g of monobasic potassium
phosphate in 250 mL of water and then adding 77 mL of 0.2 N sodium hydroxide and 500 mL of water.
10.0 g of pancreatin is added and the resulting solution is adjusted with 0.2 N sodium hydroxide or 0.2 N
hydrochloric acid to a pH of 6.8 ± 0.1 and finally diluted to 1000 mL). The size of the globules after
dilution was assessed, after keeping them in room temperature for 2 h in gastric fluid and up to 8 h in
10
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intestinal fluid at 25°C, by photon correlation spectroscopy using ZetasizerNano ZS model (Malvern
Instrument Ltd., UK). The SMEDDS were evaluated for its self emulsification of ARTin the
gastrointestinal tract; 1.2 g of formulation (i.e., 0.2 g of ART) was diluted in 250 ml of simulated gastric
and/or intestinal medium. The optimized SMEDDS which were chosen for further studies had a size of
approximately 80 nm and a poiydispersity index (PDI) less than 0.2, indicating homogeneous distribution
of size. In contrast, SMEDDS with an upper PDI of 0.5 and presenting multimodal sizes with and without
ART and SP and the globule size larger than 450 nm was rejected. No major changes were observed as a
function of time. The globule size of the emulsions is an important parameter for self-emulsifying
systems. Indeed, it influences the speed and the quantity of released and absorbed compounds.
According to one aspect of this invention, the pharmaceutical formulation is administered through various
routes including, but not limited to, oral, rectal, intravenous, intramuscular and intraperitoneal.
In an embodiment of the invention, the pharmaceutically active agent is very poorly water-soluble drug.
In another aspect of the invention, a pharmaceutical formulation can be delivered in suspension and/or
liquid suspension form.
In another aspect, it can be administered as suspension dosage form, where it contains SMEDDS of a/parteether
and SP.
Another aspect of the invention relates to a method for making drug delivery system comprising drug
loaded SMEDDS. Placebo may also be delivered according to certain embodiments of the invention.
Advantages of the invention:
1. First oral formulation containing three anti-malarial drugs a/p-arteether and SP (sulfadoxine and
pyrimethamine) in a single formulation.
2. Formulation showing synergistic effect of three anti-malarial drugs a/p-arteether and SP
(sulfadoxine and pyrimethamine) by oral route.
3. Formulation with reduced dose of all the three anti-malarial drugs a,P-arteether and SP
(sulfadoxine and pyrimethamine).
4. An a,P-arteether, sulfadoxine and pyrimethamine formulation with reduced toxicity and easy
route of administration, where all the doses will be given orally.
5. No formulation of a,P-arteether is available for oral administration. In the present invention we
are reporting the oral formulationof a,p-arteether.
6. The three drugs used, are having different log P values and preparation of their formulation in a
single oral dose is not an easy task. Further, ART is a highly lipophilic compound and has good
solubility in oils, these factors, inspired us to prepare the SMEDDS which can solve the problem
11
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associated with the ART of oral bioavailability and contribute towards absorption of it via
lymphatic route.
7. We have prepared SMEDDS with the blend of drug, oil and surfactants. These surfactants were
known to increase the permeability by disturbing the cell membrane and thus by enhancing the
absorption of poorly soluble drugs [P. Artursson, J. Karlsson, Correlation between oral drug
absorption in humans and apparent drug permeability coefficients in human intestinal epithelial
(Caco-2) cells, Biochemical and Biophysical Research Communications, 175 (1991) 880-885].
8. These SMEDDS are found to be more useful for oral treatment of MDR P. falciparum. The
major advantage of the SMEDDS are that it cured the MDR malaria without causing any toxicity,
so we can suggest that it can treat serious malaria by the oral route of administration, with a view
to reduce mortality among P. falciparum cases particularly in children, pregnant women who are
at maximum risk and can overcome the problems associated with MDR infections which cannot
be treated effectively or cured with conventional antimalarial drugs.
The following examples broadly illustrate the nature of this invention, the manner in which it is to be
performed without limiting the nature and scope of the invention.
Example 1
Solubility studies
Excess amount of ART was placed in 1 ml of vehicle (Labrafac PG). Then, the mixture was vortexed and
kept for 48h at ambient temperature in a shaking water bath to facilitate the solubilization. The samples
were centrifuged at 5000g for 20 min to remove the un-dissolved ART. The supernatant was taken and
diluted with methanol for quantification of ART by HPLC on a C18 column (250mm, 4mm, and 5|4.m,
Merck) with a mobile phase consisted of a mixture of acetonitrile:water (70:30 v/v). Chromatography was
performed at a flow rate of 1.0 ml/minute.
Example 2
Preparation of SMEEDS of SP in GNO:
SP(2.5mg) was dissolved in alcohol (0.5 ml) followed by the addition of ground nut oil(2.5 ml),
cremophor (5ml) and Span 80 (2ml) and the mixture was stirred vigorously for 20 min to achieve
complete mixing and homogenized product. The resultant solution was filtered through a membrane filter
of 0.45 |im size and filled in a suitable sterile ampoule and sealed.
Example 3
Preparation of SMEDDS of SP:
SP (25 mg) was dissolved in alcohol 0.5 ml followed by the addition of labrafac (2.5ml),
cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred vigorously for 20 min to achieve
12
s i
complete mixing and homogenized product. The resultant solution was filtered through a membrane filter
of 0.45 |im size and filled in a suitable sterile ampoule and sealed.
Example 4
Preparation of SMEDDS of SP:
SP (80 mg) was dissolved in alcohol 0.5 ml followed by the addition of labrafac (2.5ml),
cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred vigorously for 20 min to achieve
complete mixing and homogenized product. The resultant solution was filtered through a membrane filter
of 0.45 \xm size and filled in a suitable sterile ampoule and sealed.
Similarly other SMEEDS formulations of SP containing different amounts of SP were prepared.
Example 5
Preparation of SMEDDS of a/p-arteether:
a,P-arteether (25mg)was dissolved in alcohol 0.5 mlfollowed by the addition of labrafac (2.5ml),
cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred vigorously for 20 min to achieve
complete mixing and homogenized product. The resultant solution was filtered through a membrane filter
of 0.45 jam size and filled in a suitable sterile ampoule and sealed.
Example 6
Preparation of SMEDDS of a/p-arteether:
a,P-arteether (250 mg) was dissolved in alcohol (0.5 ml) and labrafac (2.5 ml), cremophor (5 ml)
and Span 80 (2 ml) and the mixture was stirred vigorously for 20 min to achieve complete mixing and
homogenized product. The resultant solution was filtered through a membrane filter of 0.45 |xm size and
filled in a suitable sterile ampoule and sealed.
Similarly other SMEEDS formulations containing different amounts of a,P-arteether were prepared.
Development of oral ART and SP self micro emulsifying drug delivery system (SMEDDS)
Example 7
Preparation of SMEDDS of a/p-arteether and SP (Formulation 1):
SP(2.5mg) was dissolved in alcohol (0.5 ml), a,P-arteether (25mg) was added to it followed by
the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 [vm size and filled in a suitable sterile ampoule and sealed.
Example 8
Preparation of SMEDDS of a/p-arteether and SP (Formulation 2):
SP (2.5mg) was dissolved in alcohol (0.5 ml), a,P-arteether (12.5mg) was added to it followed
by the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
13
^ ^
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 |a,m size and filled in a suitable sterile ampoule and sealed.
Example 9
Preparation of SMEDDS of a/p-arteether and SP (Formulation 3):
SP (2.5mg) was dissolved in alcohol (0.5 ml), a,P-arteether (7mg) was added to it followed by
the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 fim size and filled in a suitable sterile ampoule and sealed.
Example 10
Preparation of SMEDDS of a/p-arteether and SP (Formulation 4):
SP (2.5mg) was dissolved in alcohol (0.5 ml), a,P-arteether (3.5mg) was added to it followed by
the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 |j,m size and filled in a suitable sterile ampoule and sealed.
Table I:-Description of composition of various SMEDDS
Formulation
F-1
F-2
F-3
F4
Arteether
(mg)
25
12.5
7
3.5
SP
(mg)
2.5
2.5
2.5
2.5
Alcohol
(ml)
0.5
0.5
0.5
0.5
Labrafac
(ml)
2.5
2.5
2.5
2.5
Cremophore
EL (ml)
5
5
5
5
Span 80
(ml)
2
2
2
2
Example 11
Preparation of SMEDDS of a/p-arteether and SP (Formulation 4):
SP (lOmg) was dissolved in alcohol (0.5 ml), a,P-arteether (250mg) was added to it followed by
the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 jam size and filled in a suitable sterile ampoule and sealed.
14
^
Similarly other SMEEDS formulations containing different amounts of SP and a,p-arteether were
prepared.
Example 12
Preparation of SMEDDS of a/p-arteether and SP:
SP (0.25mg) was dissolved in alcohol (0.5 ml) a,P-arteether (25mg) was added to it followed by
the addition of labrafac (2.5ml), tween 80 (5ml) and Span 80 (2ml) and the mixture was stirred vigorously
for 20 min to achieve complete mixing and homogenized product. The resultant solution was filtered
through a membrane filter of 0.45 yon size and filled in a suitable sterile ampoule and sealed.
Example 13
Preparation of SMEDDS of a/p-arteether and SP:
SP2.5mg was dissolved in alcohol (0.5 ml), a,P-arteether (25mg) was added to it followed by the
addition of GNO (2.5ml), cremophor (5ml) and Span 80 (2ml) and the mixture was stirred vigorously for
20 min to achieve complete mixing and homogenized product. The resultant solution was filtered through
a membrane filter of 0.45 )a,m size and filled in a suitable sterile ampoule and sealed.
Example 14
Preparation of SMEDDS of a/p-arteether and SP:
SP(2.5mg) was dissolved in alcohol 0.5 ml, a,P-arteether (25mg) was added to it followed by the
addition of lauroglycol (2.5ml), cremophor (5ml) and Span 80 (2ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 ixm size and filled in a suitable sterile ampoule and sealed.
Example 15
Preparation of SMEDDS of a/p-arteether and SP:
SP(2.5mg) was dissolved in alcohol (0.5ml), a,P-arteether (25mg) was added to it followed by
the addition of sesame oil (2.5ml), cremophor (5ml) and Span 80 (2ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 /am size and filled in a suitable sterile ampoule and sealed.
Example 16
Preparation of SMEDDS of a/p-arteether and SP (Formulation 1):
SP (2.5mg) was dissolved in alcohol (0.5 ml), a,P-arteether (25mg) was added to it followed by
the addition of labrafac (2.5ml), cremophor (5 ml) and Span 80 (2 ml) and the mixture was stirred
vigorously for 20 min to achieve complete mixing and homogenized product. The resultant solution was
filtered through a membrane filter of 0.45 |xm size and filled in a suitable sterile ampoule and sealed.
15
^
Example 17
Self-emulsiflcation of ART and SPformulations in gastrointestinal medium:
The formulations prepared as mentioned above were checked by different parameters to see their
suitability as SeIf-emulsification system. The size and solubility of ART and SPformulations were
measured in triple distilled water, in simulated gastric fluid (prepared by dissolving 2.0 g of sodium
chloride and 3.2 g of purified pepsin, in 7.0 mL of hydrochloric acid and water up to 1000 mL) and in
simulated intestinal fluid(prepared by dissolving 6.8 g of monobasic potassium phosphate in 250 mL of
water and then adding 77 mL of 0.2 N sodium hydroxide and 500 mL of water. 10.0 g of pancreatin is
added and the resulting solution is adjusted with 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to a
pH of 6.8 ± 0.1 and finally diluted to 1000 mL)[31].
The % incorporation efficiency was checked to see the % amount of drugs taken was present in the
formulation. Average globule size (nm) indicates the emulsification efficiency and Zeta potential give
some indications for their stability. Polydispersity index (PDI) indicates the presence of different size of
globules and their closeness with the average size if PDI is less than 0.25 it indicates that the particles are
almost of the same size.
Table II: Physicochemical characterization of some SMEDDS formulations
Formulation
F-1
F-2
F-3
F-4
%Incorporation
Efficiency
99±3
98±2
99±2
99±2
Zeta potential
(mV)
-12.0±1.0
-25.3±1.0
-28.2±2.0
-25.2±2.0
Average
globule size (nm)
125±15.0
118±12.0
114±16.0
108±09.0
Poly
dispersity
0.234±0.05
0.189±0.06
0.197±0.08
0.161±0.04
Values are expressed mean ± SD; (n=3)
Table III: Self emulsification of SMEDDS formulafionsin simulated gastro-intestinal fluids
Formulation
F-1
F-2
F-3
F-4
Gastric pH 1.2 buffer
Globule size
(nm)
124.12
126.22
128.19
129.13
PDI
0.11
0.09
0.18
0.13
Intestinal pH 7.5 buffer
Globule
Size(nm)
117.23
126.73
112.28
118.41
PDI
0.16
0.29
0.14
0.11
Triple distilled water
Globule size
(nm)
118.25
139.42
115.44
116.25
PDI
0.18
0.26
0.17
0.15
16
Example 18
Synergistic antimalarial effect of a,p-arteether and SP (oral) against multi drug resistant P.
yoeliinigeriensisin Swiss mice: Swiss mice (21±2 g), from CDRI Laboratory Animal Facility Division
(approval no. IAEC/2007/117), were inoculated intraperitoneal with 2x10^ Plasmodium yoeliinigeriensis
(multi drug resistant malaria parasite) infected mouse red blood cells. New formulation containing 12.5
mg/kg of a,P-arteether and 2.5 mg/kg of SP was administered by oral route in previously infected Swiss
mice. After giving first combined dose, following four doses of only 12.5 mg/kg a,|3-arteether were
administered orally to these mice. SP in combination with a,(3-arteether was administered by oral route
for only 1 day i.e. on day 0. These doses of both the drugs were also given individually for comparison.
Other batches of infected mice were treated with first combined dose of 7mg/kg of a,p-arteether and 2.5
mg/kg of SP on day 0, following the administration of 4 doses of only a,(3-arteether (7mg/kg x 4 days) in
the same formulation. These doses of both the drugs were also given individually for comparison. One
group was treated with the formulation only to see the effect of formulation on the level of parasitaemia.
One group of mice was kept as infected control. After treatment, parasitaemia was recorded from tail
blood smears of individual mouse and their mean parasitaemia was calculated.
Theresults of present experiment suggest that oral formulation of a,P-arteether and SP dissolved together
in a new formulation, at the dose of 12.5 mg/kg in combination with 2.5mg/kg SP (SP) and then 4 doses
of oral formulation of only a,p-arteether at the same dose i.e. 12.5 mg/kg treatment produced 100 %
curative efficacy. A lower dose of a,|3-arteether i.e. 7mg/kg with same dose of SP 2.5 mg/kg on first day
and then only 7mg/kg of a,P-arteether for next 4 days were also found to be 100% curative. Further lower
dose of 3.5mg/kg of a,P-arteether with 2.5mg/kg of SP followed by 3.5mg/kg of a,(3-arteether for 4 days
was only partially curative i.e. 40%. Alone a,p-arteether at dose of 12.5, 7 and 3.5 mg/kgx5 days
produced90, 60 and 0% cure whilst alone SP (SP) at 2.5mg/kg x 1 produced 0% or no cure.
17
^
TablelV: Chemotherapeutic response of arteether a/|3 and SP together in P.yoeliinigeriensis MDR
infected swiss mice.
Formulation
ART+ FAN
X5 X 1
(mg/kg)
12.5 +2.5
7+2.5
3.5+2.5
12.5+0 -
7+0
3.5+0
25+0
0+2.5
0+80
Blank
Formulation
D4
0±0
(15/15)
0±0
(15/15)
0+0
(5/5)
0+0
(20/20)
0+0
(20/20)
0.71+
1.13
(2/10)
0+0
(15/15)
0.766+
1.127
(10/16)
0±0
(15/15)
44.14+
25.37
(0/10)
D7
0±0
(15/15)
0±0
(15/15)
0+0
(5/5)
0.025
+0.109
(19/20)
0.0005
±0.002
(19/20)
28.09+
21.67
(0/10)
0±0
(15/15)
14.53+
26.84
(4/13)
0+0
(15/15)
20.33
+9.88
(0/3)
Mean % parasitemia ± SD
DIO
0±0
(15/15)
0±0
(15/15)
0+0
(5/5)
0.0005
+0.002
(19/20)
4.77+
11.87
(15/20)
35.76+
24.01
(0/5)
0±0
(15/15)
20.26+
20.33
(0/7)
0±0
(15/15)
28.25+
9.75
(0/2)
D14
0+0
(15/15)
0+0
(15/15)
28.6+
35.26
(2/5)
0+0
(20/20)
3.94+
14.74
(12/16)
44.33
+6.01
(0/3)
0±0
(15/15)
25.57+
25.42
(0/2)
0+0
(15/15)
57.5+
22.5
(0/2)
D18
0+0
(15/15)
0+0
(15/15)
0+0
(2/5)
1.9
+8.28
(19/20)
0.0014+
0.005
(13/14)
50.6+
16.35
(0/3)
0±0
(15/15)
0.002+0
(0/1)
0+0
(15/15)
D21
0+0
(15/15)
0+0
(15/15)
0+0
(2/5)
3.25+
14.17
(19/20)
0.14+
0.51
(13/14)
33+33
(1/2)
0+0
(15/15)
0+0
(1/1)
0+0
(15/15)
D24
0+0
(15/15)
0+0
(15/15)
0+0
(2/5)
2.73+
11.9
(19/20)
3.62+
13.05
(13/14)
23.5+
23.5
(1/2)
0+0
(15/15)
0+0
(1/1)
0+0
(15/15)
D28
0+0
(15/15)
0+0
(15/15)
0+0
(2/5)
3.45+
15.03
(19/20)
0.14+
0.51
(13/14)
23.1+
23.1
(1/2)
0+0
(15/15)
0+0
(1/1)
0+0
(15/15)
% cure
100
100
40
90
60
0
100
0
100
0
18
Control 32.26±
19.79
(0/22)
37.9±
7.9
(0/3)
Dead 0
ART= a,|3-arteether in formulation; FAN= SP in same formulation
Number of mice with no parasitaemia/ no. of total live mice are given in parenthesis
Table V: Median (range) sum of FIC for the interaction of arteether- SPagainst P.yoeliinigeriensis
Drug Combination (oral)
a,P-Arteether oral+SP (SP)
Dose
7mg/kg X 5 + 2.5mg/kg x 1
Median (range) sum of FIC of
P.yoeliinigeriensis
0.311
FIC - fractional inhibitory concentration
Effect on Mean Survival Time (MST): Treated and control mice were observed for their survival for 28
days. a,P-Arteether doses 12.5 and 7mg/kg x 5 with SP (2.5mg/kg x 1) were competent to cure all the mice
and all of them survived for more than 28 days of observation time while the lower dose of a,P-arteether
(3.5mg/kg x5) with the same dose of SP kept mice survived for 22.8 days. Alone drugs a,P-arteether
(7mg/kg), SP (2.5mg/kg) showed MST of 23.7±6.66 and 11.37±5.12 days respectively. Control mice
survived for only 7.15 days (Table VI).
Thus we get the 100% curative effect with nearly one fourth of curative dose of a,p-arteether in
combination of 1/32"" of SP curative dose. This study clearly indicates the synergism between the
proposed two drugs. This is indicated with LFIC value of 0.311. EFIC of < 0.5 indicates strong
synergism. This combination will reduce the toxicity of individual drugs, if any, as their dose in
combination is drastically reduced.
19
TableVI: Mean Survival Time of P.yoeliinigeriensis MDR infected swiss mice treated with a,P-arteether
and SP formulation together and alone.
Formulations ART x 5 + FAN x 1 (mg/kg)
12.5+2.5
7+2.5
3.5+2.5
12.5+0
7+0
25+0
0+2.5
0+80
Blank Formulation
Control
Mean Survival Time ± SD
>28 days
>28 days
22.8+6.36
>28 days
23.7+6.66
>28 days
11.37+5.12
>28 days
8.9+4.39
7.15+3.94
Example 19
Synergistic antimalarial effect of a,P-arteether and SP (oral) in ground nut oil against multi drug
resistant P. yoeliinigeriensis in Swiss mice: Swiss mice (21+2 g),from CDRI Laboratory Animal Facility
Division (approval no. lAEC/ 2007/117), were inoculated intraperitonially with 2x10^ Plasmodium
yoeliinigeriensis (multi drug resistant malaria parasite) infected mouse red blood cells. Ground nut oil
formulation containing 12.5 mg/kg of a,P-arteether and 2.5 mg/kg of SP was administered by oral route in
previously infected Swiss mice. After giving first combined dose, following four doses of only 12.5
mg/kg a,P-arteether were administered to these mice. SP in combination with a,P-arteether was
administered by oral route for only 1 day i.e. on day 0. These doses of both the drugs were also given
individually for comparison. One group was treated with the ground nut formulation only to see the effect
of formulation on the level of parasitaemia. One group of mice was kept as infected control. After
treatment, parasitaemia was recorded from tail blood smears of individual mouse and their mean
parasitaemia was calculated.
Theresults of present experiment suggest that oral formulation of a,P-arteether and SP dissolved together
in ground nut oil, at the dose of 12.5 mg/kg in combination with 2.5mg/kg SP (SP) and then 4 doses of
oral GNO formulation of only a,P-arteether at the same dose i.e. 12.5 mg/kg treatment produced 80%
curative efficacy. Alone arteether at dose of 12.5 mg/kg x 5 days produced 30% cure whilst alone SP (SP)
at 2.5mg/kg x 1 produced 0% or no cure. A higher dose of arteether at 25 mg/kg x 5 in GNO produced
91% cure whilst its lower doses i.e 7 and 3.5mg/kg x 5 days produced no cure at all (Table VII).
20
i •' )»
o
Effect on Mean Survival Time (MST): Treated and control mice were observed for their survival for 28
days. a,P-Arteether dose 12.5 x 5 days with SP (2.5mg/kg x 1) was competent to cure 80% of the treated
mice and they survived for 24.8 days .a,P-Arteetheraloneat 25, 12.5, 7 and 3.5mg/kg) showed MST of
27.09±2.87, 18.29±7.08, I5.2±7.0 and 10.8±5.64 days respectively. SP alone (2.5 mg/kg x 1) could make
mice survived for 10.4±5.57 and control mice survived for only 7.15 days (Table VIII).
Thus we get the 80% curative effect with combination of arteether and SP in GNO by oral route.
Here 80% cure was observed with nearly one third of curative dose of arteether in GNO (40mg/kg
X 5days) in combination with 1/32"" of SP curative dose. This study indicates that synergism exist
between the proposed two drugs even in ground nut oil. This combination will reduce the toxicity of
individual drugs, if any, as their dose in combination is drastically reduced.
21
Table VII: Chemotherapeutic response of a,(3-arteether (GNO) and SP (Tween+ Water) together in
P.yoeliinigeriensis MDR infected swiss mice
Formulations
ART + FAN
X5 X 1
(mg/kg)
12.5+2.5
12.5+0
0+2.5
25+0
7+0
3.5+0
Vehicle (only
GNO)
Control
D4
0±0
(15/15)
0.0
(20/20)
6±
11.24
(2/10)
0.0
(11/11)
0.001 +
0.006
(19/20)
4.20+
4.69
(0/5)
43.8+
28.8
(5)
32.26+
19.79
(0/22)
D7
0.38+
1.44
(14/15)
0.006+0
.017
(15/20)
16.5+
8.73
(0/4)
0.0
(11/11)
4.68+
9.82
(9/20)
52.25+2
8.25
(0/4)
Dead
37.9+
7.9
(0/3)
Mean % parasitaemia ±
DIO
5.54+
13.89
(12/14)
13.78+
26.26
(8/17)
44.3+
11.02
(0/3)
0.0
(11/11)
16.06+
21.30
(3/11)
0.6+0
(0/1)
Dead
D14
0+0
(12/12)
3.19+
7.42
(7/10)
30.4+
12.4
(0/2)
0.11+
0.34
(10/11)
12.86+
14.62
(1/7)
30+0
(0/1)
D18
0+0
(12/12)
2.87+
7.60
(7/8)
60+0
(0/2)
0.0
(10/10)
15.22+
15.97
(1/5)
44+0
(0/1)
SD
D21
0+0
(12/12)
5.52+
12.93
(6/8)
68+0
(0/1)
0.0
(10/10)
24+19.71
(2/5)
75+0
(0/1)
D24
0+0
(12/12)
0.0
(6/6)
Dead
0.0
(10/10)
33.5+
33.68
(2/4)
Dead
D28
0+0
(12/12)
0.0
(6/6)
0.0
(10/10)
0+0
(2/2)
% cure
80
30
0
91
0
0
0
0
*Pooled data of 2-4 experiments.
22
# " *•
^
No. of surviving mice are given in parentheses; ART= arteether in Ground nut oil (GNO); FAN= SP in
Tween + water
Number of mice with no parasitaemia/ no. of total live mice are given in parenthesis
Table VIII: Mean Survival Time of P.yoeliinigeriensis MDR infected swiss mice treated
arteether a/p in GNO and SP together Vs alone.
Formulations ART x 5 + FAN x l(mg/kg)
12.5+2.5
12.5+0
0+2.5
25+0
7+0
3.5+0
Vehicle GNO only
Control
Mean Survival Time ± SD
24.8+6.43
18.29+7.08
10.4+5.57
27.09+2.87
15.2+7.0
10.8+5.64
6+0.89
7.15+3.94
Example20
Curative dose of a/p arteether (oral) in ground nut oil and SP (oral) in Tween 80 plus water against
P. yoeliinigeriensis in Swiss mice.
Experimental:Swiss mice (20+2 g),from CDRI Laboratory Animal Facility Division (approval no.
lAEC/ 2007/117)were inoculated intra peritoneal with 2x10^Plasmodium yoeliinigeriensis{mahna
parasite) infected mouse red blood cells. SP(SP) was suspended in tween 80 and then in triple distilled
water and different doses i.e., 30, 60 and 80 mg/kg were administered in previously infected Swiss mice.
SP was administered by oral route for only 1 day i.e. on day 0. a,(3-arteether was dissolved in neutralized
and sterilized groundnut oil and two doses i.e. 25 mg/kgx5 and 40mg/kgx 5 were given orally. These
doses of both the drugs were given individually. Vehicle controls for both the drugs were kept to see the
effect of vehicle only on parasitaemia. One group of mice was kept as infected control. After treatment
parasitaemia was recorded from tail blood smears of individual mouse and their mean parasitaemia was
calculated.
Theresults of experiment suggested that oral arteether dissolve in oil at the dose of 25 mg/kgx4 and 40
mg/kg x5 days treatment produced 91 and 100% curative efficacy respectively whereas SP at the single
23
« - V
0
dose of 80mg/kg also provided 100% curative efficacy. Lower doses of SP i.e. 60 and 30mg/kg produced
85 and 40 % cure respectively with MST of 25.35±6.31 and 17.33±8.38 days correspondingly (Table-
IX&X).
Thus the 100% curative dose with arteether and SP were observed as 40 x Sdays and 80 mg/kg x
Iday correspondingly.
TablelX: Curative doses of arteether and SPalone in P.yoeliinigeriensis MDR infected swiss mice
Drugs
D4 D7
a,p-arteether in GNO
25mg/kg X
5days
40mg/kg X
Sdays
Only GNO
(0.2ml) X 5
days
SP in Tweei
30 mg/kg
xl
60 mg/kg
xl
80 mg/kg
xl
Only
Tween 80
+ water
Control
o±o
(11/11)
0±0
(S/S)
43.8±28
.8
(0/5)
0±0
(11/11)
0±0
(5/5)
Dead
n 80+water
0±0
(6/6)
0±0
(20/20)
0±0
(S/S)
43.6±1
2.98
(0/6)
32.26±
19.79
(0/22)
0.85±1.85
(4/6)
0.16±
0.485
(17/20)
0±0
(5/S)
30±0
(0/1)
37.9±7.9
(0/3)
Mean % parasitaemia ± SD
DIO
0±0
(11/11)
0±0
(5/5)
23.3±34.6
(2/5)
4.44±
18.32
(17/18)
0±0
(S/S)
Dead
D14 D18
O.lli
0.34
(10/11)
0±0
(S/S)
0±0
(10/10)
0±0
(5/5)
0±0
(2/2)
0±0
(17/17)
0±0
(5/5)
0±0
(2/2)
0±0
(17/17)
0±0
(S/S)
D21
0±0
(10/10)
0±0
(S/S)
0±0
(2/2)
0±0
(17/17)
0±0
(5/5)
D24 D28
0±0
(10/10)
0±0
(5/5)
o±o
(10/10)
0±0
(5/5)
0±0
(2/2)
0±0
(17/17)
0±0
(5/5)
0±0
(2/2)
0±0
(17/17)
0±0
(5/5)
% Cure
91
100
0
40
85
100
0
0
24
Table X: Mean Survival Time of P.yoeliimgeriensis MDR infected swiss mice treated with arteether
(GNO) / SP (Tween 80 + water).
Drugs (mg/kg)
40 ART X 5 days
Only GNO
30 FAN X 1 day
60 FAN X 1 day
80 FAN X 1 day
Only Tween 80 + water
Control
Mean Survival Time ± SD (days)
>28
6±0.89
17.33±8.38
25.35±6.31
>28
7±1.41
7.15±3.94
25

¥»
15 We claim: , JUL
1. A combination kit for the treatment of multidrug resistantmalaria for a period of oneto five days
schedule wherein the kit comprising;
a. a synergistic single oral formulation of anti-malarial agents containing a/p arteether, sulfadoxine
and pyrimethamine in the form of SMEDDS;
b. an oral formulation containing a/p arteether or containing a/p arteether with one tenth dose of
sulfadoxine and pyrimethamine as mentioned in step a in the form of SMEDDS ;
c. instruction manual for the administration of the three antimalarial drugs.
2. A kit as claimed in claim 1 wherein single oral formulation in the form of SMEDDS comprising a/p
arteether in the ratio ranging between 15 to 250, sulfadoxine in the ratio ranging between 47.6 to
500 and pyrimethamine in the ratio ranging between 2.4 to 251ong with the pharmaceutically
acceptable diluents, surfactants, co-surfactants.
3. A kit as claimed in claim 1 wherein oral formulation containing a/p arteether in the form of
SMEDDS comprising a/p arteether along with the pharmaceutically acceptable diluents, surfactants,
co-surfactants wherein the ratio of a and P-arteether is 30±5:70±5.
4. A combination kit according to claim 1 wherein the instruction manual for administering anti-malarial
agents for one to five days treatment comprising:
i) instructions for administration of a synergistic oral SMEDDS formulation of anti-malarial
agents containing a,P arteether, sulfadoxine and pyrimethamine on day one;
ii) instructions for administration of oral SMEDDS formulation containing a,p arteether or
containing a/p arteether with one tenth dose of sulfadoxine and pyrimethamine (as
mentioned above in step i) per day in predetermined doses and predetermined dose
schedule for next two to five day therapy.
5. A process for preparation of a synergistic single oral SMEDDS formulation as claimed in claim
1 wherein the process steps comprising :
i) a, P-arteether, sulfadoxine and pyrimethamine are dispersed in alcohol followed by the
addition of natural glycerides or semi-synthetic glycerides and surfactant under agitation at
200-1000 rpm for 2-60 minutes at 10-40° C until homogenization.
26
» ^ >
^
,5 ja w«
11) the final mixture is vortexed vigorously for 2-60 min to achieve complete mixing and
homogenized the product until a clear solution of SMEDDS is obtained,
iii) examiningthe SMEDDS for any signs of turbidity or phase separation prior to selfemulsification
and globule size studies, and equilibrating the SMEDDS to ambient
temperature for 2-50hr and then stored at room temperature.
6. A process for preparation of an oral SMEDDS formulation of a,p arteether comprising the
steps:
(I) a,P-arteether is dispersed in alcohol followed by the addition of natural glycerides or semisynthetic
glycerides and surfactant under agitation at 200-1000 rpm for 2-60 minutes at 10-
40° C until homogenization,
(II) the final mixture is vortexed vigorously for 2-60 min to achieve complete mixing and
homogenized the product until a clear solution of SMEDDS is obtained,
(iii) examining the SMEDDS for any signs of turbidity or phase separation prior to selfemulsification
and globule size studies, and equilibrating the SMEDDS to ambient
temperature for 2-50hr and then stored at room temperature.
7. A process as claimed in claim 5 wherein the process solvent used in the preparation of the
formulation is selected from a group of primary or secondary aliphatic alcohols such as ethanol,
2-propanol.
8. A process as claimed in claim 5 wherein the diluent used is anatural glycerides or semi-synthetic
glycerides and is selected from a group consisting of naturally occurring oil, synthetic source or
combination thereof, such as Coconut oil, Corn oil. Cottonseed oil, Olive oil. Palm oil. Peanut
oil (Ground nut oil), Rapeseed oil, Safflower oil, Sesame oil, Soybean oil, Captex 355 (Glyceryl
Tricaorylate/Caprate), Captex 200 (Propylene Dicaprylate/Dicaprate Glycol), Captex 8000
(Glyceryl Tricaprylate(Tricaprylin)), Witepsol (90:10 % w/w cl2 Glyceride tri: diesters), Myritol
318 (c8/cl0 triglycerides) Isopropyl myristate (Myristic acid isopropyl ester).
9. A process as claimed in claim 5 wherein the surfactants used in the preparation is the generally
regarded as safe (GRAS) is selected from a group consisting of Tween 20, Tween 40, Tween 60,
Tween 65, Tween 80, Tween 85, Span 40, Span 60, Span65, Span 80, PEG 200, PEG 300, PEG
400, PEG 600, PEG 1500, PEG 6000, Pluronic F-68, Pluronic F-127, PLUROL, PVA, BRIJ 35,
BRIJ 56, Carbopol 934, Carbopol 940, Cremophor EL, Labrafac, Labrosol, Lauroglycol 90,
27
>•* .>
o^ic.1^^^
10
Sodium Dodecyl Sulphate, Sodium Deoxycholate, POE 40 Stearate, POE 25 Propylene Glycol
Stearate, POE 20 Oleyl Ether, Transcutol, Gelucire 44/14, Gelucire 34.
10. A process as claimed in claim 5 wherein the co-surfactants used in the preparation is generally
regarded as safe (GRAS) and is selected from a group consisting of Transcutol, glycerin, ethylene
glycol, propylene glycol, ethanol, propanol.
11. A combination kit according to claim 1 wherein the kit is useful for the treatment of malaria
including malaria caused by P. falciparum or P. F/voxadministration of a SMEDDS formulation
to a subject in need containing 15 to 250 mg of a/p arteether 47.6 to 500 mg of sulfadoxine and
2.4 to 25 mg of pyrimethamine on day one and SMEDDS formulation containing 15 to 250 mg of
a/p arteether per day in predetermined doses and predetermined dose schedule for next two to
five day therapy.