[0001] The present invention relates to a novel pharmaceutical combination for the
5 treatment of infectious diseases caused by intracellular pathogens, particularly malaria.
Specifically, the present invention relates to a novel pharmaceutical combination comprising
drugs having intra-erythrocytic effect on pathogen along with drugs having extra-erythrocytic
effect on pathogen and host cell mimetic nanoparticles for the treatment of malaria. The present
invention further relates to a novel pharmaceutical combination comprising Artemisinin or
10 Artemisinin derivatives along with low molecular weight anti-coagulant along with Erythrocyte
mimetic nanoparticles for the treatment of malaria. The present invention also relates to method
of treatment of malaria comprising administering a pharmaceutically effective amount of the said
pharmaceutical combination to a subject in need thereof.
15 BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the
present invention. It is not an admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any publication specifically or implicitly
referenced is prior art.
20 [0003] Infectious diseases caused by intracellular pathogens have been ranked as one of the
leading cause of death worldwide by the World Health Organization. Although these
intracellular pathogens are phylogenetically diverse encompassing bacteria, viruses, fungi,
parasites etc, yet they share common pathogen type-specific mechanisms during host-pathogen
interaction inside host cells. Likewise, host cell invasion and intracellular survival mechanisms
25 are used by Plasmodium to infect erythrocytes.
[0004] Malaria is an infectious disease affecting the red blood cells, caused by the
apicomplexan Plasmodium species, and is transmitted by bite of Anopheles mosquito species.
The five species P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi can infect
humans. Plasmodium falciparum is the most prevalent malaria parasite in the WHO African
30 Region, accounting for 99.7% of estimated malaria cases in 2018, as well as in the WHO SouthEast Asia Region (50%), the WHO Eastern Mediterranean Region (71%) and the WHO Western
3
Pacific Region (65%). Outside of Africa, Plasmodium vivax is the most endemic form present in
approximately 50 countries; collectively accounting for half of the world’s malaria. Although
clinical manifestations of vivax malaria are frequently less severe than falciparum malaria, yet in
poor socio-economic regions, if left untreated, malaria leads to death due to severe anemia.
5 [0005] Most of the antimalarial drugs have low bioavailability and little or no specificity for
the Plasmodiuminfected cell, requiring higher doses of the drug to be administered, leading to
drug resistance. Thus, drug resistance has limited the use of most of the currently used antimalarial drugs. Another limitation is that with resistance developing to these drugs, most of the
current antimalarial drugs have more of schizonticidal activity and are inert towards the ring
10 stages and early trophozoite blood-stage of Plasmodium. A strong immune response is required
to naturally clear away the early ring and trophozoite blood stage infection. However, some antimalarial drugs exert a down-regulating effect on the immune system, by suppressing the
expression of adhesion receptors and decreasing the production of pro-inflammatory cytokines
such as tumor necrosis factor and IL-2, which are involved in the pathogenesis of severe malaria.
15 [0006] Common anti-malarial drug Chloroquine reduces chemotaxis of human monocytes.
However, Chloroquine resistance has also been implicated as one of the major factors
responsible for therapeutic failure against hypnozoite blood stages of P.vivax and
recurrence/relapse of malaria by activation of these recrudesced hypnozoites. These unnoticed or
invisible infections probably represent a dominant majority in most endemic settings. In addition
20 to the mutations occurring in Plasmodium in Anopheles mosquitoes, the mutations occurring in
these recrudesced hypnozoites reservoir in endemic communities is another biggest challenge to
tackle with conventional therapies. In such relapsed cases, estimating the efficacy of blood
schizonticidal antimalarial therapy becomes all the more complex and difficult as the relapses
might be either homologous or heterologous to the primary infection event.
25 [0007] 8-Aminoquinolines, another class of anti-malarials are the only currently available
drugs for chemo-preventive or presumptive periodic preventive therapeutic activity against P.
vivax. Although a complete understanding of the various host associated factors limiting the
efficacy of 8-Aminoquinolines in eradicating malaria especially in South Eastern population is
still not available, yet one of the host associated factor accounting for such a lower efficacy
30 against P. vivax has been found to be natural polymorphism in the gene expressing CYP2D6
which thereby limits the metabolism of Primaquine to its active hynozoicidal metabolite. Other
4
host associated factorsthat hamper determining the true effectiveness of any given antimalarial
agent include Primaquine contraindicationin glucose-6-phosphate dehydrogenase (G6PD)
deficient patients due to risk of haemolysis, regime adherence by the patient, availability and
quality of the available drug all are factors.
5 [0008] In order to combat drug resistance and to improve antimalarial chemotherapy, it is
becoming a standard practice to use combinations of more than one antimalarial, either
simultaneously or sequentially. However, many such combinations are antagonistic, resulting in
less effective treatment and the dosage regimens are often complicated, increasing the likelihood
of patients failing to complete the treatment. Moreover, there are four species of human malaria
10 parasites each of which may react differently to any combination of antimalarial drugs. In
particular, P. vivax malaria is difficult to treat successfully because a latent form of the parasite
persists in the liver after treatment with most anti-malarial drugs.At present, Artemisinin based
combination therapies have been found to combat this drug resistance.
[0009] Artemisinin a first line drug, is used widely to treat P. falciparum caused malaria
15 (Van Hensbroek et al 1996; White 2008) due to its unique pharmacodynamic properties, such as
faster onset of action and the pleiotropic effects exerting inhibition at multiple stages of the
Plasmodium life cycle (Woodrow 2005) including gametocyte development. It is also one of
most preferred anti-malarial drugs to be used to treat P. vivax caused malaria in areas with
Chloroquine resistance (Krishna, Uhlemann, Haynes, 2004). A number of Artesmisinin
20 derivatives are available enabling it to be administered by almost any route of administration.
Artemisinin being water insoluble is preferably administered orally. Artesunate, an Artemisinin
derivative, being water soluble is administered intravenously. Although,Artesunate has a short
half-life in blood (Morris CA, 2011; Djimde A, Lefevre G, 2009) but kills blood asexual stages
due to its more rapid onset of action.
25 [0010] However, these must not be used as monotherapy, as this promotes the development
of resistance.Artemisinin and Artesmisinin derived compoundshave a number of limitations.
Artesunate has low bioavailability due to its short half-life of 3-4 hrs (Bull World Health Organ.
2000) and little or no specificity for the Plasmodium-infected cell, requiring higher doses to be
administered. Thus, drug resistance has limited the use of most of these anti-malarials including
30 Artesunate (Walker et al 2014). Another limitation is that most of the current antimalarial drugs
are effective in treatment, when used only in initial uncomplicated stages of malaria and not
5
against complicated cerebral malaria. Further, a strong immune response is required to naturally
clear away the early ring and trophozoite blood stage infection. However, Artesunate is known to
exert a downregulating effect on the immune system and exerts immunosuppressive functions
against T cell activation (L. F. Hou et al 2009; Z. Wang et al 2007; Z. S. Yang, Wang, Zhou,
5 Zuo, Li, 2006; Z. S. Yang et al. 2005) and inhibits production of mediators of pro-inflammatory
TNF alpha production by suppressing nuclear translocation of NF-kappa B (W. D. Li, Dong, Tu,
Lin 2006; Y. Li et al 2013). Thus, there is an urgent need to develop new antimalarial treatment
which tends to lower Artesunate associated resistance and also upregulates immune response.
[0011] Recently in a number of in-vitro tests, it has been demonstrated that
10 glycosaminoglycans (GAGs) due to presence of negatively charged sulphate chains can be a
frontrunner to a new class of antimalarial drugs. Glycosaminoglycans (GAGs) are covalent
modifications of proteins, mainly O-linked and consist of alternating disaccharide units of the
hexosamines Glucosamine (GlcN) or Galactosamine (Ga1N), and of the Hexuronic acids
Glucuronic acid (GlcA) or Iduronic acid (IdoA). Many sulphated GAGs like Dextran Sulphate
15 (Butcher et al 1988), Inulin Sulphate, Carrageenan from seaweed (Adams et al 2005),
CurdlanSulphate (Havlik, Rovelli and Kaneko 1994; Evans et al 1998), fucosylated Chondroitin
Sulphate (Bastos et al 2014), SulphatedCyclodextrins (Crandall et al 2007), K5 polysaccharides
(Boyle et al 2010), XylanSulphate, Tragacanth Sulfate and ScleroglucanSulphate (Boyle et al
2017) have been identified to blockP.falciparummerozoite at various stages viz invasion and
20 egress. Among these GAGs, Heparin exerts most potent antimalarial effect by blocking both the
merozoite invasion process (Butcher, Parish and Cowden 1988; Boyle et al 2017) as well as
multiple other antigens involved in egress (Glushakova et al 2017). Heparin inhibits early
essential steps of merozoite interaction to erythrocyte by targeting Merozoite Surface Protein
1(MSP1) (Boyle et al 2010), and merozoite reorientation by inhibiting formation of MSP1
25 complex with rhoptrymicroneme proteins (Baum et al 2009; Kobayashi et al 2010; Kobayashi et
al 2013).
[0012] WHO recommends Artemisinin-based combination therapies (ACTs) for the
treatment of uncomplicated malaria. Efficacy is determined by the drug partnering the
Artemisinin derivative.
30 [0013] Thus there is an urgent need to develop new therapeutic and prophylactic
combinations for use alone or in conjunction with conventional malarial chemotherapy in the
6
prophylaxis and treatment of parasite infections and, in particular, infection by the malaria
parasite. Need is also felt for new anti-malarial treatment options, which can overcome
deficiencies associated with the known arts.
5 OBJECTS OF THE INVENTION
[0014] An object of the present invention is to provide a novel treatment option for infectious
diseases caused by intracellular pathogens, particularly malaria, which can overcome
deficiencies associated with the known arts.
[0015] Yet another object of the present invention is to provide a novel pharmaceutical
10 combination for the treatment of malaria.
[0016] Another object of the present invention is to provide a pharmaceutical combination
for the treatment of malaria that acts rapidly, produces fast clinical responses to treatment and
reduces the growth of the parasites.
[0017] Another object of the present invention is to provide a pharmaceutical combination
15 for the treatment of malaria, having dual mode of action, arresting the progression of disease and
inhibits both extra-erythrocytic and intra-erythrocytic stages.
[0018] Another object of the present invention is to provide a pharmaceutical combination
having reduced risk of development of resistance.
[0019] Another object of the present invention is to provide a pharmaceutical combination
20 for the treatment of malaria that up regulates host immuneomodulatory response.
[0020] The other objects and preferred embodiments and advantages of the present invention
will become more apparent from the following description of the present invention when read in
conjunction with the accompanying examples and figures, which are not intended to limit scope
of the present invention in any manner.
25
SUMMARY OF THE INVENTION
[0021] This summary is provided to introduce a selection of concepts in a simplified form
that are further described below in Detailed Description section. This summary is not intended to
identify key features or essential features of the claimed subject matter, nor is it intended to be
30 used as an aid in determining the scope of the claimed subject matter.
7
[0022] The present invention relates to a novel pharmaceutical combination for the
treatment of infectious diseases caused by intracellular pathogens. Specifically, the present
invention relates to a novel pharmaceutical combination for the treatment of infectious diseases
caused by intracellular pathogens, particularly malaria.
5 [0023] In another aspect, the present invention relates to a novel pharmaceutical
combinationfor the treatment of malaria, comprisingdrug having intra-erythrocytic mechanism of
action, drug having extra-erythrocytic mechanism of action and host cell mimetics.
[0024] In another aspect, the present invention relates to a novel pharmaceutical
combination for the treatment of malaria, comprising drugs having intra-erythrocytic effect on
10 pathogen along with drugs having extra-erythrocytic effect on pathogen and host cell mimetic
nanoparticles.
[0025] In another aspect, the present invention relates to a novel pharmaceutical
combination for the treatment of malaria, comprisingdrugs having intra-erythrocytic effect on
Plasmodium along with drugs having extra-erythrocytic effect on Plasmodium and host cell
15 mimetic nanoparticle.
[0026] According to another aspect of the present invention, drugs having intraerythrocytic effect on Plasmodium, specifically antimalarial drugs having intra-erythrocytic
effect may be selected from but not limited to Quinine and its derivatives, Chloroquine,
Hydroxychloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine,
20 Atovaquone, Primaquine, Halofantrine, Lumefantrine, Doxycycline , Clindamycin, Artemisinin
or Artemisinin derivatives, particularlyArtesunate, Artemether, Artemotil and
Dihydroartemisinin.
[0027] According to another aspect of the present invention, antimalarial extra-erythrocytic
drugs capable of suitably modifying phenotypic activity include but are not limited to structural
25 carbohydrates like sulfated glycosaminoglycans (Hyaluronan, Chondroitin Sulphate, Dermatan
Sulphate, Heparin, KeratanSulphate), anti-coagulants (Dalteparin, Danaparoid,
Enoxaparin,Ttinzaparin, Apixaban, EdoxabanTosylate, Fondaparinux, Rivaroxaban, Betrixaban),
Cysteine Protease inhibitor, Calcium Chelator or Calcium pump Inhibitors.
[0028] In another aspect, the present invention relates to a novel pharmaceutical
30 combination for the treatment of malaria, comprising Artemisinin or Artemisinin derivatives
along withlow molecular weight anti-coagulantand erythrocyte mimetic nanoparticles.
8
[0029] In another aspect, the present invention relates to a pharmaceutical combination for
the treatment of malaria, wherein Artemisinin derivative can be selected from but not limited to
Artesunate, Artemether, Artemotil and Dihydroartemisinin and the like.
5 [0030] In a preferred aspect, the present invention relates to a pharmaceutical combination
for the treatment of malaria, wherein Artemisinin derivative is Artesunate.
[0031] In another aspect, the present invention relates to a pharmaceutical combination for
the treatment of malaria, wherein low molecular weight anti-coagulant can be selected from
Enoxaparin,Bemiparin, Certoparin, Dalteparin, Nadroparin, Parnaparin, Reviparin, Tinzaparin
10 and the like.
[0032] In a preferred aspect, the present invention relates to a pharmaceutical combination
for the treatment of malaria, wherein low molecular weight anti-coagulant is Enoxaparin.
[0033] In another aspect, the present invention relates to anti-malarial pharmaceutical
combination comprising Artesunate, Enoxaparin and Erythrocyte mimeticnanoparticles, for the
15 treatment of malaria.
[0034] In another aspect, the present invention relates to a pharmaceutical combination for
the treatment of malaria, wherein the combination can be administered concomitantly or
sequentially.
[0035] In still another aspect, the present invention relates to a pharmaceutical combination
20 for the treatment of malaria, wherein Artemisinin derivatives, low molecular weight anticoagulant and Erythrocyte mimeticnanoparticles are present in a potentiating ratio.
[0036] In yet another aspect, the present invention relates to a formulation comprising
pharmaceutical combination for the treatment ofinfectious diseases caused by intracellular
pathogens, particularly malaria, wherein the formulation comprises Artemisinin derivatives, low
25 molecular weight anti-coagulant and Erythrocyte mimetic nanoparticles along with a
pharmaceutically acceptable carrier.
[0037] In yet another aspect, the present invention relates toa pharmaceutical combination
comprising Artemisinin derivatives, low molecular weight anti-coagulant along with Erythrocyte
mimeticnanoparticles,for the treatment and/or prophylaxis of infectious diseases caused by
30 intracellular pathogens, particularly malaria, in mammals.
9
[0038] In still another aspect, the present invention relates toa pharmaceutical combination
comprising Artemisinin derivatives, low molecular weight anti-coagulant along with Erythrocyte
mimeticnanoparticles, for the manufacture of a medicament,for the treatment of infectious
diseases caused by intracellular pathogens, particularly malaria.
5 [0039] In another aspect, the present invention relates to a method of treatment of
infectious diseases caused by intracellular pathogens, particularly malaria, comprising
administering a therapeutically effective amount of the pharmaceutical combination comprising
Artemisinin derivatives, low molecular weight anti-coagulant along with Erythrocyte
mimeticnanoparticles, to a subject in need thereof.
10 [0040] In yet another aspect, the present invention relates to use of the pharmaceutical
combination comprising Artemisinin derivatives, low molecular weight anti-coagulant along
with Erythrocyte mimeticnanoparticles, for the treatmentand prophylaxis of infectious diseases
caused by intracellular pathogens, particularly malaria.
[0041] Other aspects of the invention will be set forth in the description which follows, and in
15 part will be apparent from the description, or may be learnt by the practice of the invention.
BRIEF DESCRIPTION OF DRAWINGS THE INVENTION
[0042] The following drawings form part of the present specification and are included to further
illustrate aspects of the present disclosure. The disclosure may be better understood by reference
20 to the drawings in combination with the detailed description of the specific embodiments
presented herein.
Figure 1: illustrates the Erythrocyte mimetic nanoparticles; Figure 1a depicts Erythrocyte Cell
Membrane; Figure 1b depicts Biodegradable or Non-Biodegradable
25 Nanoparticle; Figure 1c depicts Erythrocyte Cell membrane completely coated core-shell
Nanoparticle;Figure 1d depicts Erythrocyte Cell membrane partially coated core-shell
Nanoparticle Figure 1e depicts one or more part of Erythrocyte Cell membrane
partially coated core-shell nanoparticle.
Figure 2: illustrates the particle size determination; Figure 2a depicts particle size determination
30 for Chitosan nanoparticles; Figure 2b depictsparticle size determination for biomimetic Chitosan
nanoparticles.
10
Figure 3: illustrates Zeta Potential Analysis; Figure 3a depicts Zeta Potential Analysis for
Chitosan nanoparticles; Figure 3b depicts Zeta Potential Analysis for Erythrocyte mimetic
nanoparticles.
Figure 4:illustrates FT-IR Analysis; Figure 4a depicts FT-IR Analysis for Chitosan
5 Hydrochloride; Figure 4b depicts FT-IR Analysis for Chitosan nanoparticles.
Figure 5:illustrates Scanning Electron Microscope (SEM) Analysis; Figure 5a depicts SEM
Analysis for Chitosan nanoparticles at 200m; Figure 5b depicts SEM Analysis for
Erythrocyte mimetic nanoparticles at 500 nm.
Figure 6: is a graphical representation of Results for Mean Percent Parasitemiastudies in
10 experimental groups.
DETAILED DESCRIPTION
[0043] The following is a detailed description of embodiments of the disclosure. The
embodiments are in such detail as to clearly communicate the disclosure. However, the amount
15 of detail offered is not intended to limit the anticipated variations of embodiments; on the
contrary, the intention is to cover all modifications, equivalents, and alternatives falling within
the spirit and scope of the present disclosure as defined by the appended claims.
[0044] All publications herein are incorporated by reference to the same extent as if each
individual publication or patent application were specifically and individually indicated to be
20 incorporated by reference. Where a definition or use of a term in an incorporated reference is
inconsistent or contrary to the definition of that term provided herein, the definition of that term
provided herein applies and the definition of that term in the reference does not apply.
[0045] Reference throughout this specification to “one embodiment” or “an embodiment”
means that a particular feature, structure or characteristic described in connection with the
25 embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one
embodiment” or “in an embodiment” in various places throughout this specification are not
necessarily all referring to the same embodiment. Furthermore, the particular features, structures,
or characteristics may be combined in any suitable manner in one or more embodiments.
[0046] In some embodiments, the numbers expressing quantities of ingredients, properties
30 such as concentration, reaction conditions, and so forth, used to describe and claim certain
embodiments of the invention are to be understood as being modified in some instances by the
11
term “about.”Accordingly, in some embodiments, the numerical parameters set forth in the
written description and attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular embodiment.In some embodiments, the
numerical parameters should be construed in light of the number of reported significant digits
5 and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of some embodiments of the invention are
approximations, the numerical values set forth in the specific examples are reported as precisely
as practicable.The numerical values presented in some embodiments of the invention may
contain certain errors necessarily resulting from the standard deviation found in their respective
10 testing measurements.
[0047] As used in the description herein and throughout the claims that follow, the meaning
of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.
Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the
context clearly dictates otherwise.
15 [0048] Unless the context requires otherwise, throughout the specification which follow, the
word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be
construed in an open, inclusive sense that is as “including, but not limited to.”
[0049] The recitation of ranges of values herein is merely intended to serve as a shorthand
method of referring individually to each separate value falling within the range. Unless otherwise
20 indicated herein, each individual value is incorporated into the specification as if it were
individually recited herein .All methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g. “such as”) provided with respect to certain
embodiments herein is intended merely to better illuminate the invention and does not pose a
25 limitation on the scope of the invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential to the practice of the
invention.
[0050] Groupings of alternative elements or embodiments of the invention disclosed herein
are not to be construed as limitations. Each group member can be referred to and claimed
30 individually or in any combination with other members of the group or other elements found
herein. One or more members of a group can be included in, or deleted from, a group for reasons
12
of convenience and/or patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified thus fulfilling the written
description of all Markush groups used in the appended claims.
[0051] The description that follows, and the embodiments described therein, is provided by
5 way of illustration of an example, or examples, of particular embodiments of the principles and
aspects of the present disclosure. These examples are provided for the purposes of explanation,
and not of limitation, of those principles and of the disclosure.
[0052] It should also be appreciated that the present disclosure can be implemented in
numerous ways, including as a system, a method or a device. In this specification, these
10 implementations, or any other form that the invention may take, may be referred to as processes.
In general, the order of the steps of the disclosed processes may be altered within the scope of
the invention.
[0053] The headings and abstract of the invention provided herein are for convenience only
and do not interpret the scope or meaning of the embodiments.
15 [0054] The following discussion provides many example embodiments of the inventive
subject matter. Although each embodiment represents a single combination of inventive
elements, the inventive subject matter is considered to include all possible combinations of the
disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second
embodiment comprises elements B and D, then the inventive subject matter is also considered to
20 include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0055] Various terms as used herein are shown below. To the extent a term used in a claim
is not defined below, it should be given the broadest definition persons in the pertinent art have
given that term as reflected in printed publications and issued patents at the time of filing..
[0056] The term "Pharmaceutical composition" or “pharmaceutical formulation” refers to
25 the combination of one or more drug substances and one or more excipients.
[0057] The term "Drug product," "pharmaceutical dosage form," "dosage form," "final
dosage form" and the like, refer to a pharmaceutical composition that is administered to a subject
in need of treatment and generally may be in the form of tablets, capsules, sachets containing
powder or granules, pellets, liquid solutions or suspensions, patches, and the like.
30
13
[0058] The term 'pharmaceutically acceptable excipient' according to the present invention
means, but not limited to, any inactive ingredient which is required for the formulation in a
suitable dosage form. Particularly the excipient includes, but not limited to, diluents, carriers,
fillers, bulking agents, binders, disintegrants, polymer, lubricant, glidant, surface active agents,
5 stabilizers, absorption accelerators, flavoring agents, preservatives, antioxidants, buffering
agents, and any other excipient commonly used in the pharmaceutical industry.
[0059] “Subject” includes humans, non-human mammals (e.g., dogs, cats, rabbits, cattle,
horses, sheep and the like) or non-mammals (e.g., birds and the like).
[0060] The term “Artemisinin” refers to a sesquiterpene lactone obtained from sweet
10 wormwood, Artemisia annua, which is used as an antimalarial for the treatment of multi-drug
resistant strains of P.falciparum malaria. It has a role as an antimalarial and a plant metabolite. It
is a sesquiterpene lact one and organic peroxide.
[0061] The term “low molecular weight anti-coagulant” also known as “low molecular weight
heparins” refers toa class of anticoagulant medications used in the prevention of blood clots and
15 treatment of venous thromboembolism (deep vein thrombosis and pulmonary embolism) and in
the treatment of myocardial infarction. These low molecular weight Heparins consist of short
chains of polysaccharide and are defined as heparin salts having an average molecular weight of
less than 8000 Da for which at least 60% of all chains have a molecular weight less than 8000
Da.
20 [0062] The present invention relates to a novel pharmaceutical combination for the
treatment of infectious diseases caused by intracellular pathogens, particularly malaria.
Specifically, the present invention relates to a novel pharmaceutical combination for the
treatment of malaria.
[0063] In one embodiment, the present invention relates to a novel pharmaceutical
25 combination comprising pathocidal drug, pathostatic drug and host cell mimetics.
[0064] In another embodiment, the present invention relates to a novel pharmaceutical
combination for the treatment of infectious diseases caused by intracellular pathogens,
particularly malaria, comprisingdrug having intra-erythrocytic mechanism of action, drughaving
extra-erythrocytic mechanism of action and host cell mimetics.
30 [0065] In another embodiment, the present invention relates to a novel pharmaceutical
combination for the treatment of infectious diseases caused by intracellular pathogens,
14
particularly malaria, comprising drugs having intra-erythrocytic effect on pathogen along with
drugs having extra-erythrocytic effect on pathogen and host cell mimetic nanoparticles.
[0066] In another embodiment, the present invention relates to a novel pharmaceutical
combination for the treatment of infectious diseases caused by intracellular pathogens,
5 particularly malaria, comprisingdrugs having intra-erythrocytic effect on Plasmodium along with
drugs having extra-erythrocytic effect on Plasmodiumand host cell mimetic nanoparticle.
[0067] According to one embodiment of the present invention, drugs having intraerythrocytic effect on pathogen may include one or more drugs that inhibit, arrest, retard, stop or
kill the activity and/or growth of one or more intracellular life cycle stage of the pathogen.
10 [0068] According to another embodiment of the present invention, drugs having intraerythrocytic effect on Plasmodium, specifically antimalarial drugs having intra-erythrocytic
effect may be selected from but not limited to quinine and its derivatives, Chloroquine,
Hydroxychloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine,
Atovaquone, Primaquine, Halofantrine, Lumefantrine, Doxycycline , Clindamycin, Artemisinin
15 or Artemisinin derivatives, particularlyArtesunate, Artemether, Artemotil and
Dihydroartemisinin.
[0069] According to another embodiment of the present invention, drugs having extraerythrocytic effect on pathogen act as a receptor to modify the phenotypic activity by specific
binding to ligands which are expressed on the surface of pathogen and/or pathogen infected host
20 cells.
[0070] According to another embodiment of the present invention, antimalarial extraerythrocytic drugs capable of suitably modifying phenotypic activity include but are not limited
to structural carbohydrates like sulfated glycosaminoglycans (Hyaluronan, Chondroitin Sulphate,
Dermatan Sulphate, Heparin, KeratanSulphate), anti-coagulants (Dalteparin, Danaparoid,
25 Enoxaparin, Tinzaparin, Apixaban, EdoxabanTosylate, Fondaparinux, Rivaroxaban, Betrixaban),
Cysteine Protease Inhibitor, Calcium Chelator or Calcium pump Inhibitors.
[0071] More specifically, according to the present invention, antimalarial extraerythrocytic drugs capable of suitably modifying phenotypic activity is a sulfated
glycosaminoglycan moiety particularly, a Heparin sulfate moiety. Most preferably, according to
30 the present invention, antimalarial extra-erythrocytic drugs capable of suitably modifying
phenotypic activity is an anti-coagulant, specifically, low molecular weight Heparin compound.
15
In a preferred embodiment, the antimalarial extra-erythrocytic drug capable of suitably
modifying phenotypic activity isEnoxaparin, or a functional fragment thereof.
[0072] The present invention also encompasses all the functionally equivalent analogues of
structural carbohydrates useful as antimalarial extra-erythrocytic drugs capable of suitably
5 modifying phenotypic activity.
[0073] According to another embodiment of the present invention, the host cell mimetic
nanoparticle is a host cell membrane functionalized nanoparticle that acts as a receptor to modify
the phenotypic activity by non-specific generalized binding to ligands which are expressed on
the surface of pathogen and/or pathogen infected host cells.
10 [0074] Specifically, the antimalarial host cell mimetic nanoparticle according to the
embodiment of the present invention capable of modifying phenotypic activity include but are
not limited to core-shell Erythrocyte mimetic nanoparticles or fused Erythrocyte mimetic
nanoparticles. The core-shell Erythrocyte mimetic nanoparticlesrefers to nanoparticles wherein
Erythrocyte membrane partially or completely surrounds or envelops the nanoparticle core
15 whereas in the fused Erythrocyte mimetic nanoparticles, one or more domain of Erythrocyte cell
membrane is fused along with the nanoparticle excipient to form one continuous layer.
[0075] According to an embodiment of the present invention, the antimalarial host cell
mimetic nanoparticle is depicted in Figure 1. Figure 1a depicts Erythrocyte Cell Membrane;
Figure 1b depicts Biodegradable or Non-Biodegradable Nanoparticle; Figure 1c depicts
20 Erythrocyte Cell membrane completely coated core-shell nanoparticle; Figure 1d depicts
Erythrocyte Cell membrane partially coated core-shell nanoparticle Figure 1e depictsone or
more part of Erythrocyte Cell membrane partially coated core-shell Nanoparticle.
[0076] According to the embodiments of the present invention, the nanoparticle may be
completely or partially biodegradable or non-biodegradable and can be selected from but not
25 limited to carbon-based nanoparticles, ceramic nanoparticles, metal nanoparticles, semiconductor
nanoparticles, synthetic polymeric nanoparticles, natural polymeric nanoparticles and/or lipid
based nanoparticles.
[0077] In another embodiment of the present invention, the antimalarial host cell mimetic
nanoparticle is prepared fromlow molecular weight polymer, preferably low molecular weight
30 Chitosan having molecular weight in the range of 50,000 – 1,90,000 Da.
16
[0078] In yet another embodiment of the present invention, the antimalarialhost cell
mimetic nanoparticle is prepared from water soluble salt of low molecular weight Chitosan. In a
preferred embodiment of the present invention, the water soluble salt of low molecular weight
Chitosan is Chitosan Hydrochloride.
5 [0079] In another embodiment, the present invention relates to a novel pharmaceutical
combination for the treatment of infectious diseases caused by intracellular pathogens,
particularly malaria, comprising Artemisinin or Artemisinin derivatives along withlow molecular
weight anti-coagulantand Erythrocyte mimeticnanoparticles.
[0080] In another embodiment, the present invention relates toa pharmaceutical
10 combination for the treatment of infectious diseases caused by intracellular pathogens,
particularly malaria, wherein Artemisinin derivative can be selected from but not limited to
Artesunate, Artemether, Artemotil and Dihydroartemisinin and the like.
[0081] In a preferred embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein Artemisinin derivative is Artesunate.
15 [0082] In another embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein low molecular weight anti-coagulant can be
selected from Enoxaparin, Bemiparin, Certoparin, Dalteparin, Nadroparin, Parnaparin,
Reviparin, Tinzaparin and the like.
[0083] In a preferred embodiment, the present invention relates to a pharmaceutical
20 combination for the treatment of malaria, wherein low molecular weight anti-coagulant is
Enoxaparin.
[0084] In another embodiment, the present invention relates to anti-malarial
pharmaceutical combination comprising Artesunate, Enoxaparin and Erythrocyte
mimeticnanoparticles, for the treatment of infectious diseases caused by intracellular pathogens
25 particularly malaria.
[0085] In another embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein the combination can be administered
concomitantly or sequentially.
[0086] In one embodiment, the present invention relates to a pharmaceutical combination
30 for the treatment of malaria, wherein the combination is administered concomitantly.
17
[0087] In another embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein the combination is administered sequentially.
[0088] In yet another embodiment, the present invention relates to a pharmaceutical combination
for the treatment of malaria, wherein the combination is administered such that a therapeutically
5 effective amount of Artesunateis administered concomitantly along with administering a
therapeutically effective amount of Erythrocyte mimetic nanoparticle and sequentially
administering a therapeutically effective amount of Enoxaparin.
[0089] In still another embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein the combination is administered such that a
10 therapeutically effective amount of Artesunateis administered concomitantly along with
administering a therapeutically effective amount of Enoxaparin and sequentially administering a
therapeutically effective amount of Erythrocyte mimetic nanoparticle.
[0090] In another embodiment, the present invention relates to a pharmaceutical combination for
the treatment of malaria, wherein the combination is administered such that a therapeutically
15 effective amount of Erythrocyte mimetic nanoparticle is administered concomitantly along with
administering a therapeutically effective amount of Enoxaparin and sequentially administering a
the rapeutically effective amount of Artesunate.
[0091] Most preferably Enoxaparin and Artesunate are administered concomitantly followed by
administration of Erythrocyte mimetic nanoparticle.
20 [0092] In still another embodiment, the present invention relates to a pharmaceutical
combination for the treatment of malaria, wherein Artemisinin derivatives, low molecular weight
anti-coagulant and Erythrocyte mimetic nanoparticles are present in a potentiating ratio.
[0093] The term 'potentiating ratio' is used herein to indicate that Artesunate, Enoxaparin and
Erythrocyte mimeticnanoparticle are present in a ratio such that the antimalarial activity of the
25 combination is greater than that of either Artesunate, Enoxaparin and Erythrocyte
mimeticnanoparticlealone or of the additive activity that would be predicted for the combination
based on the activities of the individual components. Thus the individual components act
synergistically in combination provided they are present in a potentiating ratio.
[0094] A significant antimalarial activity is exhibited by combining Artesunate,
30 Enoxaparin and Erythrocyte mimeticnanoparticles in a potentiating ratio. The combination
18
according to the present invention demonstrates therapeutic efficacyfor the treatment and/or
prophlyaxis of Plasmodium species caused malaria.
[0095] In another embodiment, the present invention relates to a pharmaceutical combination for
the treatment of malaria, wherein the ratio of Artesunate: Enoxaparin: Erythrocyte
5 mimeticnanoparticle, by weight of composition is in the range of 1: 0.1 to 100: 0.05 to 200.
[0096] According to a preferred embodiment of the present invention, a potentiating ratio of the
pharmaceutical combination, which is successfully used to treat malaria, in animals is 1:20:20 by
weight of the total combination of Artesunate: Enoxaparin: Erythrocyte mimetic nanoparticle.
The same has been demonstrated by in-vivo experiments as shown in the Examples. The exact
10 potentiating ratio of the pharmaceutical combination that will lead to therapeutic efficacy in
humans can be determined by carrying out the relevant experimentation/ clinical trials in
humans.
[0097] More preferably, in animal models, Enoxaparin and Artesunate are administered
concomitantly in the ratio of 20:1 followed by administration of Erythrocyte mimetic
15 nanoparticle.
[0098] The amount of a combination of separate Artesunate, Enoxaparin and Erythrocyte
mimeticnanoparticle required to be effective as an antimalarial agent will, of course, vary and is
ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered
include the route of administration and nature of the formulation, the mammal's bodyweight, age
20 and general condition and the nature and severity of the disease to be treated.
[0099] It should be understood that the dosages referred to above are calculated in terms of
the drugs per se.
[00100] In one embodiment, the pharmaceutical combination drug, for the treatment of
malaria, can exhibit one or more properties of Attach, Attune and Attack. The term “attach” with
25 respect to the pharmaceutical combination drug, for the treatment of malaria, refers to a drug that
can bind with the pathogen by interacting with the surface antigens of the pathogen. This
interaction can either be reversible or irreversible. Further, this interaction can be based on
electrostatic forces between positively charged domains of the host and negatively charged
domains of the pathogen and drug. Also this interaction can be covalent bonding wherein the
30 drug molecule binds irreversibly with the surface antigen of the pathogen. The term “attune”
with respect to the pharmaceutical combination drug, for the treatment of malaria, refers to a
19
drug that can render the pathogen ineffective to infect the host cell. One of the possible ways is a
result of receptor lock and key mechanism wherein the drug molecule blocks the availability of
the active sites present on the surface antigenic receptor of the pathogen. This can be attributed
to configureational changes in the surface antigens of the pathogen as a result of interaction with
5 the drug molecule. The term “attack” with respect to the pharmaceutical combination drug, for
the treatment of malaria, refers to a drug that is able to kill/stop/inhibit/retard the growth of the
pathogen. One of the mechanisms is interfering directly with the biological growth. Another
mechanism is by increasing the residence time period of the pathogen in the blood stream
thereby making the pathogen exposed to an attack by the host immune cells.
10 [00101] The novel anti-malarial pharmaceutical combination according to the embodiments
of the present invention can exhibit one or more therapeutic roles.
[00102] According to one embodiment of the present invention, the novel anti-malarial
pharmaceutical combination can inhibit the invasion of the host cell by the pathogen.
[00103] According to another embodiment of the present invention, the novel anti-malarial
15 pharmaceutical combination can directly kill the pathogen.
[00104] According to yet another embodiment of the present invention, the novel antimalarial pharmaceutical combination promotes phagocytic uptake of the pathogen by the
macrophages.
[00105] According to still another embodiment of the present invention, the novel anti20 malarial pharmaceutical combination promotes activation ofhost immune response against the
pathogen.
[00106] According to another embodiment of the present invention, the novel anti-malarial
pharmaceutical combination confers immunity against the pathogen.
[00107] According to yet another embodiment of the present invention, the novel anti25 malarial pharmaceutical combination reduces the parasite load, inhibiting the effect of parasites
on various host cells, thus ameliorating the clinical symptoms and/or any other condition
resulting from pathophysiological processes occurring in malaria or its treatment.
[00108] The novel pharmaceutical combination according to the embodiments of the present
invention demonstrates enhanced antimalarial effect at very low dose of Artesunate. Based on
30 the animal studies, it was found that the combination of Enoxaparin and Erythrocyte mimetic
Nanoparticle exerts approximate same efficacy as exerted by Artesunate alone when
20
administered at a dose of 3 mg/kg body weight. Surprisingly, it was found that the synergistic
combination of using Artesunate at very low doses of 0.3mg/kg alongwith Enoxaparin and
Erythrocyte mimetic nanoparticle, resulted in an 8-fold rapid decline in parasite proliferation, as
described in Table 2. The use of Enoxaparin and Biomimetic nanoprticles thus led to drastically
5 reducing the amount of Artesunate needed, which would enable slowing the development of
resistance against Artemisinin.
[00109] The novel pharmaceutical combination according to the embodiments of the present
invention demonstrates dual mode of action arresting the progression of disease. Most of the
currently used antimalarial drugs/combinations have inhibitory effect only on the intra10 erythrocytic stages of life cycle of P. falciparum, resulting in severe anemia due to enhanced
destruction of infected RBCs in late stage malaria that often leads to death. The novel
pharmaceutical combination according to the present invention exerts inhibitory effects at both
extra-erythrocytic and intra-erythrocytic stages. The anti-coagulant Enoxaparin in combination
with Artesunate offers a novel combination for treatment of malaria and associated conditions.
15 [00110] The novel pharmaceutical combination according to the embodiments of the present
invention demonstrates reduced risk of development of resistance to combination. Enoxaparin
and host cell mimetic nanoparticles possess unique ability to inhibit merozoite interaction with
erythrocytes by simultaneously targeting the multiple antigens present on the surface of
merozoites. From in-vitro and in-vivo studies, it has been found out that the probability of
20 multiple molecules to simultaneously acquire resistance to low molecular weight anti-coagulant
is very low and thus the risk for emergence of parasites resistant to anti-coagulant is also
extremely low.
[00111] The novel pharmaceutical combination according to the embodiments of the present
invention upregulates host immunomodulatory response. For a robust and protective immune
25 response, activation of both cell mediated and humoral immunity is required. However,
treatment with Artesunate alone downregulates the host immune response by decreasing the
number of splenic and circulatory B-cells. Further, it also reduces the production of mediators of
pro-inflammatory cytokines. The use of pharmaceutical combination according to the present
invention demonstrates that the simultaneous administration of Enoxaparin and Erythrocyte
30 mimetic nanoparticle upregulates the host immune response and increases the number of splenic
and circulatory B cells required for generation of an effective immune response.
21
[00112] The novel pharmaceutical combination according to the embodiments of the present
invention prevents recurrence of blood stage infection. Artesunate treatment even at very high
doses of 100mg/kg invariably results in recurrence of infection. However, treatment of infected
mice with the combination comprising Artesunate along with Enoxaparin and Erythrocyte
5 mimetic nanoparticles not only inhibitserythrocyte reinvasion by merozoites but also prevent
recurrence of infection.
[00113] The novel pharmaceutical combination according to the embodiments of the present
invention is a novel therapy for treatment against infectious diseases caused by intracellular
pathogens, particularly malaria, exhibiting excellent antimalarial therapeutic efficacy and
10 reducing the growth of the parasites. The drug combination treatment also allows development of
natural immunity that provides protection against subsequent secondary infection.
[00114] In yet another embodiment, the present invention relates to a formulation
comprising pharmaceutical combination along with a pharmaceutically acceptable carrier, for the
treatment ofinfectious diseases caused by intracellular pathogens, particularly malaria.
15 [00115] In a preferredembodiment, the present invention relates to a formulation comprising
pharmaceutical combination of Artesunate, Enoxaparin and Erythrocyte mimetic nanoparticles
along with a pharmaceutically acceptable carrier, for the treatment of infectious diseases caused
by intracellular pathogens, particularly malaria.
[00116] According to another embodiment of the present invention, the pharmaceutical
20 formulations may further comprise combination of active ingredients, namely, Artesunate,
Enoxaparin and Erythrocyte mimeticnanoparticle, together with one or more pharmaceutically
acceptable carriers thereof and optionally along with other therapeutic and/or prophylactic
ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible
with the other ingredients of the formula and not deleterious to the recipient thereof.
25 [00117] Pharmaceutical formulations include those suitable for oral, topical (including
dermal, buccal and sublingual), rectal and parenteral (including subcutaneous, intradermal,
intramuscular and intravenous), administration as well as administration by naso-gastric tube.
The formulation may, where appropriate, be conveniently presented in discrete dosage units and
may be prepared by any of the methods well known in the art of pharmacy. All methods include
30 the step of bringing into association the active ingredients with liquid carriers or finely divided
solid carriers or both and then, if necessary, shaping the product into the desired formulation.
22
[00118] Pharmaceutical formulations suitable for oral administration wherein the carrier is a
solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets
each containing a predetermined amount of the active ingredients. A tablet may be made by
compression or molding, optionally with one or more accessory ingredients. Compressed tablets
5 may be prepared by compressing in a suitable machine the active compounds in a free-flowing
form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent,
lubricating agent, surface-active agent or dispersing agent. Molded tablets may be made by
molding an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may
optionally be scored. Capsules may be prepared by filling the active ingredients, either alone or
10 in admixture with one or more accessory ingredients, into the capsule shells and then sealing
them in the usual manner. Cachets are analogous to capsules wherein the active ingredients
together with any accessory ingredient(s) are sealed in a rice paper envelope. Formulations for
oral administration include controlled release dosage forms e.g. tablets wherein the active
ingredients are formulated in an appropriate release controlling matrix, or are coated with a
15 suitable release controlling film. Such formulations may be particularly convenient for
prophylactic use.
[00119] The combination of Artesunate, Enoxaparin and Erythrocyte mimetic nanoparticle
may also be formulated as dispersible granules, which may for example be suspended in water
before administration, or sprinkled on food. The granules may be packaged e.g. in a sachet.
20 [00120] Formulations suitable for oral administration wherein the carrier is a liquid may be
presented as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oilin-water liquid emulsion.
[00121] The active ingredients may also be formulated as a solution or suspension suitable
for administration via a naso-gastric tube.
25 [00122] Pharmaceutical formulations suitable for rectal administration wherein the carrier is
a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa
butter and other materials commonly used in the art. The suppositories may be conveniently
formed by admixture of the active combination with the softened or melted carrier(s) followed
by chilling and shaping in moulds.
30 [00123] Pharmaceutical formulations suitable for parenteral administration include sterile
solutions or suspensions of the active combination in aqueous or oleaginous vehicles. Injectible
23
preparations may be adapted for bolus injection or continuous infusion. Such preparations are
conveniently presented in unit dose or multi-dose containers which are sealed after introduction
of the formulation until required for use. Alternatively, the active ingredients may be in powder
forms which are constituted with a suitable vehicle, such as sterile, pyrogen-free water, before
5 use. The combination of Artesunate, Enoxaparin and Erythrocyte mimeticnanoparticlemay also
be formulated as dispersible granules, which may for example be suspended in water before
administration, or sprinkled on food.
[00124] Formulations suitable for oral administration wherein the carrier is a liquid may be
presented as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil10 in-water liquid emulsion.
[00125] The combination of Artesunate, Enoxaaprin and Erythrocyte
mimeticnanoparticlemay also be formulated as a long-acting depot preparation, which may be
administered by intramuscular injection or by implantation e.g. subcutaneously or
intramuscularly. Depot preparations may include, for example, suitable polymeric or
15 hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly
convenient for prophylactic use.
[00126] In one embodiment, the present invention provides a process for the preparation of
a pharmaceutical formulation comprising Artesunate, Enoxaparin and Erythrocyte
mimeticnanoparticles along with a pharmaceutically acceptable carrier.
20 [00127] It should be understood that in addition to the aforementioned carrier ingredients
the pharmaceutical formulations for the various routes of administration described above may
include, as appropriate one or more additional carrier ingredients such as diluents, buffers,
flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including
anti-oxidants) and the like, and substances included for the purpose of rendering the formulation
25 isotonic with the blood of the intended recipient.
[00128] In yet another embodiment, the present invention relates toa pharmaceutical
combination comprising Artemisinin derivatives, low molecular weight anti-coagulant along
with Erythrocyte mimeticnanoparticles,for the treatment and/or prophylaxis of infectious
diseases caused by intracellular pathogens, particularly malaria.
30 [00129] In yet another embodiment, the present invention relates toa pharmaceutical
combination comprising Artesunate, Enoxaparin along with Erythrocyte mimetic
24
nanoparticles,for the treatment and/or prophylaxis of infectious diseases caused by intracellular
pathogens, particularly malaria.
[00130] In still another embodiment, the present invention relates to a pharmaceutical
combination comprising Artemisinin derivatives, low molecular weight anti-coagulant along
5 with Erythrocyte mimetic nanoparticles, for the manufacture of a medicament, for the treatment
of infectious diseases caused by intracellular pathogens, particularly malaria.
[00131] In another preferred embodiment, the present invention relates to a pharmaceutical
combination comprising Artesunate, Enoxaparin along with Erythrocyte mimetic nanoparticles,
for the manufacture of a medicament, for the treatment of infectious diseases caused by
10 intracellular pathogens, particularly malaria.
[00132] In another embodiment, the present invention relates to a method of treatment of
infectious diseases caused by intracellular pathogens, particularly malaria comprising
administering a therapeutically effective amount of the pharmaceutical combination comprising
Artemisinin derivatives, low molecular weight anti-coagulant along with Erythrocyte mimetic
15 nanoparticles, to a subject in need thereof.
[00133] In a preferred embodiment, the present invention relates to a method of treatment of
infectious diseases caused by intracellular pathogens, particularly malaria, comprising
administering a therapeutically effective amount of the pharmaceutical combination comprising
Artesunate, Enoxaparin along with Erythrocyte mimetic nanoparticles, to a subject in need
20 thereof.
[00134] Thus, in a preferred embodiment the present invention provides a method for the
treatment and/or prophylaxis of infectious diseases caused by intracellular pathogens,
particularly malaria, which comprises administering concomitantly a therapeutically effective
amount Enoxaparin and Artesunate in the ratio 20:1 and sequentially administering a
25 therapeutically effective amount of Erythrocyte mimetic nanoparticle.
[00135] In yet another embodiment, the present invention relates to use of the
pharmaceutical combination comprising Artemisinin derivatives, low molecular weight anticoagulant alongwith Erythrocyte mimetic nanoparticles, for the treatmentand prophylaxis of
infectious diseases caused by intracellular pathogens, particularly malaria.
30 [00136] In yet another embodiment, the present invention relates to use of the
pharmaceutical combination comprising Artesunate, Enoxaparin along with Erythrocyte
25
mimeticnanoparticles, for the treatment of infectious diseases caused by intracellular pathogens,
particularly malaria.
[00137] In yet another embodiment, the present invention relates to use of the
pharmaceutical combination where in Enoxaparin and Artesunateare administered to a subject in
5 need thereof, in the ratio 20:1 followed by sequential administration of with Erythrocyte
mimeticnanoparticle, for the treatment and/or prophylaxis of infectious diseases caused by
intracellular pathogens, particularly malaria.
[00138] While the foregoing describes various embodiments of the disclosure, other and
further embodiments of the disclosure may be devised without departing from the basic scope
10 thereof. The scope of the invention is determined by the claims that follow. The invention is not
limited to the described embodiments, versions or examples, which are included to enable a
person having ordinary skill in the art to make and use the invention when combined with
information and knowledge available to the person having ordinary skill in the art.
[00139] The present invention is further explained in the form of following examples.
15 However, it is to be understood that the following examples are merely illustrative and are not to
be taken as limitations upon the scope of the invention.
[00140] Source of starting Materials: Artesunate was purchased commercially from Edinburg
Pharmaceuticals, Manimajra, India while Enoxaparin (Clexane) was obtained commercially from
Healthcare Medical Centre, Mumbai, India. Erythrocyte mimeticnanoparticles were synthesized
20 as per procedure described in Example 1. Plasmodium berghei (NK 65) strain, used for
evaluation of antimalarial activity, as described in detail in Example 3, was obtained from the
library of strains maintained in the Parasitology Lab, Department of Zoology, Panjab University,
Chandigarh.
[00141] Example 1: Preparation of Erythrocyte Mimetic nanoparticles
25 [00142] Methodology: Preparation of Erythrocyte membrane coated Chitosan
nanoparticles
[00143] Step-1:Preparation of Chitosan nanoparticles
[00144] Low molecular weight Chitosan was dissolved overnight in an aqueous solution
of acetic acid to form a final concentration of 0.5mg/ml having pH 3.6. The resulting Chitosan
30 solution was filtered using pore size 0.45 µm, Millipore, USA to remove insoluble particles.
26
[00145] Chondroitin sulphate was dissolved in ultrapure water at a concentration of 1.5
mg/mL and also filtered using syringe filter; pore size 0.22 µm.
[00146] In varying ratios, Chitosan and Chondroitin sulphate were mixed by magnetically
stirring. Briefly, five milliliters of Chitosan solution in a 50 mL round-bottom flask was
5 preheated in a water bath at 60 ◦C for 10 min, the flask was then placed on the magnetic stirrer
stirring at 700 rpm, and 5 ml of Chondroitin sulphate solution was added to the Chitosan
solution. To initiate the nanoparticle synthesis, the pH of the resulting solution was adjusted from
3.6 to 4.7–6.2 using 20 wt% aqueous Sodium hydroxide solution. The reaction was carried out
for 10 min and theresulting suspensionwas centrifuged at 12000rpm for 30 mins at 4oC and re10 suspended in water.
Step-2:Preparation of RBC-Membrane Derived Vesicles
RBC membranes were collected in pellet by centrifuging haemolysed RBCs.
Step- 3: Fusion of RBC-Membrane-Derived Vesicles with nanoparticles
RBC membrane cloaking was accomplished by dispersing and fusing RBC membrane vesicles
15 with Chitosan nanoparticlesvia sonication using a bath sonicator for 2 min.
Step- 4: Freeze Drying and Lyophilization
The Erythrocyte mimeticnanoparticles were repeatedly washed with deionized water and
freeze-dried for 48h at −80 °C to obtain the powdered Erythrocyte membrane coated Chitosan
nanoparticles.
20 [00147] Example 2: Physicochemical Characterization of Chitosan and Erythrocyte
mimeticnanoparticles
[00148] Size Characterization: Chitosan nanoparticles were characterized by analyzing
their size (with appropriate dilutions using triple distilled water) using laser diffraction
(Zetasizer, Malvern Instruments, UK). Appropriate dilutions of the dispersion were made for
25 particle size determination. As shown in Figure 2a, Size of Chitosan nanoparticles was found to
be 232nm. Size of Erythrocyte mimetic nanoparticle as shown in Figure 2bwas found to be
302nm.
[00149] Zeta potential of Chitosan nanoparticles(with appropriate dilutions using triple
distilled water) was determined using laser diffraction (Zetasizer, Malvern Instruments, UK). As
30 shown in Figure 3a zeta potential of Chitosan nanoparticles was found to be -30mV.The zeta
potential of Erythrocyte mimetic nanoparticle was found to be -21.2 mVas shown in Figure 3b.
27
[00150] FTIR Spectra Analysis: The IR spectroscopy was used to elaborate the structure
and stereochemistry of the bulk material and the nanoparticles. Analysis of polymer and
nanoparticles was done using Perkin Elmer Spectrum Version 10.03.08.
[00151] Figure 4a shows the FT-IR spectra of Chitosan hydrochloride. The
characterization peaks in the Chitosan spectrum are 3399 cm-1
5 (–OH and NH2 stretching), 2926
cm-1
(C-H stretching) 1629.87 cm-1
(amide I band), 1523.79 cm-1
(amide II band), and 1154.6 cm1
(C-O stretching). Shown in Figure 4b is the FTIR spectrum of the Chitosan nanoparticles
characterized by appearance of a band at 1058 cm-1
assigned to NH3SO3 stretching, an evidence
of the interaction between both polymers.
10 [00152] Scanning Electron Microscopy: Morphology of Chitosan nanoparticles was
examined using Scanning Electronic Microscope (Hitachi , Japan). As shown in Figures 5a and
5b, nanoparticles were polyhedral in shape.
[00153] Example 3: In-vivo studies and results
[00154] Step 1: Artesunate intravenous injection was prepared by dissolving 60mg
15 Artesunate was dissolved in 1ml of (5%) Na2HCO3.The solution, thus obtained was diluted with
0.9% Normal saline to desired dosage and volume.
[00155] Enoxaparin intravenous injection was prepared by diluting 60mg Enoxaparinwith
0.9% Normal saline to desired dosage.
[00156] 10 mg of Erythrocyte mimetic nanoparticles(obtained as per example 1) were
20 dissolved in 0.9% Normal saline to desired dosage.
[00157] Step 2: Female Swiss mice weighing 22-26g were obtained from the Central
animal facility, NIPER, Mohali and used as experimental models. They were maintained on a
standard pellet diet and water ad libtum. Plasmodium berghei (NK 65) strain was used for
evaluation of antimalarial activity and was maintained inthemice by intraperitoneal inoculation
of 1×106
25 infected erythrocytes to naïve mice (Santiyanont 1985). After challenge, mean
percentparasitemia was checked by preparing Giemsa stained thin blood smears on glass slides
through tail vein incision .The ethical clearance was obtained from Institutional Animal Ethics
Committee of Panjab University (PU/45/99/CPCSEA/IAEC/2019/311) and experiments were
conducted according to Committee for the Purpose of Control And Supervision of Experiments
30 on Animals (45/GO/ReBi/S/99/CPCSEA) guidelines. Mean percent parasitemia of the animals
was calculated as follows.
28
Mean percent parasitemia= Infected RBCs *100
Total no. of RBCs
Table 1
Serial
No.
Groups of Animals
(Abbreviation)
Controls / Treatments
1 Normal Control (N.C) Without Infection
2 Infected Control (I.C) Infected+ Distilled Water
3 Vehicle control (V.C) Infected + Normal Saline
4 Postitive Control (P.C) Infected and Treated with Artesunate(30mg/kg for 4 days)
orally
5 Treatment Group 1
(T-1)
Infected and Treated with Artesunate (0.3mg/kg b.w.)
intravenously
6 Treatment Group 2
(T-2)
Infected and Treated with Enoxaparin (6mg/kg b.w.)
intravenously
7 Treatment Group 3
(T-3)
Infected and Treated intravenously with combination of
Artesunate (0.3mg/kg b.w.) + Enoxaparin (6mg/kg b.w.)
8 Treatment Group 4
(T-4)
Infected and Treated intravenously with combination of
Artesunate (0.3mg/kg b.w) + Enoxaparin (6mg/kg b.w) +
Erythrocyte mimetic nanoparticle (6mg/kg b.w)
[00158] Eight groups (1 - 8) having 6 mice each (female, weighing 22-26 gm) were used
for present study (Table 1). All groups except N.C, were injected with 1×106
5 P. bergheiinfected
erythrocytes on day zero (D0). Treatment was continued for 4 days (Day 0 - Day 3). Drugs to T1,T-2,T-3 and T-4 groups of animals were administered intravenously
(0.025ml/mouse/injection). However to P.C group of animals, the drug was administered orally
to a total volume of 100µl/mice, (* P.C treated animals being the standard positive control
10 group*). The dosage of drugs to be administered intravenously was fixed at Enoxaparin (6mg/kg
body weight), Artesunate (0.3 mg/kg of body weight) and Erythrocyte mimetic nanoparticles (6
mg/kg of body weight) once a day starting from first day of post inoculation (pi) for 4 days (D0-
D3) to monitor the efficacy and potency of said combination. Dosage of orally administrable
29
Artesunate was fixed at 30mg/kg of bodyweight to be administered in a total volume of
0.1ml/mouse. Parasitemia was checked on 7
th daypost inoculation (D6 pi).
[00159] Statistical analysis
[00160] Resulting data is presented as mean and standard deviation (SD). Statistical
5 evaluation of differences between the experimental groups was determined by the Student’s t-test
with the level of significance of p<0.0005 using Graph Pad Software (San Diego, California,
USA). The results are depicted in Table 2 and illustrated graphically in Figure 6.
Table 2
Serial
No.
Group (Abbreviation) Controls / Treatments Mean percent
parasitemia
On Day 7
1 Normal Control (N.C) Without Infection 0
2 Infected Control (I.C) Infected+ Distilled Water 37.63 ± 1.95
3 Vehicle control (V.C) Infected + Normal Saline 36.64 ± 4.1
4 Postitive Control (P.C) Infected and Treated with
Artesunate(30mg/kg for 4 days)
orally
16.34 ± 1.75
5 Treatment Group 1
(T-1)
Infected and Treated with
Artesunate (0.3mg/kg b.w.)
intravenously
36.25 ± 5.2
6 Treatment Group 2
(T-2)
Infected and Treated with
Enoxaparin (6mg/kg b.w.)
intravenously
10.06 ± 0.698
7 Treatment Group 3
(T-3)
Infected and Treated intravenously
with combination of Artesunate
(0.3mg/kg b.w.) +
Enoxaparin (6mg/kg b.w.)
9.23 ± 0.404
8 Treatment Group 4
(T-4)
Infected and Treated intravenously
with combination of Artesunate
(0.3mg/kg b.w) + Enoxaparin
2.1± 0.290
30
(6mg/kg b.w) + Erythrocyte
mimetic nanoparticle (6mg/kg b.w)
[00161] Course of parasitemia in experimental groups: Maximum infection of 37.63%
± 1.95 was observed in infected control (I.C) by 7th day post inoculation. In Artesunate
monotherapy (P.C treated animals), the infection was reduced to 16.34±1.75% by day 7 whereas
5 in T-1 treated animals, there was no significant reduction in parasitemia (p<0.0005). In
Enoxaparin monotherapy (T-2 treated animals), there was significant reduction in parasitemia
compared to T-1 treated animals (p<0.0005). However in T-3 treated animals, there was no
significant reduction in parasitemia compared to T-2 treated animals (p<0.0005). Surprisingly,
in mice treated with combination of low dose Artesunate, Enoxaparin and Erythrocyte mimetic
10 nanoparticle (T-4 treated animals), there was drastic reduction in parasitemia of 8 fold order on
day 7 compared to P.C. treated animals (p<0.0005).
[00162] It is thus clearly evident from the animal studies that combination of Enoxaparin,
Erythrocyte mimetic nanoparticle and Artesunate (at low concentrations) is an effective
antimalarial treatment option.
15 [00163] The novel pharmaceutical combination according to the embodiments of the
present invention demonstrates enhanced antimalarial effect at very low dose of Artesunate. The
use of Enoxaparin and Erythrocyte mimetic nanoparticles thus led to surprisingly reduce the
amount of Artesunate needed as indicated in Table 2, which would enable slowing the
development of resistance against Artemisinin derivatives.
20 [00164] The foregoing examples are merely illustrative and are not to be taken as
limitations upon the scope of the invention. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such changes and modifications may be
made without departing from the scope of the invention.
25 ADVANTAGES OF THE PRESENT INVENTION
[00165] The present invention provides a novel pharmaceutical combination for the
treatment of malaria.
31
[00166] The present invention provides a pharmaceutical combination for the treatment of
malaria that acts rapidly, produces fast clinical responses to treatment and reduces the growth of
the parasites.
[00167] The present invention provides a pharmaceutical combination for the treatment of
5 malaria, having dual mode of action arresting the progression of disease and inhibits both extraerythrocytic and intra-erythrocytic stages.
[00168] The present invention provides a pharmaceutical combination having reduced risk
of development of resistance.
[00169] The present invention provides a pharmaceutical combination for the treatment of
10 malaria that upregulates host immunomodulatory response

We Claim:

1. A novel pharmaceutical combination for the treatment of infectious diseases caused by
intracellular pathogens, particularly malaria, comprising drugs having intra-erythrocytic
5 effect on pathogen along with drugs having extra-erythrocytic effect on pathogen and
host cell mimetic nanoparticles, wherein the pathogen is Plasmodium.
2. The pharmaceutical combination as claimed in claim 1, whereindrugs having intraerythrocytic effect on Plasmodium are selected from quinine and its derivatives,
Chloroquine, Hydroxychloroquine, Amodiaquine, Pyrimethamine, Proguanil,
10 Sulfonamides, Mefloquine, Atovaquone, Primaquine, Halofantrine, Lumefantrine,
Doxycycline, Clindamycin, Artemisinin or Artemisinin derivatives,
particularlyArtesunate, artemether, artemotil and dihydroartemisinin.
3. The pharmaceutical combination as claimed in claim 2, whereindrug having intraerythrocytic effect on Plasmodium isArtesunate.
15 4. The pharmaceutical combination as claimed in claim 1, whereindrugs having extraerythrocytic effect onPlasmodium are selected fromstructural carbohydrates like sulfated
glycosaminoglycans (Hyaluronan, Chondroitin Sulphate, Dermatan sulphate, Heparin,
KeratanSulphate), anti-coagulants (Dalteparin, Danaparoid, Enoxaparin, Tinzaparin,
Apixaban, EdoxabanTosylate, Fondaparinux, Rivaroxaban, Betrixaban), Cysteine
20 Protease Inhibitor, Calcium Chelator andCalcium Pump Inhibitors.
5. The pharmaceutical combination as claimed in claim 4, where in drug having extraerythrocytic effect onPlasmodium is anti-coagulant, specifically, low molecular weight
anti-coagulant selected from Enoxaparin,Bemiparin, Certoparin, Dalteparin, Nadroparin,
Parnaparin, Reviparin and Tinzaparin.
25 6. The pharmaceutical combination as claimed in claim 5, wherein drug having extraerythrocytic effect on Plasmodium is low molecular weight anti-coagulant Enoxaparin.
7. The pharmaceutical combination as claimed in claim 1, where in the Host cell mimetic
nanoparticle is a host cell membrane functionalized nanoparticle selected from core-shell
Erythrocyte mimetic nanoparticleor fused Erythrocyte mimetic nanoparticle.
30
33
8. The pharmaceutical combination as claimed in claim 7, wherein the nanoparticle is
completely or partially biodegradable or non-biodegradable and can be selected from
carbon-based nanoparticles, ceramic nanoparticles, metal nanoparticles, semiconductor
nanoparticles, synthetic polymeric nanoparticles, natural polymeric nanoparticles and/or
5 lipid based nanoparticles.
9. The pharmaceutical combination as claimed in claim 8, wherein Biomimetic nanoparticle
is prepared from low molecular weight polymer, preferably low molecular weight
Chitosan having molecular weight in the range of 50,000 – 1,90,000 Da.
10. The pharmaceutical combination as claimed in claim 9, wherein low molecular weight
10 chitosan is a water soluble salt of low molecular weight chitosan, preferably Chitosan
Hydrochloride.
11. Anovel pharmaceutical combination for the treatment of infectious diseases caused by
intracellular pathogens, particularly malaria, comprising Artemisinin or Artemisinin
derivatives along withlow molecular weight anti-coagulantandErythrocyte mimetic
15 nanoparticles.
12. Anovel pharmaceutical combination for the treatment of infectious diseases caused by
intracellular pathogens, particularly malaria, comprising Artesunate, Enoxaparin and
Erythrocyte mimetic nanoparticles.
13. The pharmaceutical combination as claimed in claims 1-12, whereinthe combination can
20 be administered concomitantly or sequentially.
14. The pharmaceutical combination as claimed in claim 11, wherein Artemisinin
derivatives, low molecular weight anti-coagulant and Erythrocyte mimetic nanoparticles
are present in a potentiating ratio.
15. The pharmaceutical combination as claimed in claim 12, where in Enoxaparin and
25 Artesunate are administered concomitantly followed by administration of Erythrocyte
mimeticnanoparticle.
16. The pharmaceutical combination as claimed in claim 12, wherein the potentiating ratio of
Artesunate: Enoxaparin: Erythrocyte mimetic nanoparticle, by weight of composition is
in the range of 1: 0.1 to 100: 0.05 to 200.
30
34
17. Apharmaceutical formulation for the treatment of infectious diseases caused by
intracellular pathogens, particularly malaria comprising the pharmaceutical combination
as claimed in claims 1, 11 and 12, along with a pharmaceutically acceptable carrier.
18. The pharmaceutical formulations as claimed in claim 17, wherein the formulation is
5 suitable for oral, topical (dermal, buccal and sublingual), rectal and parenteral
(subcutaneous, intradermal, intramuscular and intravenous) and naso-gastric tube
administration.
19. The pharmaceutical combination as claimed in claims 1, 11 and 12, for the manufacture
of a medicament, for the treatment of infectious diseases caused by intracellular
10 pathogens, particularly malaria.
20. A method of treatment of infectious diseases caused by intracellular pathogens,
particularly malaria, comprising administering a therapeutically effective amount of the
pharmaceutical combination as claimed in claims 1, 11 and 12, to a subject in need
thereof.
15 21. The method of treatment as claimed in claim 20, comprising administering to a subject in
need thereof, a therapeutically effective amount Enoxaparin and Artesunate in the ratio
20:1 concomitantly and sequentially administering a therapeutically effective amount of
Erythrocyte mimetic nanoparticle.
22. Use of the pharmaceutical combination as claimed in claims 1, 11 and 12, for the treat
20 mentand prophylaxis of infectious diseases caused by intracellular pathogens, particularly
malaria.
23. Use of the pharmaceutical combination as claimed in claim 22, where in Enoxaparin and
Artesunateare administered to a subject in need thereof, in the ratio 20:1 followed by
sequential administration of with Erythrocyte mimetic nanoparticle.