[0001] The present invention relates to a novel pharmaceutical kit for producing an
5 immune response against infectious diseases caused by intracellular pathogens, particularly
malaria. Specifically, the present invention relates to a novel pharmaceutical kit comprising
atleast one unit dosage of pathogenic inoculum and atleast one unit dosage of drugs having extraerythrocytic effect on pathogen, for producing an immune response against malaria. The present
invention also relates to a pharmaceutical kit for preparation of medicament, for producing an
10 immune response against malaria.
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
15 or relevant to the presently claimed invention, or that any publication specifically or implicitly
referenced is prior art.
[0003] 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
20 humans. Plasmodium falciparum is the most prevalent malaria parasite in the WHO African
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
Pacific Region (65%). Outside of Africa, Plasmodium vivax is the most endemic form present in
approximately 50 countries; collectively accounting for half the world’s malaria. Although
25 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.
[0004] Intracellular pathogens are organisms that are capable of growing and reproducing
inside host cells. Intracellular pathogens cause persistent infections by evading immune
response, residing and multiplying inside the host cells. Although, these intracellular pathogens
30 are phylogenetically diverse and engage in diverse immune evasion and persistence strategies,
3
they share common pathogen type-specific mechanisms during host-pathogen interaction inside
host cells.
[0005] Immunity against intracellular pathogens can develop naturally. A chronic malaria
infection facilitates the acquisition of protective immunity against superinfections of this
5 parasite. However, this immunity is incomplete as individuals with such immunity still develop
malaria on subsequent infection. Besides chronic infection, repeated infection in humans living
in holo or hyperendemic areas, mainly in Africa and in some places of Papua-New Guinea, it is
only after repeated infections of P. falciparum that a progressive modulation of the immune
response is induced, eventually leading to an anti-parasite immunity characteristic of
10 premonition. However, this form of premonition immunity does not ensure development of
neutralizing antibodies and sterilizing immunity is developed only over a period of time ranging
to about 20 years.
[0006] It is well known that clinical immunity is acquired in endemic areas after a number of
years and a sufficient number of naturally acquired infections (Snow et al., Parasitol Today, 11,
15 188–190, 1995). Epidemiological studies in the above areas have helped to define three clinical
periods: a short period of 0-5 years where mortality can occur, a longer period of 0-15/20 years
where morbidity is frequent (though decreasing in frequency with age); thereafter a longer period
of premonition where the disease in any form is usually absent. Immunologically also, such
premonition immunity is partial, species specific and bi-phasic. In the first phase, the hosts still
20 get infected but do not develop clinical symptoms. Immunologically, this phase is characterized
by acquisition of in vivo protective antibodies. However, these antibodies are short lived. These
antibodies are lost once a person gets isolated from those malarial antigens by leaving the
endemic area (Langhorne et al., Nat Immunol 2008, 9, 725–32). On the contrary, the second
phase which is preventive or limitation of parasite multiplication, takes long to develop. This
25 second phase is heavily dependent on parasite exposure—more the exposure the host gets, lesser
the time this immunity takes to develop. Immunologically, this phase is characterized by
acquisition of in vivo protective antibodies and generation of T cell responses, majorly CD4 T
cells. Thus, in case of malaria development of natural immunity developed against a primary
infection does not always confer protection against any subsequent infection.
30 [0007] Immunity to pathogens can be developed with help of vaccines also. Traditionally
vaccines can be classified into live-attenuated vaccines, whole-inactivated vaccines. Live
4
attenuated vaccines are known to generate the best immune response by providing continual
antigenic stimulation giving sufficient time for memory cell production. However, the
development of a live attenuated vaccine by traditional continuous passaging either in-vitro
tissue cultures or in-vivo or a combination of both until the avirulent form can be isolated, is both
5 empirical and time-consuming. Immunization with radiation attenuated sporozoite forms has
consistently been shown to induce 90% protection in rodents and humans in experimental set-ups
(Friesen et al., Vaccine, 2011, 29(40), 7002-8; Hoffman et al., TLJ Infect Dis. 2002, 185(8),
1155-64). In case of malaria, producing sporozoites in such large numbers is a challenge in itself
and thus practically unfeasible.
10 [0008] Many vaccines also contain adjuvant. The term ―adjuvant‖ means ―to help/aid‖.
Adjuvants were initially used to enhance their immune modulating effects (Geeraedtset al.,
PLoSPathog. 2008, 4(8), e1000138). Homologous and heterologous protective immunity has
been reported in mice following vaccination with killed parasites/parasite lysate (including
merozoites) (McCarthy et al, Hum Vaccin, 6 (2010), pp. 114-123) when co-administered with
15 complete Freund's Adjuvant. In another study the protective efficacy of low doses (100 and
1000) of frozen/thawed pRBC in the adjuvant CpG-ODN has also been reported [Close et al., J
Clin Invest, 120 (2010), pp. 2967-2978). However, development of vaccine-adjuvant strategy
requires the identification and testing of adjuvants appropriate for use in humans (Close et al.,
Biomed Res Int, 2013 (2013), p. 282913) as Complete Freund’s adjuvant is not suitable for
20 administration to humans. Issues encountered with the selection and utilization of these
compounds includes: limited access to novel or existing proprietary adjuvants; unacceptable
safety profile of the final formulation; and a lack of understanding of the type of immune
response that is required.
[0009] In the scenarios of challenging pandemic and in non-endemic areas, this traditional
25 approach cannot be brought into use and would be of limited practical utility. With the advent of
modern recombinant based technology subunit vaccines, vector based vaccines or virus-like
particles have been attempted to generate an immune response. However the major drawback of
recombinant technology based-vaccines is the inability to replicate significantly and completely
antigenic diversity of target epitopes and the often-observed poor immunogenicity of the soluble
30 parasite-derived proteins used. Also, the antigenic variations occurring in the parasite in due
5
course of time lessen the efficacy of these vaccines. Against this background, it is reasonable that
a whole-parasite approach may perform better.
[0010] Antimalarial immunity can also be conferred by fine balancing between antimalarial
treatment regime and persistence of sub-patent clinical infection. The idea that using medicines
5 together with naturally acquired infection could result in the induction of protective immunity
has long been explored. An inadequate ant malarial treatment prolongs the presence of malaria
parasites in the host and this may serve as a prerequisite for the acquisition of protective
immunity against re-infection. A number of chemo-preventive approaches namely,
Chemoprophylaxis, Intermittent preventive treatment (IPT) and Controlled human malaria
10 infections (CHMI) have been applied for the development of immunity against Plasmodium.
Chemoprophylaxis involves mass administration of a full therapeutic course drug in such a way
that its blood concentration is maintained above the minimum inhibitory level to whole
population at risk. This can be done either directly, when a curative dose of the antimalarial drug
is given to an entire population, or indirectly, when the antimalarial is added to food, usually to
15 salt. Several studies conducted since the late 1950s, show that mass drug administration (MDA)
does substantially reduce the incidence of clinical malaria and parasite prevalence but that the
impact of MDAs was transitory (von Seidlein et al, Trends Parasitol, 2003, 19, 452–460).
[0011] In Intermittent preventive treatment (IPT), complete antimalarial treatment is
followed at specified time points without parasite screening. IPT restricts the use of antimalarials
20 to specific risk groups at specified time points. The idea is that parasite exposure will be less to
undesired sub-therapeutic drug concentrations while concomitantly allowing a more effective
generation of natural immunity in the intermittent periods between two doses of IPT
(Schellenberg et al., M., Lancet 2005, 365, 1481–1483; Schultz et al., Am J Trop Med Hyg,
1994, 51, 515–522).The WHO recommends IPT with sulfadoxine–pyrimethamine (SP) for areas
25 with high or moderate transmission to pregnant women (IPTp), and subsequently extended to
infants and children.
[0012] Controlled human malaria infection (CHMI) has been demonstrated to induce fully
protective immune responses (Sauerwein et al., Nat Rev Immunol, 2011, 11, 57–64). In this
mode, healthy malaria-naıve volunteers in malaria-endemic areas are exposed to natural infection
30 by Plasmodium-infected mosquito bites, whilst taking prophylactic drugs. An efficient sterile
protective immunity is known to be induced in rodents by inoculation of intact sporozoites
6
alongwith chloroquine, azithromycin, pyrimethamine or primaquine, chemoprophylaxis
(Belnoue et al., J Immunol., 2004, 172, 2487–2495; Borrmann et al., CurrOpinImmunol, 2011,
23, 500–508).Similarily, in an analogous clinical study, an efficient method was achieved by
exposing malaria-naıve volunteers to 12-15 P.falciparuminfected mosquito-bites once a month
5 for 3 months along with azithromycin or chloroquine (Dunne et al., J Infect Dis 2005, 191,
1582–1588), azithromycin and chloroquine (Nakornchaiet al., Acta Trop, 2006, 100, 185–191),
mefloquine (van Eijk et al., Cochrane Database Syst Rev, 2011, (2), CD006688),
dihydroartemisinin–piperaquine (Sinclair et al.,Cochrane Database Syst Rev 2009 (3),
CD007483). An effective sterile immunitycapable to generate a protective immune response was
10 observed on subsequent challenge with infected mosquitoes. Further cellular immune responses,
more specifically multifunctional effector memory T-cells have been associated with markers of
protection (Roestenberg et al., N Engl J Med, 2009, 361, 468–477; Roestenberg et al., Lancet,
2011, 377, 1770–1776; Teirlinck et al., PLoSPathog2011, 7, e1002389).However, the current
chemoprophylaxis based strategies use drugs that act intracellularly. Moreover, resistance to
15 these drugs has been reported, thereby questioning the protective efficacy of these drugs.
[0013] Thus there is an urgent need to develop new vaccination strategies to induce immune
response against Plasmodium. Need is felt for vaccines in the form of pharmaceutical kits that
are capable of reducing the time needed to acquire active immunity, provide long lasting
immunity and give reproducible results.
20
OBJECTS OF THE INVENTION
[0014] An object of the present invention is to provide a pharmaceutical kit for producing an
immune response against infectious diseases, which can overcome deficiencies associated with
the known arts.
25 [0015] Another object of the present invention is to provide a pharmaceutical kit for
producing an immune response against malaria.
[0016] Yet another object of the present invention is to provide a pharmaceutical kit for
producing an immune response against malaria, preferably without administration of an
adjuvant.
7
[0017] Another object of the present invention is to provide a pharmaceutical kit for
producing an immune response against malaria that provides protection against any subsequent
secondary infection.
[0018] Another object of the present invention is to provide a pharmaceutical kit that
5 produces fast clinical responses to treatment and reduces the growth of the parasites.
[0019] Another object of the present invention is to provide a pharmaceutical kit for
generating an immune response against malaria and having dual mode of action, by arresting the
progression of disease and inhibiting both extra-erythrocytic and intra-erythrocytic stages.
[0020] Another object of the present invention is to provide a pharmaceutical kit having
10 reduced risk of development of resistance.
[0021] Another object of the present invention is to provide a pharmaceutical kit that
upregulates host immunomodulatory response.
[0022] 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
15 conjunction with the accompanying examples and figures, which are not intended to limit scope
of the present invention in any manner.
SUMMARY OF THE INVENTION
[0023] This summary is provided to introduce a selection of concepts in a simplified form
20 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
used as an aid in determining the scope of the claimed subject matter.
[0024] The present invention relates to a novel pharmaceutical kit for producing an
immune response against infectious diseases caused by intracellular pathogens. Specifically, the
25 present invention relates to a novel pharmaceutical kit for the production of an immune response
against infectious diseases caused by intracellular pathogens, particularly malaria.
[0025] In another aspect, the present invention relates to a novel pharmaceutical kit for the
production of an immune response against malaria, the kit comprising pathogenic inoculum and
drug(s) having extra-erythrocytic mechanism of action against the pathogen.
30 [0026] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
production of an immune response against malaria, wherein the pathogen is Plasmodium.
8
[0027] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
production of an immune response against malaria, wherein the pathogenic inoculum is
Plasmodium pathogenic inoculum.
[0028] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
5 production of an immune response against malaria, wherein Plasmodium pathogenic inoculum is
selected from isolated, purified or unpurified mixture of liver stage Plasmodium parasites
infected erythrocytes or blood stage Plasmodium parasite infected erythrocytes.
[0029] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
production of an immune response against malaria, whereinliver stage Plasmodium parasites
10 includesporozoites, intact or lysate of sporzoite infected hepatocytes.
[0030] According to another aspect of the present invention, pathogenic inoculum capable
of suitably modifying immune response include but is not limited to isolated or purified bloodstage malaria parasites such as merozoitesschizonts, trophozoites or rings, intact or lysate of
blood stage malaria parasite infected erythrocytes, combination of isolated or purified blood15 stage malaria parasites such as merozoitesschizonts, trophozoites, rings, intact or lysate of blood
stage malaria parasite infected erythrocytes (pRBC) hereafter referred to as complete blood stage
Plasmodium pathogenic inoculum.
[0031] In a preferred aspect of the present invention, the pathogenic inoculum capable of
suitably modifying immune response isa complete blood stage Plasmodium pathogenic
20 inoculum.
[0032] In another embodiment, the present invention relates to a novel pharmaceutical kit
for the production of an immune response against malaria, wherein drug(s) having extraerythrocytic mechanism of action are selected from but not limited to structural carbohydrates
like sulfated glycosaminoglycans.
25 [0033] In another embodiment of the present invention, sulfated glycosaminoglycans are
selected from but not limited to Hyaluronan, Chondroitin Sulphate, Dermatan Sulphate, Heparin,
heparin sulphate, KeratanSulphate and the like.
[0034] According to a preferred embodiment of the present invention, the drug having
extra-erythrocytic mechanism of action is a sulfated glycosaminoglycan moiety particularly, a
30 Heparin sulfate moiety.
9
[0035] According to another preferred embodiment of the present invention, drug having
extra-erythrocytic mechanism of action isan anti-coagulant, specifically, low molecular weight
Heparin compound.
[0036] In another embodiment of the present invention, low molecular weight anti5 coagulant is selected from Enoxaparin, Bemiparin, Certoparin, Dalteparin, Nadroparin,
Parnaparin, Reviparin, Tinzaparin and the like.
[0037] According to a most preferred embodiment of the present invention, the drug
having extra-erythrocytic mechanism of action is Enoxaparin, or a functional fragment thereof.
[0038] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
10 production of an immune response against malaria,wherein kit comprises complete blood-stage
Plasmodium pathogenic inoculum and Enoxaparin.
[0039] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
production of an immune response against malaria,wherein components of the kit can be
administered concomitantly or sequentially.
15 [0040] In one embodiment, the present invention relates to a novel pharmaceutical kit
comprising atleast one dosage unit of complete blood stage Plasmodium pathogenic inoculum
and atleast one dosage unit of Enoxaparin, for the production of an immune response against
infectious diseases caused by intracellular pathogens particularly malaria.
[0041] In another aspect, the present invention relates to a novel pharmaceutical kitfor the
20 production of an immune response against malaria,wherein kit comprises one or two doses of
pathogenic inoculum and one or multiple doses of Enoxaparin.
[0042] In another aspect, the present invention relates to a novel pharmaceutical kit for the
production of an immune response against malaria, wherein the pathogenic inoculum is a ―low
dose‖ pathogenic inoculum, capable of inducing a sub-patent infection.
25 [0043] In another aspect, the present invention relates to a novel pharmaceutical kit for the
production of an immune response against malaria,wherein administration of components of the
kit to a subject in need thereof, is useful for prophylaxis of infectious diseases caused by
intracellular pathogens, particularly malaria.
[0044] In another aspect, the present invention relates to a novel pharmaceutical
30 kitcomprising low molecular weight anti-coagulant along with complete blood-stage
Plasmodium pathogenic inoculum, for the preparation of medicament for the production of an
10
immune response againstinfectious diseases caused by intracellular pathogens, particularly
malaria.
[0045] In another aspect, the present invention relates to a method of producing an immune
response against infectious diseases caused by intracellular pathogens, particularly malaria,
5 comprising administering a therapeutically effective amount of the components of
pharmaceutical kit, to a subject in need thereof.
[0046] In another aspect, the present invention relates touse of then ovel pharmaceutical kit
comprising blood stage Plasmodium pathogenic inoculum and low molecular weight anticoagulant, for the production of an immune response against malaria, and prophylaxis of
10 infectious diseases caused by intracellular pathogens, particularly malaria.
[0047] Other aspects of the invention will be set forth in the description which follows, and in
part will be apparent from the description, or may be learnt by the practice of the invention.
BRIEF DESCRIPTION OF DRAWINGS THE INVENTION
15 [0048] 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
to the drawings in combination with the detailed description of the specific embodiments
presented herein.
Figure 1: illustrates a Course of Parasitemia in various experimental groups on Primary
20 Challenge (expressed as Mean Percent Parasitemia).
Figure 2: illustrates a KAPLAN-MEIER Survival Plot illustrating animal survival in various
experimental groups on Primary Challenge.
Figure 3: illustrates a Course of Parasitemia in various experimental groups on Secondary / ReChallenge (expressed as Mean Percent Parasitemia).
25 Figure 4: Illustrates a KAPLAN-MEIER Survival Plot illustrating animal survival in various
experimental groups on Secondary / Re- Challenge.
DETAILED DESCRIPTION
[0049] The following is a detailed description of embodiments of the disclosure. The
30 embodiments are in such detail as to clearly communicate the disclosure. However, the amount
of detail offered is not intended to limit the anticipated variations of embodiments; on the
11
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.
[0050] 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
5 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.
[0051] Reference throughout this specification to ―one embodiment‖ or ―an embodiment‖
means that a particular feature, structure or characteristic described in connection with the
10 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.
[0052] In some embodiments, the numbers expressing quantities of ingredients, properties
15 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
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
20 numerical parameters should be construed in light of the number of reported significant digits
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
25 contain certain errors necessarily resulting from the standard deviation found in their respective
testing measurements.
[0053] 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
30 context clearly dictates otherwise.
12
[0054] 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.‖
[0055] The recitation of ranges of values herein is merely intended to serve as a shorthand
5 method of referring individually to each separate value falling within the range. Unless otherwise
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
10 embodiments herein is intended merely to better illuminate the invention and does not pose a
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.
[0056] Groupings of alternative elements or embodiments of the invention disclosed herein
15 are not to be construed as limitations. Each group member can be referred to and claimed
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
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
20 description of all Markush groups used in the appended claims.
[0057] The description that follows, and the embodiments described therein, is provided by
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.
25 [0058] 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
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.
30 [0059] The headings and abstract of the invention provided herein are for convenience only
and do not interpret the scope or meaning of the embodiments.
13
[0060] 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
5 embodiment comprises elements B and D, then the inventive subject matter is also considered to
include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0061] 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..
10 [0062] ―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).
[0063] The term ―Inoculum‖ refers to a substance introduced into the body to create or
increase the body's resistance or immunity to a disease. Determining the optimal amount of
inoculum dose is an important component of rational vaccine design. The inoculum dose, i.e.,
15 amount of antigen or live attenuated pathogen is important in determining immune response.
Higher levels although is known to a better stimulation of the immune response but might cause
more severe side effects.
[0064] The term ―low dose pathogenic inoculums‖ refers to administered dose wherein the
resultant parasite density is sufficiently low that the parasite cannot be detected microscopically.
20 In relation to malaria, low dose is typically referred to as a sub-patent infection.
[0065] By ―low-dose‖ in the context of blood-stage malaria parasites is meant an
administered dose wherein the resultant parasite density is sufficiently low that the parasite
cannot be detected on a blood smear. Low dose may also typically refer to a sub-patent infection.
[0066] The term ―low molecular weight anti-coagulant‖ also known as ―low molecular weight
25 heparins‖ refers toa class of anticoagulant medications used in the prevention of blood clots and
production of an immune response against venous thromboembolism (deep vein thrombosis and
pulmonary embolism) and in the production of an immune response against 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
30 at least 60% of all chains have a molecular weight less than 8000 Da.
14
[0067] The present invention relates to a novel pharmaceutical kit for producing an
immune response against infectious diseases caused by intracellular pathogens. Specifically, the
present invention relates to a novel pharmaceutical kit for the production of an immune response
against infectious diseases caused by intracellular pathogens, particularly malaria.
5 [0068] In another embodiment, the present invention relates to a novel pharmaceutical kit
for the production of an immune response against malaria, the kit comprising pathogenic
inoculum and drug(s) having extra-erythrocytic mechanism of action against the pathogen.
[0069] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein the pathogen is
10 Plasmodium.
[0070] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein the pathogenic inoculum is
Plasmodium pathogenic inoculum.
[0071] In another embodiment, the present invention relates to a novel pharmaceutical
15 kitfor the production of an immune response against malaria, wherein Plasmodium pathogenic
inoculum is selected from isolated, purified or unpurified mixture of liver stage Plasmodium
parasites infected erythrocytes or blood stage Plasmodium parasite infected erythrocytes.
[0072] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein liver stage Plasmodium
20 parasites include sporozoites, intact or lysate of sporzoite infected hepatocytes.
[0073] According to another embodiment of the present invention, pathogenic inoculum
capable of suitably modifying immune response include but is not limited to isolated or purified
blood-stage malaria parasites such as merozoitesschizonts, trophozoites or rings, intact or lysate
of blood stage malaria parasite infected erythrocytes, combination of isolated or purified blood25 stage malaria parasites such as merozoitesschizonts, trophozoites, rings, intact or lysate of blood
stage malaria parasite infected erythrocytes (pRBC) hereafter referred to as complete blood stage
Plasmodium pathogenic inoculum.
[0074] In a preferred embodiment of the present invention, the pathogenic inoculum
capable of suitably modifying immune response isa complete blood stage Plasmodium
30 pathogenic inoculum.
15
[0075] The present invention also encompasses all the functional life cycle stages of
Plasmodium for pathogenic inoculum capable of suitably modifying immune response.
[0076] According to one embodiment of the present invention, drugs having extraerythrocytic effect on pathogen may include one or more drugs that inhibit, arrest, retard, stop or
5 kill the activity and/or growth of one or more intracellular life cycle stage of the pathogen.
[0077] In another embodiment, the present invention relates to a novel pharmaceutical kit
for the production of an immune response against malaria, wherein drug(s) having extraerythrocytic mechanism of action against the Plasmodium pathogen are selected from but not
limited to structural carbohydrates like sulfated glycosaminoglycans.
10 [0078] In another embodiment of the present invention, sulfated glycosaminoglycans are
selected from but not limited to Hyaluronan, Chondroitin Sulphate, Dermatan Sulphate, Heparin,
heparin sulphate, KeratanSulphate and the like.
[0079] According to a preferred embodiment of the present invention, the drug having
extra-erythrocytic mechanism of action is a sulfated glycosaminoglycan moiety particularly, a
15 Heparin sulfate moiety.
[0080] According to another preferred embodiment of the present invention, drug having
extra-erythrocytic mechanism of action isan anti-coagulant, specifically, low molecular weight
Heparin compound.
[0081] In another embodiment of the present invention, low molecular weight anti20 coagulant is selected from Enoxaparin, Bemiparin, Certoparin, Dalteparin, Nadroparin,
Parnaparin, Reviparin, Tinzaparin and the like.
[0082] According to a most preferred embodiment of the present invention, the drug
having extra-erythrocytic mechanism of action is Enoxaparin, or a functional fragment thereof.
[0083] The present invention also encompasses all the functionally equivalent analogues of
25 structural carbohydrates useful as antimalarial drugs having extra-erythrocytic mechanism of
action.
[0084] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein kit comprises complete
blood-stage Plasmodium pathogenic inoculum and low molecular weight anti-coagulant.
16
[0085] In a preferred embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria,wherein kit comprises complete
blood-stage Plasmodium pathogenic inoculum and Enoxaparin.
[0086] In another embodiment, the present invention relates to a novel pharmaceutical
5 kitfor the production of an immune response against malaria, wherein components of the kit can
be administered concomitantly or sequentially.
[0087] In one embodiment, the present invention relates to a novel pharmaceutical kit
comprising atleast one dosage unit of complete blood stage Plasmodium pathogenic inoculum
and atleast one dosage unit Enoxaparin, for the production of an immune response against
10 infectious diseases caused by intracellular pathogens particularly malaria.
[0088] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein kit comprises one or two
unit doses of complete blood-stage Plasmodium pathogenic inoculum and one or multiple unit
doses of Enoxaparin.
15 [0089] In another embodiment, the present invention relates to a novel pharmaceutical
kitfor the production of an immune response against malaria, wherein kit comprises unit doses of
Enoxaparin in the range of one to seven.
[0090] In another embodiment, the present invention relates to a novel pharmaceutical kit
for the production of an immune response against malaria, wherein the pathogenic inoculum is a
20 ―low dose‖ pathogenic inoculum, capable of inducing a sub-patent infection.
[0091] According to another embodiment of the present invention, low dose pathogenic
inoculum is capable of suitably modifying immune response by inducing a T-cell response and
preferably not inducing B-cells to produce detectable levels, or only low levels, of antibodies. A
low level of antibody production preferably refers to a level that would not be sufficient to
25 protect an animal against the malaria parasite.
[0092] In another embodiment, the present invention relates to a pharmaceutical kit for the
production of an immune response against malaria, wherein the unit dosage of complete blood
stage Plasmodium pathogenic inoculum and unit dosage of Enoxaparin can be administered
concomitantly or sequentially on the first day, followed by administering only the unit dosage of
30 Enoxaparin for the rest of days till the completion of protocol.
17
[0093] In yet another embodiment, the present invention relates to a pharmaceutical kit for
the production of an immune response against malaria, wherein the unit dosage of complete
blood stage Plasmodium pathogenic inoculum and unit dosage of Enoxaparin is administered
sequentially on the first day followed by administering only the unit dosages of Enoxaparin for
5 the rest of days till the completion of protocol.
[0094] According to one embodiment of the present invention, the protocol followed for
production of immune response is the classical 4-day suppressive test of Peters (Peters, W. et al.,
1965 Drug resistance in Plasmodium bergheiVincke and Lips, 1948. I. Chloroquine resistance.
Exp. Parasitol.17:80-89) according to which the test compound (Enoxaparin) is administered to
10 malaria-infected mice for 4 days. This procedure has been proposed by the WHO as the first-line
primary screen for in vivo testing of potential antiplasmodial compounds.
[0095] In still another embodiment, the present invention relates to a pharmaceutical kit for
the production of an immune response against malaria, wherein complete blood stage
Plasmodium pathogenic inoculum and low molecular weight anti-coagulant are present in a
15 potentiating amount.
[0096] The term 'potentiating amount' indicates that complete blood stage Plasmodium
pathogenic inoculum and Enoxaparin are present in an amount such that the combined
antimalarial activity of the components of the kit is greater than that of either complete blood
stage Plasmodium pathogenic inoculums or Enoxaparin alone or of the additive activity that
20 would be predicted for the kit based on the activities of the individual components. Thus the
individual components act synergistically in kit provided they are present in a potentiating
amount.
[0097] According to one embodiment of the present invention, significant antimalarial activity is
exhibited by combining complete blood stage Plasmodium pathogenic inoculum and Enoxaparin
25 in a potentiating amount. The kit according to the present invention demonstrates immunepotentiating efficacy for the production of an immune response and/or prophlyaxis against
Plasmodium species.
[0098] In another embodiment, the present invention relates to a pharmaceutical kit for the
production of an immune response against malaria, wherein the potentiating amount of blood
stage Plasmodium pathogenic inoculumis no more than 107
pRBC such as including 106
, 105
30 ,
104
, 103
, 102
or 10 pRBC. Preferred low dose of blood stage Plasmodium pathogenic inoculum is
18
in the range including 101
-106
, 101
-105
, 101
-104
, 101
-103
, 101
-102
, 102
-106
, 102
-105
, 102
-104
, 102
-
103
, 103
-106
, 103
-105
, 103
-104
, 104
-106
and 104
-105
pRBC per unit dosage kit.
[0099] According to a preferred embodiment of the present invention, a potentiating
amount of blood stage Plasmodium pathogenic inoculum, which is successfully used to generate
an immune response, is 103
5 pRBC per unit dosage kit.
[00100] In another embodiment, the present invention relates to a pharmaceutical kit for the
production of an immune response against malaria, wherein the amount of Enoxaparin per unit
dosage, is in the range of be 0.1 mg/ kg of body weight to 1000 mg kg of body weight,
preferably in the range of 1mg/kg of body weight to 200 mg/kg of body weight.
10 [00101] According to a preferred embodiment of the present invention, the amount of
Enoxaparin per unit dosage in the pharmaceutical kit, which is successfully used to treat malaria,
is 6mg/kg of body weight.
[00102] The amount of complete blood stage Plasmodium pathogenic inoculum and
Enoxaparin per unit dosage in the kit, required to be effective as an antimalarial agent can vary,
15 if required and is ultimately at the discretion of the medical or veterinary practitioner. The factors
to be considered include the route of administration, the mammal's bodyweight, age, and general
condition and the nature and severity of the disease to be treated.
[00103] According to one embodiment of the present invention, for producing an immune
response in mammals, and especially humans, the components of the kit namely, complete Blood
20 stage Plasmodium pathogenic inoculum and low molecular weight anticoagulant, specifically
Enoxaparin are administered as a pharmaceutical formulation in admixture with a
pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard
to the intended route of administration and standard pharmaceutical practice.
[00104] In accordance with embodiments of the present invention, components of the
25 pharmaceutical kit may be administered intravenously, subcutaneously, buccally, rectally,
dermally, nasally, tracheally, bronchially, topically, by any other parenteral route, or via
inhalation, in the form of a pharmaceutical preparation comprising the low molecular weight
anticoagulant in a pharmaceutically acceptable dosage form. Depending on a number of factors
specific to the patient to be treated, as well as the route of administration, the compositions may
30 be administered at varying doses.
19
[00105] Preferred modes of delivery are systemic. Preferably, compositions of the
pharmaceutical kit may be administered parenterally, more preferably intravenously.
Pharmaceutical kits suitable for parenteral administration include prefilled vials containing
sterile solutions or suspensions of the active kit in aqueous or oleaginous vehicles. Injectible
5 preparation 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 kit 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 use. The
kit of Enoxaparin and Low dose plasmodium infected erythrocytes may also be formulated as
10 dispersible granules, which may for example be suspended in water before administration, or
sprinkled on food.
[00106] In one embodiment, the components of the pharmaceutical kit, for the production of
an immune response against malaria, can exhibit one or more properties of Attach, Attune and
Attack. The term ―attach‖ with respect to the components of the pharmaceutical kit, for the
15 production of an immune response against malaria, refer 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 drug molecule binds irreversibly
20 with the surface antigen of the pathogen. The term ―attune‖ with respect to the components of
the pharmaceutical kit, for the production of an immune response against malaria, refers to a
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
25 to configurational changes in the surface antigens of the pathogen as a result of interaction with
the drug molecule. The term ―attack‖ with respect to the components of the pharmaceutical kit,
for the production of an immune response against 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
30 pathogen in the blood stream thereby making the pathogen exposed to an attack by the host
immune cells.
20
[00107] The pharmaceutical kit for the production of an immune response against malaria,
according to the embodiments of the present invention can exhibit one or more therapeutic roles.
[00108] According to one embodiment of the present invention, pharmaceutical kit for the
production of an immune response against malaria can inhibit the invasion of the host cell by the
5 pathogen.
[00109] According to another embodiment of the present invention, pharmaceutical kit for
the production of an immune response against malaria can directly kill the pathogen.
[00110] According to yet another embodiment of the present invention, the novel antimalarial pharmaceutical kit promotes phagocytic uptake of the pathogen by the macrophages.
10 [00111] According to still another embodiment of the present invention, pharmaceutical kit
for the production of an immune response against malaria promotes activation of host immune
response against the pathogen.
[00112] According to another embodiment of the present invention, pharmaceutical kit for
the production of an immune response against malaria confers immunity against the pathogen.
15 [00113] According to yet another embodiment of the present invention, pharmaceutical kit
for the production of an immune response against malaria 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
production of an immune response.
20 [00114] The novel pharmaceutical kit according to the embodiments of the present invention
demonstrates enhanced antimalarial immune response at potentiating amounts of complete
blood-stage Plasmodium pathogenic inoculum. Based on the animal studies, it was found that the
kit of present invention comprising low dose of complete blood-stage Plasmodium pathogenic
inoculum and Enoxaparin, exerts approximate same immune response as exerted by high doses
25 of complete blood-stage Plasmodium pathogenic inoculumand Enoxaparin. Surprisingly, it was
found that the components of the kit demonstrate synergistic mechanism of action resulting in
100% survival on primary challenge, as described in Table 1.
[00115] The inventive ingenuity of the present invention lies in the fact that combination of
Enoxaparin and low dose of complete blood-stage Plasmodium pathogenic inoculum surprisingly
30 generates a potent protective immune response comparable to that generated by using
Enoxaparin and high dose complete blood-stage Plasmodium pathogenic inoculum.
21
[00116] The novel pharmaceutical kit 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/kits have inhibitory effect only on the intra-erythrocytic stages of life cycle of
P. falciparum, resulting in severe anemia due to enhanced destruction of infected RBCs in late
5 stage malaria that often leads to death. The novel pharmaceutical kit according to the present
invention exerts inhibitory effects at both intra-erythrocytic and extra-erythrocytic stages.
Synergistic combination of Enoxaparin with complete blood-stage Plasmodium pathogenic
inoculum in the kit according to the present invention produces an immune response against
malaria and associated conditions.
10 [00117] The novel pharmaceutical kit according to the embodiments of the present invention
demonstrates reduced risk of development of resistance. Enoxaparin possess unique ability to
inhibit Plasmodium interaction with host cells by simultaneously targeting the multiple antigens
present on the surface of Plasmodium. From in-vitro and in-vivo studies, it has been found out
that the probability of multiple molecules to simultaneously acquire resistance to low molecular
15 weight anti-coagulant is very low and thus the risk for emergence of parasites resistant to anticoagulant is also extremely low.
[00118] The novel pharmaceutical kit according to the embodiments of the present invention
upregulates host immunomodulatory response. For a robust and protective immune response,
activation of both cell mediated and humoral immunity is required. It has been known in the
20 literature that production of an immune response with various anti-malarial drugsalone
downregulates the host immune response by decreasing the number of splenic and circulatory Bcells. Further, they also reduce the production of mediators of pro-inflammatory cytokines. Also,
most of the vaccines require tobe administered alongwith an adjuvant to increase the immune
enhancing property. Surprisingly, the pharmaceutical kit according to the present invention
25 demonstrates that the combined administration of Enoxaparin and complete blood-stage
Plasmodium pathogenic inoculum upregulate the host immune response without the use of an
adjuvant and increases the number of splenic and circulatory B cells required for generation of
an effective immune response.
[00119] The novel pharmaceutical kit according to the embodiments of the present invention
30 prevents recurrence of blood stage infection. Earlier studies using anti-malarial drug
Artesunateeven at very high doses of 100mg/kg invariably results in recurrence of infection.
22
Surprisingly, the kit according to the present invention comprising Enoxaparin and complete
blood-stage Plasmodium pathogenic inoculum produces an immune response against malaria,
which not only inhibits erythrocyteinvasion by merozoites but also prevents recurrence of
infection.
5 [00120] The novel pharmaceutical kit according to the embodiments of the present invention
can be used as a novel therapy for chemoprophylaxis against infectious diseases caused by
intracellular pathogens, particularly malaria. The kit according to the present invention exhibits
excellent antimalarial therapeutic efficacy and reduces the growth of the parasites. The
production of an immune response, thus produced also allows development of natural immunity
10 that provides protection against subsequent secondary infections.
[00121] According to one embodiment of the present invention, the pharmaceutical kit of
the present invention may further comprise other components like instruction manual on how to
use the kit, accessories required to administer the components of the kit etc.
[00122] According to another embodiment of the present invention, the pharmaceutical kit
15 of the present invention comprises unit dosages in the form of preloaded syringes or vials.
[00123] Pharmaceutical kits suitable for parenteral administration include sterile solutions or
suspensions of the active kit in aqueous or oleaginous vehicles. Injectible preparation 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 kit until required
20 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 use. The kit of Enoxaparin and Low
dose plasmodium infected erythrocytes may also be formulated as dispersible granules, which
may for example be suspended in water before administration, or sprinkled on food.
[00124] According to one embodiment of the present invention, the kit comprising
25 Enoxaparin and complete blood-stage Plasmodium pathogenic inoculum may also be formulated
as a long-acting depot preparation, which may be administered by extramuscular injection or by
implantation e.g. subcutaneously or extramuscularly. Depot preparation may include, for
example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting
kits are particularly convenient for prophylactic use.
30 [00125] In another embodiment, the present invention relates to a novel pharmaceutical kit
for the production of an immune response against malaria, wherein administration of
23
components of the kit to a subject in need thereof, is useful for prophylaxis against infectious
diseases caused by intracellular pathogens, particularly malaria.
[00126] In another embodiment, the present invention relates to a novel pharmaceutical
kitcomprising low molecular weight anti-coagulant along with complete blood-stage
5 Plasmodium pathogenic inoculum,for the preparation of medicament for the production of an
immune response againstinfectious diseases caused by intracellular pathogens, particularly
malaria.
[00127] In another embodiment, the present invention relates to a novel pharmaceutical
kitcomprising low molecular weight anti-coagulant along with complete blood-stage
10 Plasmodium pathogenic inoculum,for the preparation of medicament for the production of an
immune response against infectious diseases caused by intracellular pathogens, particularly
malaria.
[00128] In another embodiment, the present invention relates to a method of production of
an immune response against infectious diseases caused by intracellular pathogens, particularly
15 malaria comprising administering a therapeutically effective amount of the pharmaceutical kit
comprising Enoxaparin along with complete blood-stage Plasmodium pathogenic inoculum, to a
subject in need thereof.
[00129] In a preferred embodiment, the present invention relates to a method of production
of an immune response against infectious diseases caused by intracellular pathogens, particularly
20 malaria, comprising administering the components of the pharmaceutical kit comprising
Enoxaparin along with complete blood-stage Plasmodium pathogenic inoculum, to a subject in
need thereof.
[00130] In a preferred embodiment, the present invention relates to a method of production
of an immune response and/or prophylaxis of infectious diseases caused by intracellular
25 pathogens, particularly malaria, comprising administering the components of the pharmaceutical
kit comprising Enoxaparin along with complete blood-stage Plasmodium pathogenic inoculum,
to a subject in need thereof.
[00131] In yet another embodiment, the present invention relates to use of the
pharmaceutical kit comprising low molecular weight anti-coagulant along with complete blood30 stage Plasmodium pathogenic inoculum, for the production of an immune response and
prophylaxis of infectious diseases caused by intracellular pathogens, particularly malaria.
24
[00132] In yet another embodiment, the present invention relates to use of the
pharmaceutical kit comprising Enoxaparin along with complete blood-stage Plasmodium
pathogenic inoculum, for the production of an immune response and prophylaxis of infectious
diseases caused by intracellular pathogens, particularly malaria.
5 [00133] In yet another embodiment, the present invention relates to use of the
pharmaceutical kit wherein complete blood-stage Plasmodium pathogenic inoculum are
administered to a subject in need thereof followed by sequential administration of Enoxaparin,
for the production of an immune response and/or prophylaxis of infectious diseases caused by
intracellular pathogens, particularly malaria.
10 [00134] 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
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
15 information and knowledge available to the person having ordinary skill in the art.
[00135] The present invention is further explained in the form of following examples.
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.
[00136] Source of starting Materials: Artesunate was purchased commercially from Edinburg
20 Pharmaceuticals, Manimajra India; Enoxaparin (Clexane) was obtained commercially from
Healthcare Medical Centre, Mumbai, India. Enoxaparin intravenous injection was prepared by
diluting 60mg Enoxaparin with 0.9% Normal saline to desired dosage.Plasmodium berghei (NK
65) strain, used for challenging the mice and evaluation of antimalarial activity, was obtained
from the library of strains maintained in the Parasitology Lab, Department of Zoology, Panjab
25 University, Chandigarh.RBCs were collected from blood samples obtained from mice infected
with P. berghei.
[00137] Example 1: Determination of 106
and 103
number of infected RBCs
[00138] Infected RBCs were collected and counted as per the standard literature procedure
described by Helena Helmby and Brian de Souza in Methods in Malaria Research 6th Edition,
30 Cox FEG. 1988,Major animal models in malaria research, In: Wernsdorfer WH, McGregor I,
eds; Malaria: principles and practice of malariology, Vol. 2, Edinburgh: Churchill Livingstone;
25
pp. 1503- 1543 and Methods In Malaria Research 6th Edition. The counted cells were used
further in process of infection of the animal models.
[00139] Example 2: In-vivo studies and results
[00140] Step 1: Female Swiss mice weighing 22-26g were obtained from the Central animal
5 facility, NIPER, Mohali and used as experimental models. They were maintained on a standard
pellet diet and water ad libitum. Plasmodium berghei (NK-65) strain was used for evaluation of
antimalarial activity and was maintained inthemice by intraperitoneal inoculation of 1×106
infected erythrocytes to naïve mice (Santiyanont 1985). After challenge, mean percent
parasitemia was checked by preparing Giemsa stained thin blood smears on glass slides through
10 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
on Animals (45/GO/ReBi/S/99/CPCSEA) guidelines. Mean percent parasitemia and Survival
rate of the animals was calculated as follows:
15 Mean percent parasitemia= Infected RBCs *100
Total no. of RBCs
Survivalrate = Survived animals *100
Total no. of animal
[00141] Step 2: Primary Challenge
20 [00142] Six groups (1 - 6) having 6 mice each (female, weighing 22-26 gm) were used for
present study (Table 1). All groups except N.C and T-2, were inoculated with 1×106
P.
bergheiparasitized erythrocytes on day zero (D0). T-2 treated mice were inoculated with 1×103
P.
bergheiparasitized erythrocytes on day zero (D0). Treatment was continued for 4 days (Day 0 -
Day 3). Drugs to T-1 and T-2 groups of animals were administered intravenously
25 (0.025ml/mouse/injection). However to PC group of animals, the known drug Artesunatewas
administered orally to a total volume of 100µl/mice, PC treated animals being the standard
positive control group. The dosage of drugs to be administered intravenously was fixed at
Enoxaparin 6mg/kg body weight and Artesunate 30mg/kg body weight, once a day starting from
first day of post inoculation (pi) for 4 days to monitor the efficacy and potency of prepared
30 formulation.
26
Table 1
Serial
No.
Groups of Animals
(Abbreviation)
Controls / Treatments
1 Normal Control (NC) Without Infection
2 Infected Control (I.C) Infected+ Distilled Water
3 Vehicle control (V.C) Infected + Normal Saline
4 Positive Control (P.C) Infected and Treated with *Artesunate 30mg/kg for
4 days, orally
5 Treatment Group1 (T-1) Infected and Treated with Enoxaparin (6mg/kg
b.w.)intravenously
6 Treatment Group2 (T-2) Infected with lower inoculum and Treated with
Enoxaparin (6mg/kg b.w.) intravenously
*Artesunate was used as comparator as well as Positive control
[00143] Step 3: Statistical analysis
5 [00144] Resulting data of above experiments is presented as mean and standard deviation
(SD). Statistical 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 GraphPad Software (San
Diego, California, USA). The results are depicted in Table 2 and illustrated graphically in
Figure 1 and 2.
10 Table 2
Serial No. Group
(Abbreviation)
Mean percent parasitemia Survival Percent
7
th day (D6) 28th day
(D27)
28th day (D27)
1 Normal Control
(NC)
0 0 100
2 Infected Control
(I.C)
37.63 ± 1.95 -------- 0
3 Vehicle control 36.64 ± 4.1 -------- 0
27
(V.C)
4 Positive Control
(P.C)
16.04 ± 2.4 1.2 ± 0.6 100
5 Treatment Group 1
(T-1)
10.1 ± 0.9 0 66.66
6 Treatment Group 2
(T-2)
7.68± 1.26 0 100
[00145] Course of parasitemia in experimental groups
[00146] Table 2 provides information about course of parasitemia in different
experimental groups on primary challenge and the same has been represented graphically in
Figure 1. Maximum infection of 37.63% ± 9.5 was observed in infected control (IC) by 7th 5 day
post inoculation. In Artesunate monotherapy (PC treated animals), the infection was reduced to
16.04±2.4% by day 7 whereas in Enoxaparin monotherapy (T-1 treated animals), there was
significant reduction in parasitemia compared to PC treated animals (p<0.0005). In mice infected
with lower inoculums 1×103
P. bergheiparasitized erythrocytes and treated with Enoxaparin
10 (6mg/kg b.w.) intravenously (T-2 treated animals), there was 7.68% ± 1.26 parasitemia on day 7.
However by day 28, infection in T-1 and T-2 treated mice was completely resolved whereas in
PC group of treated mice, 1.2 ± 0.6 % infection due to recrudescence was observed.
[00147] Survival Analysis
[00148] Figure 2 provides information about number of animals surviving in different
15 experimental groups on primary challenge. In I.C and V.C group, all the animals died between
Day 14 -19. All the animals were severely anaemic and heavily infected. However, in PC and T2 mice 100% survival rate was found by Day 28. In T-1 treated group survival was found to be
66%.
[00149] Example 3: Secondary / Re – challenge
20 [00150] After a span of 83 days from the day of initial challenge (D0), all the mice which
recovered from primary challenge were used for present study (Table 3). They were maintained
on a standard pellet diet and water ad libitum. Plasmodium berghei (NK-65) strain was used for
evaluation of antimalarial activity (Santiyanont 1985). All the surviving mice were re-inoculated
28
with 1×106
infected erythrocytes. Mean percent parasitemia was checked by preparing Giemsa
stained thin blood smears on glass slides through tail vein incision.
Mean percent parasitemia and Percent survival of the animals was calculated as follows:
Mean percent parasitemia= Infected RBCs *100
5 Total no. of RBCs
Survival rate = Survived animals *100
Total no. of animal
Table 3: List of the different groups of animals survived from previous study
Serial
No.
Groups of Animals
(Abbreviation)
Number of Animals
survived / Total animals
after Primary Challenge in
Experiment 1
Controls / Treatments
1 Normal Control (NC) 6 / 6 Infected (106
)
2
Positive Control (P.C) 2 / 6 Infected (106
)
3 Treatment Group 1
(T-1)
4 / 6
Infected (106
)
No treatment
4
Treatment Group 2
(T-2)
6/ 6 Infected (106
)
No treatment
10 [00151] Study design for rechallenging the animals surviving after primary challenge
[00152] All groups were injected with 1×106
P. bergheiparasitized erythrocytes on day 83
(D83). No drug to any animal in different groups was given.
Statistical analysis: Resulting data is presented as mean and standard deviation (SD). Statistical
evaluation of differences between the experimental groups was determined by the Student’s t-test
15 with the level of significance of p<0.0005 using Graph Pad Software (San Diego, California,
USA). The results are depicted in Table 4 and illustrated graphically in Figure 3 and 4.
Table 4
Serial Group (Abbreviation) Mean percent parasitemia Survival Percent
29
No. Day 90 Day 104 Day 104
1 Normal Control (N.C) 26.53 ± 2.3 ----- 0
2 Positive Control (P.C) 31.64 ± 1.97 ----- 0
3 Treatment Group 1(T-1) 0.72 ± 0.2 0 100
5 Treatment Group 2 (T-2) 0.345 ± 0.21 0 100
[00153] Course of parasitemia in experimental groups
[00154] Table 4 provides information about course of parasitemia in different
experimental groups on rechallenge and the same has been represented graphically in Figure 3.
In N.C group of mice 26.53 ± 2.3 % infection was observed on day 90th(7th 5 day post inoculation).
In Artesunate monotherapy (PC treated animals), there was 31.64%±1.97 infection on day 90th
.
Surprisingly, in Enoxaparin monotherapy (T-1 treated animals) and in T-2 group mice infected
with lower inoculums 1×103
P. bergheiparasitized erythrocytes and treated with Enoxaparin
(6mg/kg b.w.) intravenously (T-2 treated animals), there was significant reduction in parasitemia
on day 90th 10 compared to PC treated animals (p<0.0005).
[00155] Survival Analysis: Figure 4 provides information about number of animals
surviving in different experimental groups on rechallengeIn NC and PC group, the animals died
between days 84-97. All the animals were severely anaemic and heavily infected. Surprisingly,
in all T-1 and T-2 treated groups, 100% survival rate was found by Day 104.
15 [00156] From the above data, it isclear that animals in T-2 group have a better survivability
and a protective immune response as compared to T-1 group mice. It is thus clearly evident from
the animal studies that administration of Enoxaparin along with lower inoculum of parasite is an
effective option for inducing an immune response.
[00157] The foregoing examples are merely illustrative and are not to be taken as limitations
20 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
30
ADVANTAGES OF THE PRESENT INVENTION
[00158] The present invention provides a pharmaceutical kit for producing an immune
response against infectious diseases, which can overcome deficiencies associated with the known
arts.
5 [00159] The present invention provides a pharmaceutical kit for producing an immune
response against malaria.
[00160] The present invention provides a pharmaceutical kit for producing an immune
response against malaria, preferably without administration of an adjuvant.
[00161] The present invention provides a pharmaceutical kit for producing an immune
10 response against malaria that provides protection against any subsequent secondary infection.
[00162] The present invention provides a pharmaceutical kit that produces fast clinical
responses to treatment and reduces the growth of the parasites.
[00163] The present invention provides a pharmaceutical kit for generating an immune
response against malaria and having dual mode of action, by arresting the progression of disease
15 and inhibiting both extra-erythrocytic and intra-erythrocytic stages.
[00164] The present invention provides a pharmaceutical kit having reduced risk of
development of resistance.
[00165] The present invention provides a pharmaceutical kit that upregulates host immunemodulatory response.

We Claim:

1. A novel pharmaceutical kit for producing an immune response against infectious
diseases caused by intracellular pathogens, particularly malaria;
5 Wherein the kit comprises pathogenic inoculum and drug(s) having extra-erythrocytic
mechanism of action against the pathogen;
characterized in that the pathogenic inoculum is Plasmodium pathogenic inoculum.
2. The kit as claimed in claim 1, wherein Plasmodium pathogenic inoculum is selected
from isolated, purified or unpurified mixture of liver stage Plasmodium parasites infected
10 erythrocytes or blood stage Plasmodium parasite infected erythrocytes.
3. The kit as claimed in claim 1, wherein blood-stage Plasmodium parasites include
isolated or purified blood-stage malaria parasites selected from merozoitesschizonts,
trophozoites or rings; intact or lysate of blood stage malaria parasite infected erythrocytes;
combination of isolated or purified blood-stage malaria parasites selected from
15 merozoitesschizonts, trophozoites or rings or complete blood stage Plasmodium pathogenic
inoculum selected from intact or lysate of blood stage malaria parasite infected erythrocytes
(pRBC), preferably complete blood stage Plasmodium pathogenic inoculum.
4. The kit as claimed in claim 1, whereindrug(s) having extra-erythrocytic mechanism
of action against the Plasmodium pathogen isan anti-coagulant, specifically, low molecular
20 weight Heparin compound selected from Enoxaparin,Bemiparin, Certoparin, Dalteparin,
Nadroparin, Parnaparin, Reviparin and Tinzaparin, preferably the anti-coagulant is
Enoxaparin.
5. The kit as claimed in claim 1, wherein kit comprises complete blood-stage
Plasmodium pathogenic inoculum and low molecular weight anti-coagulant.
25 6. The kit as claimed in claim 1, whereinkit comprises complete blood-stage
Plasmodium pathogenic inoculum and Enoxaparin.
7. The kit as claimed in claim 1, whereincomponents of the kit can be administered
concomitantly or sequentially.
8. The kit as claimed in claim 6, wherein kit comprisesatleast one dosage unit of
30 complete blood stage Plasmodium pathogenic inoculum and atleast one dosage unit of
Enoxaparin.
32
9. The kit as claimed in claim 6, wherein kit comprises one or two unit doses of
complete blood-stage Plasmodium pathogenic inoculum and one or multiple unit doses of
Enoxaparin, preferably unit doses of Enoxaparin in the range of one to seven doses.
10. The kit as claimed in claims 1 and 6, wherein the Plasmodium pathogenic inoculum
5 is a ―low dose‖ pathogenic inoculum, capable of inducing a sub-patent infection.
11. The kit as claimed in claims 1 and 6, whereinthe unit dosage of complete blood
stage Plasmodium pathogenic inoculum and unit dosage of Enoxaparin can be administered
concomitantly or sequentially on the first day, followed by administering only the unit
dosage of Enoxaparin for the rest of days till the completion of protocol.
10 12. The kit as claimed in claims 1 and 6, wherein the unit dosage of complete blood
stage Plasmodium pathogenic inoculum and unit dosage of Enoxaparin is administered
sequentially on the first day followed by administering only the unit dosages of Enoxaparin
for the rest of days till the completion of protocol.
13. The kit as claimed in claims 1 and 6, wherein complete blood stage Plasmodium
15 pathogenic inoculum and low molecular weight anti-coagulant are present in a potentiating
amount.
14. The kit as claimed in claim 13, wherein the potentiating amount of blood stage
Plasmodium pathogenic inoculum is in the range of 101
-106
, 101
-105
, 101
-104
, 101
-103
, 101
-
102
, 102
-106
, 102
-105
, 102
-104
, 102
-103
, 103
-106
, 103
-105
, 103
-104
, 104
-106
or 104
-105
pRBC
20 per unit dosage kit.
15. The kit as claimed in claim 14, wherein the potentiating amount of blood stage
Plasmodium pathogenic inoculum is103
pRBC per unit dosage kit.
16. The kit as claimed in claims 1 and 6, wherein the amount of Enoxaparin per unit
dosage, is in the range of 0.1mg/kg of body weight to 1000mg/kg of body weight.
25 17. The kit as claimed in claims 1 and 6, wherein kit demonstrates dual mode of action
arresting the progression of disease by exerting inhibitory effects at both intra-erythrocytic
and extra-erythrocytic stages.
18. The kit as claimed in claim 1, wherein kit further comprises other components
selected from instruction manual on how to use the kit and accessories required to administer
30 the components of the kit.
33
19. A novel pharmaceutical kit for producing an immune response against infectious
diseases caused by intracellular pathogens, particularly malaria, wherein the kit comprises
complete blood-stage Plasmodium pathogenic inoculum and Enoxaparin, in a potentiating
ratio.
5 20. A novel pharmaceutical kit comprising low molecular weight anti-coagulant along
with complete blood-stage Plasmodium pathogenic inoculum, for the preparation of
medicament, for the production of an immune response against infectious diseases caused by
intracellular pathogens, particularly malaria.
21. Anovel pharmaceutical kit comprising Enoxaparin along with complete blood-stage
10 Plasmodium pathogenic inoculum,for the preparation of medicament for the production of an
immune response against infectious diseases caused by intracellular pathogens, particularly
malaria.
22. A method of production of an immune response against infectious diseases caused
by intracellular pathogens, particularly malaria comprising administering a therapeutically
15 effective amount of the components of pharmaceutical kit comprising Enoxaparin along with
complete blood-stage Plasmodium pathogenic inoculum, to a subject in need thereof.
23. A method of production of an immune response and/or prophylaxis of infectious
diseases caused by intracellular pathogens, particularly malaria, comprising administering the
components of the pharmaceutical kit comprising Enoxaparin along with complete blood20 stage Plasmodium pathogenic inoculum, to a subject in need thereof.
24. Use of the pharmaceutical kit comprising low molecular weight anti-coagulant
along with complete blood-stage Plasmodium pathogenic inoculum, for the production of an
immune response and prophylaxis of infectious diseases caused by intracellular pathogens,
particularly malaria.
25 25. Use of the pharmaceutical kit comprising Enoxaparin along with complete bloodstage Plasmodium pathogenic inoculum, for the production of an immune response and
prophylaxis of infectious diseases caused by intracellular pathogens, particularly malaria.
26. Use of the pharmaceutical kit as claimed in claim 25, wherein complete bloodstage Plasmodium pathogenic inoculum are administered to a subject in need thereof
30 followed by sequential administration of Enoxaparin, for the production of an immune
34
response and/or prophylaxis of infectious diseases caused by intracellular pathogens,
particularly malaria.