Tuftsin-bearing liposomes in treatment of macrophage-based infections

12 Pages·2016·0.1 MB·English

L
Advanced Drug Delivery Reviews 41 (2000) 135–146
www.elsevier.com/locate/drugdeliv
Tuftsin-bearing liposomes in treatment of macrophage-based
infections
Ajay K. Agrawala,*, C.M. Guptab
aPolyMASC Pharmaceuticals Plc,Fleet Road,London NW32EZ,UK
bCentral Drug Research Institute,Lucknow226001,India
Received 25 April 1999; accepted 1 October 1999
Abstract
The use of liposomes as drug carriers in treatment of various diseases has been explored extensively for more than 20
years. ‘Conventional’ liposomes, when administered in vivo by a variety of routes, rapidly accumulate in the mononuclear
phagocytesystem(MPS).TheinherenttendencyoftheliposomestoconcentrateinMPScanbeexploitedinenhancingthe
non-specifichostdefenceagainstinfectionsbyentrappinginthemthemacrophagemodulators,andascarriersofantibiotics
intreatmentofintracellularinfectionsthatresideinMPS.Thismustfurtherbeenhancedbygraftingontheliposomesurface
the ligands, e.g. tuftsin, that not only binds specifically to the MPS cells but also enhances their natural killer activity.
Keeping this in view, we designed and developed tuftsin-bearing liposomes as drug carriers for the treatment of
macrophage-basedinfectionsandoutlinethesestudiesinthisoverview. (cid:211) 2000ElsevierScienceB.V.Allrightsreserved.
Keywords: Drug delivery system; Liposome; Tuftsin; Immunomodulation; Mononuclear phagocyte system; Malaria; Leishmaniasis;
Tuberculosis; Aspergillosis
Contents
1. Introduction............................................................................................................................................................................ 135
2. Tuftsin-bearingliposomes........................................................................................................................................................ 136
3. Malaria................................................................................................................................................................................... 138
4. Leishmaniasis......................................................................................................................................................................... 139
5. Fungalinfections..................................................................................................................................................................... 140
6. Tuberculosis........................................................................................................................................................................... 142
Acknowledgements...................................................................................................................................................................... 143
References.................................................................................................................................................................................. 143
1. Introduction drugs to patients to achieve maximum efficacy with
minimal side effects has been one of the most
The search for improved ways of administering ‘sought-after’ dreams of clinicians for years. How-
ever, in actual practice, application of drugs in
therapeutic and preventive medicine is marred by
*Corresponding author. Tel: [phone]; fax: 144-
171-431-7594. their indiscriminate action and inability toreach only
0169-409X/00/$–see front matter (cid:211) 2000 Elsevier Science B.V. All rights reserved.
PII: S0169-409X(99)00061-7
136 A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146
the areas in need of treatment. In spite of the vivo, rapidly accumulate in the MPS [9–12]. The
enormous advances made in molecular biology and majorsitesoftheiraccumulationareKupffercellsof
pharmacology over the last two decades, develop- the liver and the resident macrophages of the spleen.
ment of new, more selective drugs is still a very About70to80%ofintravenouslyinjectedliposomes
expensive, time consuming and often uncertain pro- are concentrated into the Kupffer cells of the liver, 5
cess.While the drugs are available to combat a wide to 8% into macrophages in the spleen, and even
range of genetic, malignant, and infectious diseases, fewer into phagocytic cells in bone marrow
their efficacy is often compromised by their inability [8,13,14]. The rate and the site of the uptake of the
to reach the intended site at an appropriate con- liposomes by the MPS are influenced by the injected
centration. Consequently, much attention has been dose, blood flow, local tissue damage, and inter-
focussed on an alternative approach, namely the use action of liposomes with the serum proteins [13,15].
of drug delivery systems, which are expected to The interaction of serum proteins with the particles
optimise the action of the drugs already in existence depends on the physicochemical properties of the
by transporting or facilitating their release where particles,i.e.charge,size,hydrophobicity,andfluidi-
they are needed. ty of the particle surface [13,15]. This tendency of
Successful homing of drugs to the target depends the liposomes to localise in MPS can be turned into
on the design of the carrier as little can be done to an advantage in the treatment of a variety of
influence the target and its surroundings. In a typical infectionsintwoways:(1)AstheMPScellsplayan
drug targeting sequel, the carrier-drug unit would important role in non-specific host defence against
preserve its integrity, avoid interception by normal infections in general, liposomes can be used to
cells, penetrate interposing membranes, selectively activate them by encapsulating in them, thus enhanc-
recognise and associate with the target before the ing non-specific resistance to infections caused by
drug is released in the target area. Selective drug various micro-organisms [11,12]. (2) Liposomal
delivery to specific foci in the body have two vital encapsulation of antiparasitic and antimicrobial
benefits. First, it ensures optimal interaction of the agents can result in enhancement of their therapeutic
drug with the site(s) of its action at the right rate and efficacy against intracellular infections involving the
frequency. Secondly, on reducing the drug dose as MPS [9–12]. This must potentially be achieved by
well as by increasing the target specificity, the grafting on the liposome surface the ligands, e.g.
potential for any side effects is greatly diminished. tuftsin, that not only bind specifically to the MPS
A large number of macromolecular, cellular, and cells but also stimulate them non-specifically against
syntheticcarriershavebeenexploredwiththeaimof infections.
perfecting drug delivery systems which would local- In the early 1980s, our research group embarked
isedrugstothedesiredsitesinvivo,thusminimising on a project of drug delivery in macrophages. Over
the side effects [1–3]. Amongst these, liposomes the years we have successfully used tuftsin-bearing
bearing cell-specific recognition molecules (e.g., liposomes [16–23] in our laboratory as carriers for
antibody,lectin,glycolipidetc.)ontheirsurfacehave immunomodulator/drug homing to macrophages. In
received wide attention as vehicles for site-specific this review we present the overview of the tuftsin-
delivery of drugs and enzymes in vivo [4–7]. bearing liposomes and their usefulness in the treat-
Liposomes, soon after their discovery in the mid ment of macrophase-based infections.
sixties, have been studied extensively, and employed
in virtually every aspect of biotechnology including
gene delivery. Liposomes are best suited as carriers
for drugs and enzymes as they are formed from
naturally occurring phospholipids and therefore pos- 2. Tuftsin-bearing liposomes
sess low inherent toxicity, immunogenicity, and high
drug to carrier ratio [1,8]. Tuftsin is a natural macrophage activator tetra-
Despite their resemblance to the cell surface, peptide (Thr–Lys–Pro–Arg) which is a part of the
‘conventional’ liposomes, when administered in Fc-portion of the heavy chain of the leukophilic
A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146 137
immunoglobulin G (residues 289–292). The tetra-
peptideisreleasedphysiologicallyasthefreepeptide
fragment after enzymatic cleavage [24]. Two en-
zymes are responsible for the production of tuftsin
from leucokinin; tuftsin-endocarboxypeptidase, a
specific enzyme, cleaves the heavy chain at the
Arg–Glu bond between residues 292 and 293, and
the membrane enzyme leucikinase acts on the bound
leucokinin-S to cleave it at the amino end of
threoninebetweenresidues288and289.Thepeptide
is known to bind specifically to macrophages, mono-
Fig.1. HydrophobicderivativeofthetetrapeptideThr–Lys–Pro–
cytes and PMN leukocytes, and possesses a broad
Arg (tuftsin) (reproduced with permission [16]).
spectrum of activities related primarily to the im-
mune system function [24,25]. These include poten-
tiation of various cell functions, such as phago- thus formed liposomes in all the studies described
cytosis, pinocytosis, motility, immunogenic re- below.
sponse, bactericidal and tumoricidal activities [25]. We subsequently demonstrated that the tuftsin
The features of tuftsin, coupled with its low toxicity, (Fig. 1) could enhance non-specific defence against
make the tetrapeptide a promising candidate for infections by activating the macrophages [21]. The
immunotherapy [26–28]. Tuftsin’s capacity to aug- biological activity of the peptide was due to the
ment cellular activation is mediated by specific induction of the macrophage respiratory burst and
receptorsthathavebeenidentified,characterised,and activated macrophages exhibited enhanced levels of
isolated from rabbit peritoneal granulocytes [29]. NADPH oxidase, O2, H O and myeloperoxidase
2 2 2
Tuftsin and many of its analogs have been chemical- (MPO). Both O2 and H O are known to damage
2 2 2
ly synthesised and studied extensively for structure– proteins, nucleic acids and membranes, sufficiently
function relationships [30,31]. to kill the cell or even the whole organism. Never-
Grafting of tuftsin on the liposome surface would theless, for macrophages hypohalous acids, which
therefore enable us not only in homing the liposom- are produced by the action of MPO on H O , have
2 2
ized drug to macrophages but also to stimulate these been identified as the major killer agents [34].
cells non-specifically against infections. Structure– Theincorporationof .10mol%tuftsinintheegg
function studies of tuftsin indicate that its binding PC/cholesterol (2:1; mol/mol) liposomes was not
and consequent MPS activation is dependent upon possible as the resulting mixtures could not be
rather strict conservation of its molecular structure. dispersed even by prolonged sonication [16]. On the
Thus, modifications of the peptide at its N-terminus other hand, the liposomes containing ,5 mol%
or within the chain, lead to a significant reduction or tuftsin were only poorly bound to PMN leukocytes.
even loss of biological activity and also its ability to Therefore, it is absolutely important to have 7–8
bind to PMN leukocytes [32]. However, it has been mol% tuftsin in the liposomes for their optimal
shown that extending the peptide at its C-terminus effect. The leakage rates of 6-CF from egg PC/
does not affect the biological activity [33]. As tuftsin cholesterol/tuftsin liposomes in buffer, pH 7.4 at
is a hydrophilic molecule, it would preferentially 378C were about 2–4%/h. Incidentally, this leakage
reside in the aqueous compartment of liposomes and rate was dramatically enhanced upon incorporating
willthusloseitsabilitytoenhancetheinteractionsof another tuftsin derivative, Thr–Lys–Pro–Arg–NH–
liposomes with phagocytes via its receptor. We, C H in the liposomes bilayer [16]. This was
18 37
therefore, facilitated its grafting on the liposome probably due to the binding of the dye with the
surface by attaching a sufficiently long hydrocarbon positively charged Arg residue in this analogue.
fatty acyl residue to the C-terminus through an Since this amino acid residue should be aligned just
ethylenediamine spacer arm (Thr–Lys–Pro–Arg– at the bilayer interface, the effect of its binding with
NH–(CH ) –NH–CO–C H )(Fig. 1)and used the 6-CF on the liposomes permeability must have
2 2 15 31
138 A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146
been mediated through perturbation of the egg PC malarial parasite employs to resist the drug action is
headgroup packing in the liposomes bilayer [35]. by enhancing the drug efflux from the infected cells,
The binding/uptake of egg PC/cholesterol/tuftsin thus preventing the drug accumulation to the toxic
liposomes to PMN leukocytes was saturable, time levels within the cytosol [39]. A protein likely to be
dependent and the cell-bound liposomes were appar- involved in chloroquine resistance has recently been
ently taken up by the cells presumably by receptor- identified which poses some important questions/
mediated endocytosis without losing their structural answers [40].
integrity. That these liposomes were actively endo- A number of studies have been performed using
cytosed by the cells was further suggested by the liposomes as carriers for drugs, and vaccines against
observation that lowering of the incubation tempera- malarial infection [41–46]. Experimental liposome-
ture to 08C inhibited the uptake [16]. We also based vaccines against malaria and other infections
examined the specificity of the interaction of these havebeenshowntobesafeandhighlyimmunogenic
liposomes with other blood cells, i.e. erythrocytes, in human trials [47]. Analysis of the intracellular
lymphocytesandfoundthatvirtuallynobindingwith trafficking patterns of the liposomal antigens reveals
erythrocytes was observed but there appeared to be that after being phagocytosed by macrophages,
some binding with lymphocytes which slightly in- liposomal antigen readily escapes from the endo-
creased with time, which we concluded was primari- somes into the cytoplasm of the macrophages [47].
ly due to the presence of PMN leucocytes/mono- Mice vaccinated with liposomised recombinant pro-
cytes as contamination in the lymphocyte prepara- teins have recently been reported to resist a lethal
tion. malaria infection. The protection induced with lipo-
somes adjuvants was as good as or better than that
achieved with Freund’s adjuvant [48].
We have previously shown that binding of the
3. Malaria liposomes to red blood cells was considerably en-
hanced (20–25-fold) by covalently attaching F(ab9)
2
Malaria is still considered the most prevalent and fragmentstotheirsurface[49,50].Ithasfurtherbeen
devastating disease world wide which affects about shown that chloroquine encapsulated in these lipo-
300–500 million people and claims 1.5–2.7 million somes was very effective in suppression of the
human lives. Furthermore, one-third of the world chloroquine susceptible and resistant Plasmodium
human population lives in areas which are infested berghei infections, both in terms of reduction in
with the disease [36]. Numerous efforts have been parasitaemia and prolongation in the survival times
made towards the development of effective vaccines [51,52]. We further demonstrated that by attaching
against malaria as an effective vaccine may elicit a F(ab9) fragments of the monoclonal MAb F ,
2 10
protective immune response in individuals of diverse specific to Plasmodium berghei-infected erythro-
genetic makeup and could complement other strate- cytes, to the liposomes loaded with chloroquine,
gies for prevention and control of this serious public eventhechloroquineresistantmalarialinfectionscan
health problem in future. Although these studies be cured [53]. This biologic response may further be
provided important knowledge of the nature of the enhanced by enhancing the non-specific host resist-
protective host immunological mechanisms and their ance against malarial infection [17] as the activated
respective target antigens but there is no effective macrophages have been shown to kill the in-
malaria vaccine as yet on the horizon, and only the traerythrocytic malarial parasites in vitro [54].
chemotherapy remains the major practical method It has been reported that pre-treatment of mice
for managing all forms, i.e. ‘exoerythocytic’ and with tuftsin or one of its derivatives, rendered them
‘erythrocytic’ stages, of infection. Moreover, the at least partially resistant to lethal Plasmodium
situation is aggravating as the malarial parasites are berghei infections [17]. A pre-treatment dose of 50
rapidly developing resistance to the existing antima- mg/animal per day of tuftsin for more than 1 day
larial drugs when given in classical pharmaceutical seemed to be necessary to achieve this partial
form [37,38]. One of the mechanisms that the protection, since the mortality rate in animals pre-
A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146 139
treated with a single dose (200 mg) on day 3 or with urea stibamine have been found to be more potent in
thelowerdoses(10or25)mgonday0to3wasvery comparisontonormalliposome-encapsulateddrugor
similar to that observed in the control animals. the free drug [60,61].
The above effect of tuftsin may further be in- We encapsulated sodium stibogluconate, one of
creased by incorporating it in the liposomes bilayer. the major front line drugs for leishmaniasis, in
For this case, both the mortality and parasitaemias in tuftsin-bearing liposomes and demonstrated that the
the animals that received pre-treatment with the efficacy of the drug was markedly enhanced (at least
liposomal tuftsin were significantly decreased, as 200 times) against Leishmania donovani infections
compared to those pre-treated with saline or control inhamsters[19].Freedrug,whengivenat10mg/kg
liposomes [17]. per day dose for five consecutive days produces over
The mean survival times of mice pre-treated with 90% inhibition of the infection on day 28 post-
50 and 100 mg doses of liposomal tuftsin were about treatment. However, a single treatment at 250 mg or
16 and 19 days, respectively, which were greater 500 mg/kg drug for one day only was very effective
than that observed with free tuftsin [17]. Since (P,0.01)afterencapsulatingintuftsin-bearinglipo-
suppressionofthehostimmuneresponseseemstobe somes. At a 500 mg/kg dose, the drug encapsulated
a regular feature in almost all parasitic diseases [55], in the liposomes showed much better (P,0.01)
agents such as tuftsin may help in bringing the effects than that observed with free drug or tuftsin
immune status back to normal, which in turn could free liposomal drug on day 28 post-treatment. But
further enhance the efficacy of chemotherapy. this difference was reduced (P,0.01 on day 7 but
.0.05 on day 28 post-treatment) by reducing the
drug dose to 250 mg/kg, although at this dose the
liposomised drug exhibited the maximum antileis-
4. Leishmaniasis hmanial activity. On further decreasing the liposom-
ised drug dose, no difference was obtained between
Leishmaniasis is caused by the hemoflagellate the effects of drug encapsulated in tuftsin-free and
protozoan and represents four major clinical tuftsin-bearing liposomes. The failure to observe the
syndromes; visceral, cutaneous, mucocutaneous, and enhanced antileishmanial effect ofthe drug loaded in
diffuse cutaneous leishmaniasis. It is estimated that tuftsin-bearing liposomes, as compared to that in the
world wide more than 12 million people are infected tuftsin-free liposomes, at low drug doses is possibly
and approximately 350 million are at risk. The most due to dilution of the tuftsin-bearing liposomes
devastating clinical form, visceral leishmaniasis below their optimal concentration required to ob-
(kalaazar),iscaused byLeishmaniadonovani which serve optimal activation of the host’s macrophages
causes disseminated disease, characterised by fever, [16].However,optimisationofparameterslike,route
hepatosplennomegaly, anemia, leukopenia and hy- of administration, surface charge, cholesterol con-
perglobinaemia. If untreated, the disease is usually tent, and single vs. multiple treatment regimens,
fatal. treatment at lower parasite burden may further
The major front line drugs available for leis- improve the efficacy. The improvement in the thera-
hmaniasis are toxic. Several doses need to be given peutic efficacy of the liposomised drug may result
over a prolonged period of time and development of from the respiratory burst-inducing activity of tuf-
drug resistance is becoming a problem. Since leis- tsin, in addition to the effect of targeted drug
hmaniasis affects the MPS cells, a number of studies delivery of the drug to the macrophages [19,21].
have been conducted to exploit liposomes as drug We also studied the course of the infection in
vehiclesforthetreatmentofleishmaniasis[9,56–58]. animals pre-treated with both free and liposomal
The efficacy of the several liposomal stibanate tuftsin which considerably enhances their resistance
formulations against L.donovani has been shown to to leishmania infection. However, the inhibitory
be better than the free drug [59]. Different sugar- effectonthisinfectionwasgreater(P,0.01)onday
bearingliposomes,containingdrugsviz.pentamidine 30 post-infection for single treatment when tuftsin
isethionate and its methoxy derivative, hamycin, and was incorporated in the liposomal bilayer. Moreover,
140 A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146
a single treatment with tuftsin or liposomal tuftsin tioninliposomesdidnotshowanytoxicityevenat5
was more effective than the multiple treatment [19]. mg/kg (Agarwal and Gupta, unpublished observa-
The susceptibility of the peritoneal macrophages, tions). These results were consistent with the earlier
derived from the pre-treated animals to L.donovani studies, showing an increase in the LD of Amp B
50
infection was examined in vitro. These experiments after liposomisation [63]. Over 95% efficacy in
demonstrated that the infection of macrophages was reducing the spleen parasite burden was observed at
markedly reduced when these cells were derived a dose of 5 mg/kg even after 30 days post-infection.
from the liposomal tuftsin pre-treated animals. Also, Besides, reducing the parasite load in the spleen, this
the parasite multiplication inside such macrophages preparationwasalsoeffectiveineliminatingtheliver
was considerably decreased, as compared to the and bone marrow parasite load.
macrophages of untreated animals [19]. The Tuft-Lip-Amp B were more effective than the
Successful management of leishmaniasis cases has Lip-Amp B against leishmaniasis even at a low
been reported to be difficult due to increased resist- single dose of 0.5 mg/kg. The tissue distribution
ance of the parasite to the front line drugs, like experiments demonstrated clearly the higher and
antimonials [62]. Although the second front line faster uptake of Tuft-Lip-Amp B liposomes from the
drug, amphotericin B (Amp B), is quite effective as circulation with most of the liposomes being cleared
an antileishmanial agent, it suffers from severe side from the circulation within 1 h after administration
effects. The toxic effects of Amp B can be sig- as compared to Lip-Amp B (Agarwal and Gupta,
nificantly minimised, keeping intact the antileishma- unpublished observations). The increased uptake of
nial property, by encapsulating this drug in the Tuft-Lip-Amp B observed in infected organs sug-
liposomes [20,63]. gested that the drug distribution in the biophase was
To examine whether this property of Amp B was also influenced by infection besides the liposomisa-
further enhanced by its incorporation in the tuftsin- tion. Although liposomisation of Amp B did not
bearing liposomes, we evaluated the efficacies of significantly alter its distribution in the macrophage
Amp B incorporated in tuftsin-free liposomes (Lip- rich organs, except when tuftsin was grafted on the
Amp B) and tuftsin-bearing liposomes (Tuft-Lip- liposomessurface,butitdidleadtoadecreaseinthe
Amp B) against L.donovani infections in vitro. The drug concentration in the kidney of these animals. It
antileishmanial activity of Lip-Amp B, at all the may thus be suggested that the higher efficacy of
tested Amp B concentrations, was higher than that Lip-Amp B observed could partly be due to the
observed with free Amp B in identical conditions. reduced drug concentration in the kidney, which
This activity further increased when tuftsin was should not only lead to the decreased drug toxicity
grafted on the surface of Lip-Amp B liposomes. The but also an increased drug tolerance. In case of the
difference observed between the antileishmanial tuftsin-bearing liposomes, the higher efficacy ob-
activities of Lip-Amp B and Tuft-Lip-Amp B was served was perhaps because of both the increased
very significant (P,0.01) at Amp B concentrations drug tolerance and enhanced uptake of liposomised
lower than 10 mg/ml, but it became smaller at the drug by the macrophage rich organs of infected
higher Amp B concentrations. These results clearly mice. Tuftsin-bearing liposomes also increased the
indicate that encapsulation of Amp B in liposomes drug accessibility to areas like bone marrow which
helps not only in reducing the drug toxicity but also are otherwise inaccessible to free Amp B, and were
in increasing the drug efficacy against L.donovani the main cause of relapse of the leishmanial in-
infections in vitro (Agarwal and Gupta, unpublished fection.
observations).
Theinvitroresultswerevalidatedbyobservingan
enhancement of drug efficacy at all the tested drug 5. Fungal infections
doses in vivo. Besides enhancing the efficacy,
liposomisation of the drug also led to a marked Fungal infections continue to be a major problem
decrease in the drug toxicity.While the animals died in management of immunocompromised patients.
at a 2 mg/kg dose of free Amp B, drug administra- The presence of any fungal disease in humans
A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146 141
commonly implies that the host defence systems by formation of specific pores composed of small
have been compromised due to some reason. Thus, aggregates of Amp B and sterol. These defects cause
thefungalinfectionsoftenrepresenttheopportunistic depolarisationofthemembrane,andconsequentlyan
infections which are the major cause of morbidity in increase in the membrane permeability to protons
immunocompromised/deficient human subjects. The and monovalent cations. The leakage of metabolites
clinical effects of the fungal disease in the immuno- from cells leads to cell death.
compromised host vary widely according to the The usefulness of the liposomes as vehicles for
nature of the underlying disorder as well as the Amp B has been demonstrated in fungal diseases,
fungus involved. In general, these diseases are very such as candidiasis, histoplasmosis and cryptococ-
likely to be progressive in nature, and often dissemi- cosis by reducing its toxicity and increasing its
nated and life threatening. Patients with acute therapeutic index [70–74]. Recently, targeted deliv-
leukaemia, especially following hospitalisation and ery to lung tissues, the organs first infested by many
administration of antibiotics, are prone to various fungi,viainhaledliposomalAmpBaerosolhasbeen
fungal infections and their early diagnosis in cancer shown to be a more effective approach [75]. The
patients still remains elusive. liposomal preparation permitted the use of high
Chemotherapy with antifungal agents is a priori amountsofdrugwhichinfreeformwouldhavebeen
indication for all systemic and superficial fungal quite toxic. Earlier, it was thought that the decreased
infections in humans.Various antifungal chemothera- toxicity of liposomal Amp B was due to the altered
peutic agents available include; polyenes, azoles, disposition of the drug. However, no association
allylamines, morpholines, flucytosine, griseofulvin, between these alterations and decreased toxicity was
iodides,hydroxy–stibamine,andimidazoleclassesof found [76]. Moreover, no significant difference in
drugs [64–66]. As elimination of fungi from the organ distribution was observed between the lipo-
tissues of normal healthy subjects is often associated someand freeAmp B[77].Both invitroand invivo
with stimulation of the cell-mediated immune de- results show liposomal Amp B to be less cytotoxic.
fences which involve activation of the mononuclear The toxicity to cells is dependent on the attainment
phagocytes by sensitised T cells [67], treatment with of a sufficient level of Amp B in the aqueous phase.
antifungal drugs along with the agents that provoke Liposomes composed of phospholipids with satu-
macrophages/monocytes should find useful applica- rated acyl chains are non-toxic whereas Amp B
tion in general in fungal chemotherapy. Am- intercalated in liposomes composed of phospholipids
photericin B (Amp B), a polyene antibiotic, has been containing unsaturated acyl chains are almost as
widely used in clinical practice to treat various toxic as Amp B [78]. The reduction in toxicity of
systemic fungal infections, e.g. candidiasis, histop- liposomal Amp B is also evident in the presence of
lasmosis, aspergillosis, etc. [64–66,68]. Side effects sterols in liposomes [79]. This may be attributed to
associated with Amp B, vary from mild headache, the tighter lipid packing in liposomes giving rise to
chills and fever to severe hemolytic anemia and an increased order and stability and thus decreasing
acute nephritis, limiting its fuller exploitation as a the flux of Amp B molecules across the membranes.
proper therapeutic measure. Nephrotoxicity is the Following the promising clinical results, three
mostseriousproblemassociatedwithAmpBtherapy formulations containing Amp B (Abelcet, Amphocil,
and its severity may necessitate interruption of and AmBisome) are currently available on the mar-
treatment. ket [12].
Chemical derivatisation of amphotericin B has With a view to further increase the scope of
been done to eliminate its toxic side reactions, but liposomised Amp B (Lip-Amp B), we incorporated
withoutmuchsuccess[69].ThetoxicityofAmpBto Amp B in tuftsin-free liposomes (Lip-Amp B) and
cells originates from its binding to sterols present in tuftsin-bearing liposomes (Tuft-Lip-Amp B) and
the cellular membranes; ergosterol in the case of then studied their efficacy against human Aspergillus
fungalcells,andcholesterolinmammaliancells.The fumigatus infections in mice. The result of these
binding of Amp B to membrane sterols results in studies showed that the % survival of the A.
disorganisation of the cellular membranes, possibly fumigatus-infected mice considerably increased (70–
142 A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146
75%) by treating them with Tuft-Lip-Amp B, as Advancesinantimycobacterialchemotherapyhave
compared to theanimals that received treatment with brought tuberculosis from a life threatening situation
Lip-Amp B [22]. Also the survived animals from the to a potentially curable one. Still, the most important
Tuft-Lip-Amp B treated group were virtually free of factor in the treatment of tuberculosis is prolonged
any fungal load whereas animals treated with Lip- chemotherapy, for a minimum period of 6–12
Amp B still had some residual infection [22]. These months, which is often associated with serious and
resultsstronglyindicatethattheefficacyofTuft-Lip- undesirable side effects, e.g. hepatotoxicity [83–85].
Amp B against A.fumigatus infection considerably Also, undesired effects may be caused by the high
increased perhaps due to activation of macrophages/ levels of antitubercular drugs in blood required to
monocytes, the key line of host defence against achieve an effective intracellular drug concentration.
pathogenic fungi [67]. To further examine the va- It is, therefore, desirable to develop an approach
lidity of the belief, we first treated the animals with which would allow the use of lower drug doses by
drug-free tuftsin-bearing liposomes prior to the in- delivering the agent to the infected cells, thereby
fection and then after challenging them with lethal improving efficacy and potentially reducing the
doses of A. fumigatus, the infected animals were toxicity.
treated with Lip-Amp B and Tuft-Lip-Amp B. In Liposomes could resolve some of the above
these experiments, no difference between the ef- problems by serving as carriers for site specific
ficacies of Lip-Amp B and Tuft-Lip-Amp B were and/or sustained delivery of antitubercular drugs.
observed, demonstrating that the higher efficacy of ‘Passive targeting’ of the liposomes to MPS [9–12]
Tuft-Lip-Amp B, as compared to Lip-Amp B, was could be utilised as the causative organism,
primarily due to macrophage activation affected by Mycobacterium tuberculosis, resides and proliferates
tuftsin [22]. Further, the tuftsin incorporation in the primarily within the mononuclear phagocytes, which
Lip-Amp B did not affect the drug toxicity, as we normally serve as the first line of defence against
observed almost identical LD value (ca. 7 mg/kg) infections [86].
50
for both Lip-Amp B and Tuft-Lip-Amp B [22]. Rifampicin, isoniazid, streptomycin,
These studies thus clearly demonstrate that the Tuft- pyrazinamide, thioacetazone, p-aminosalicyclic acid,
Lip-Amp B formulation was significantly better than ethambutol, and streptomycin are amongst the major
the Lip-Amp B preparation in treatment of the front line drugs in the treatment of tuberculosis
systemic fungal infections due to its higher efficacy [83,85,87]. Most of these drugs though possess
andcomparabletoxicity,suggestingthattheadminis- strong antitubercular activity, but each of these
tration of macrophage activators along with Amp B exhibit several undesirable effects, thus further em-
in liposomes should greatly improve the efficacy of phasising the necessity of suitable vehicles which
fungal chemotherapy. may help in diminishing the undesirable effects of
these drugs without compromising their efficacy. A
number of studies have demonstrated that the effica-
6. Tuberculosis cy of these antitubular drugs is increased in their
liposomised forms [88–91].
Tuberculosis is a single most infection which Liposomes besides delivering the drug to the
results in the largest number of deaths world-wide; infected site could also act as drug reservoirs to
nearly 3 million people are killed every year [80]. provide a slow and sustained release of the drug.
The association of tuberculosis with HIV infection This would not only reduce the cost of the treatment
has significantly exacerbated the situation in de- but may also shorten the duration of the treatment,
veloped and developing nations [81]. HIV infection the two major drawbacks associated with the chemo-
is the highest risk factor identified so far for latent therapy of tuberculosis.
tuberculosis infection to progress to an active dis- We demonstrated the usefulness of the liposomes
ease. This infection also increases the risk to new as vehicles for refampicin (RFP) in treatment of
tuberculosis infection that will progress to disease Mycobacterium tuberculi infections in mice [23].
[81,82]. The drug acts on DNA-dependent RNA polymerase
A.K.Agrawal,C.M.Gupta / Advanced Drug Delivery Reviews41(2000)135–146 143
inthebacterialcelltoblocktheproteinsynthesisand enlarging the scope of RFP-loaded liposomes in the
kills the micro-organism. There is little or no cross treatment of a variety of mycobacterial infections.
resistance between RFP and other antimycobacterial
drugs [92]. The sterilizing activity of RFP is high
because of its ability to kill the semidormant bacilli.
Acknowledgements
The drug is thus comparatively safe, but its half-life
in circulation is relatively short (ca. 3 h) and most of
We thank Drs. R.K. Jain, A. Puri, A. Singhal, A.
thedrugfollowingoraladministrationismetabolized
Agarwal, M. Owais, P.Y. Guru, S. Chandra and N.B.
and excreted, leaving only a limited amount avail-
Singh for their contribution in the work.
able for activity against the mycobacterium [93].
Thus, a daily dose of 600 mg of RFP is required for
effective treatment, which often leads to toxic side
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