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Current Drug Delivery
ISSN: 1567-2018
Current Drug Delivery
Volume 3, Number 1, January 2006
Contents
Topical Non-Invasive Delivery Technologies
for the Deliveryof Genetic Material
Guest Editor: Marianna Foldvari
Editorial
Novel Methods for the Non-Invasive Administration
of DNA Therapeutics and Vaccines Pp. 3-15
Sylvia van Drunen Littel-van den Hurk
[Abstract]
DNA Delivery for Vaccination and Therapeutics
Through the Skin Pp. 17-38
Marianna Foldvari, Shawn Babiuk and Ildiko Badea
[Abstract]
Non-Invasive Immunization on the Skin Using
DNA Vaccine Pp. 29-35
Zhengrong Cui, Annie Dierling and Marianna Foldvari
[Abstract]
Topical DNA Vaccination with DNA/Lipid Based
Complex Pp. 37-45
Myeong Jun Choi, Jong Heon Kim and Howard I. Maibach
[Abstract]
Bacterial Invasin: Structure, Function,
and Implication for Targeted Oral Gene Delivery Pp.
47-53
R. Noelle Palumbo and Chun Wang
[Abstract]
Advances in Noninvasive Pulmonary Gene Therapy
Pp. 55-63
Charles L. Densmore
[Abstract]
Minimally Invasive Cutaneous Delivery
of Macromolecules and Plasmid DNA Via Microneedles
Pp. 65-75
Sion A. Coulman, David Barrow, Alexander Anstey, Chris
Gateley, Anthony Morrissey, Nicolle Wilke, Chris Allender,
Keith Brain and James C. Birchall
[Abstract]
Sequence and Time Dependence of Transfection
Efficiency of Electrically-Assisted Gene Delivery to Tumors
in Mice Pp. 77-81
Maja Cemazar, Darja Pavlin, Simona Kranjc, Alenka Grosel,
Suzana Mesojednik and Gregor Sersa
[Abstract]
Topical DermaVir Vaccine Targeting Dendritic Cells
Pp. 83-88
Julianna Lisziewicz, Laurene Kelly and Franco Lori
[Abstract]
Gene Delivery into Human Skin In
Vitro Using Biphasic Lipid Vesicle Pp.
89-93
Marianna Foldvari, Praveen Kumar, Martin King, Ravinder
Batta, Deborah Michel, Ildiko Badea and Mary Wloch
[Abstract]
General Articles
Thermotropic and Spectroscopic Behavior of Skin:
Relationship with Percutaneous Permeation Enhancement
Pp. 95-113
Kumar Babita, Vinod Kumar, Vikas Rana, Subheet Jain and
Ashok Kumar Tiwary
[Abstract]
Polylactide-Co-Glycolide Microparticles
with Surface Adsorbed Antigens as Vaccine Delivery Systems
Pp. 115-120
Manmohan Singh, Jina Kazzaz, Mildred Ugozzoli, Padma Malyala,
James Chesko and Derek T. O’Hagan
[Abstract]
Particulate and Vesicular Drug Carriers
in the Management of Tuberculosis Pp. 121-128
Rachna Rastogi, Yasmin Sultana, Asgar Ali and Mohammed
Aqil
[Abstract]
Abstracts
[Back
to top]
Editorial
The knowledge of the genome and proteome will result in
enormous opportunities in the identification of new therapeutic
molecules which will ultimately have a major impact on human
health. The new types of pharmaceuticals will be DNA and RNA
pieces, small peptides, large proteins and recombinant or
subunit vaccines. The science and technology of the delivery
and targeting of the ‘products’ of the
genome and proteome are the next crucial links in the development
of new approaches for the treatment and prevention of diseases.
Developing non-invasive delivery approaches for macromolecules
in general is a major challenge. This is conspicuously reflected
in the articles in this issue dealing with DNA delivery methods.
To replace direct injection using needles or reimplanting
ex vivo transfected keratinocytes or dermal fibroblasts,
novel delivery methods for oligo- and polynucleotides may
include less invasive procedures such as microprojectile techniques,
electroporation and topically applied formulations. There
is also effort to develop non-viral delivery systems in order
to avoid the many disadvantages of viral vectors.
This issue of Current Drug Delivery contains a collection
of articles focusing on non-invasive delivery of DNA for therapeutic
and vaccine purposes.
The reviews by von drunen Littel-van den Hurk, Foldvari
et al., Cui et al. and Choi and Maibach in this issue
provide extensive coverage of the various administration methods
of DNA into the body, the differences between the requirements
for therapeutic and vaccine DNA and the effect of formulation
design on DNA delivery. There are specific examples for dermal,
oral, pulmonary and electrically-assisted delivery technologies
(Lisziewicz et al., Wang et al., Densmore, Cemazar et
al. and Foldvari et al.).
There are reports on several novel technologies for non-invasive
DNA delivery: silicon microneedles, DNA-nanoparticles, biphasic
vesicles (Biphasix™) and an invasin-based gene targeting
system.
Birchall et al. demonstrated the ability of microfabricated
silicon microneedle arrays to create micron-sized channels
through the stratum corneum of excised human skin and the
resulting ability of the conduits to facilitate localized
delivery of charged macromolecules and plasmid DNA. Lisziewicz
et al. have developed a non-viral delivery system where
the plasmid DNA, encoding appropriate retroviral genes, is
encapsulated within pathogenlike nanoparticles. Topical application
of these nanoparticles on abraded skin resulted in suppressed
viral replication and increased survival time in HIV-infected
macaques. Foldvari et al. demonstrated high level
of plasmid DNA delivery after topical application on intact
human skin using biphasic vesicles and evaluated the quantitative
aspects of DNA delivery. Wang et al. described a
Yersinia protein, invasin, that binds to a subset of β1
integrin receptors located on the apical membrane of intestinal
M-cells, as potential delivery/targeting agents. By coupling
invasin to a micro/nanoparticle carrier, the natural transport
mechanism can be utilized for the oral delivery of therapeutic
genes and gene-based vaccines.
This issue intends to give the readers an excellent overview
on issues associated with non-invasive delivery of DNA and
some recent developments on potential technologies. It is
also intended to reflect on the necessity of further significant
efforts into delivery system development for macromolecular
therapeutics. Even though the pharmaceutics and the engineering
of delivery technologies does not seem (to some) as dazzling
as the sequencing of the genome or the compiling of the proteome,
it is probably the most crucial task necessary for turning
genes and proteins into therapeutic products for humans and
animals.
Marianna Foldvari
Guest Editor – Current Drug Delivery
College of Pharmacy and Nutrition
University of Saskatchewan
110 Science Place, Saskatoon
Sask. S7N 5C9, Canada
[Back to top]
Novel Methods for the Non-Invasive Administration
of DNA Therapeutics and Vaccines
Sylvia van Drunen Littel-van den Hurk
To develop a successful protein therapeutic, effective DNA
delivery technologies are required that induce high and sustained
levels of protein production in appropriate targets sites,
whereas robust and long-lasting immune responses need to be
induced by a DNA-based vaccine. Vectors for gene therapy and
DNA vaccines must be resistant to degradation and attack by
the immune system, have a satisfactory safety profile, and
be able to express the therapeutic protein for the desired
period of time. Effective non-viral vectors, which can express
the proteins of interest at high levels, are available. However,
since most of the DNA delivered in vivo is degraded before
it can enter the nucleus, proper formulation and delivery
are critical to the development of effective gene-based therapeutics
and vaccines. These systems must be safe for human and veterinary
clinical applications and yet ensure that the DNA survives
the extra- and intracellular environment and is capable of
entering the appropriate cellular compartments. In this review
various poten-tial and proven non-invasive chemical, mechanical,
physical and biological DNA delivery systems for therapeutic
and vaccine applications are discussed. A few of these approaches
have been evaluated and proven to be promising in target species.
Others, which promise to be less invasive, have only just
started to be explored.
[Back to top]
DNA Delivery for Vaccination and Therapeutics Through
the Skin
Marianna Foldvari, Shawn Babiuk and Ildiko Badea
Cutaneous gene therapy and DNA vaccination are potential
applications of plasmid delivery methods where a gene for
an antigen or a therapeutic protein is inserted in the plasmid
and applied to the skin. However, the delivery of the DNA
plasmid is a major challenge due to the unusual physicochemical
properties of the DNA, the tissue and cellular barriers and
expression difficulties. Even though the skin is the most
accessible organ of the body and it is an ideal target for
gene therapy, the delivery of plasmid DNA across the skin
is very difficult due to the specific barrier function of
the stratum corneum and the inconsistent transfection rate
of keratinocytes and other epidermal cells. To date there
is no gene delivery system that was shown to be optimal for
cutaneous gene therapy. In order to develop an efficient non-viral
delivery vehicle we need to design a system that provides
the combined properties of effective DNA condensation, cuta-neous
permeation, cellular transfection and sufficiently sustained
expression.
This paper reviews the formulation approaches and delivery
methods for DNA through the skin in the context of the bar-riers
both at the tissue and cellular levels for both vaccine and
gene therapy applications.
[Back to top]
Non-Invasive Immunization on the Skin Using DNA Vaccine
Zhengrong Cui, Annie Dierling and Marianna Foldvari
Skin has evolved to protect not only by acting as a physical
barrier, but also by its role in our powerful immune system.
As a frontline of the host’s defense against pathogens,
skin is well equipped for immune surveillance. For example,
compared to many other tissues, the epidermis of the skin
contains a high population of Langerhans cells, which are
very potent immature dendritic cells. Thus, targeting antigens
to the skin epidermis should be able to efficiently induce
strong immune responses. However, the forbidden barrier posed
by the stratum corneum layer of the epidermis prevents effective
entrance of antigens into the epidermis. Nevertheless, non-invasive
immunization onto the skin has proven in the last several
years to be a viable immunization modality. DNA vaccine is
a vaccine made of bacterial plasmid DNA encoding an antigen
of interest. Upon uptake of the plasmid, host express and
process the encoding antigen, and then mount immune responses
against it. DNA vaccine is advantageous over many other types
of vaccines. The feasibility of non-invasive immunization
onto the skin with DNA vaccine has been confirmed. Although
the potency of the immune response has proven to be weak,
many skin stratum corneum disrupting chemical and physical
approaches and DNA vaccine carriers/adjuvants that significantly
enhance the resulting immune response have been reported.
In addition, research on elucidating the mechanism of immune
induction from non-invasively, topically applied DNA vaccine
has also been carried out. With further improvement and optimization,
non-invasive immunization onto the skin with DNA vaccine should
be able to elicit reliable and efficacious immune response
to a variety of antigens.
[Back to top]
Topical DNA Vaccination with DNA/Lipid Based Complex
Myeong Jun Choi, Jong Heon Kim and Howard I. Maibach
Topical DNA vaccines have been shown to elicit both broad
humoral and cellular immune response in vivo. The
skin is an attractive site for the delivery DNA antigens for
DNA vaccination. However, due to skin’s barrier properties,
the penetration of DNA and the applications of topical vaccination
are limited. To improve permeability of stratum corneum and
the potency of topical DNA vaccines, efficient delivery systems
are needed. Topical vaccination has been achieved using topical
application of naked DNA with or without tape stripping and
DNA/lipid based complex such as liposomes, niosomes, Transfersomes®,
or microemulsion. All methods resulted in significant enhancement
in humoral and cellular immune response over naked DNA alone.
To develop more cost-effective and needle free vaccines, skin
targeted immunizations are required. This overview focuses
on the comparison of the potency of topical DNA vaccine between
naked DNA and DNA-lipid based complex.
[Back to top]
Bacterial Invasin: Structure, Function, and Implication
for Targeted Oral Gene Delivery
R. Noelle Palumbo and Chun Wang
The mucosal surface of the gastrointestinal (GI) tract is
the first line of defense against foreign pathogens and toxins
ingested orally. The content of the GI tract is constantly
being sampled by the immune system through specialized epithelial
cells known as M-cells, which are present in the Peyer’s
patches of the gut, providing a thin covering over lymphoid
tissue. In this way, once a harmful entity is found an immune
response can be activated to eliminate the threat. Many bacterial
pathogens, such as Yersinia, Listeria, Salmonella,
and Shigella, have evolved ways of exploiting M-cells
to gain entrance to the body. The Yersinia species
is of particular interest since its extracellular protein
invasin provides one of the most direct and efficient manners
of host cell invasion. Invasin binds to a subset of β1
integrin receptors lo-cated on the apical membrane of intestinal
M-cells, thereby facilitating the bacteria’s entry into
the cells and the lym-phatic system underneath. This mechanism
is highly specific and effective, making the invasin protein
a very attractive modality for use in the oral delivery of
molecules that include therapeutic genes and gene-based vaccines.
This article provides a brief overview of the molecular structure
and properties of the Yersinia invasin as related
to the protein’s ability to facilitate binding and entry
into M-cells. Also discussed are several innovative approaches
that demonstrate the use of invasin as an effective targeting
agent for biological and synthetic gene carrier systems, and
the future pros-pect of developing invasin-based oral gene
delivery formulations.
[Back to top]
Advances in Noninvasive Pulmonary Gene Therapy
Charles L. Densmore
One of the most noninvasive approaches to drug delivery
is via inhalation. The delivery of genes via aerosol holds
promise for the treatment of a broad spectrum of pulmonary
disorders and offers numerous advantages over more invasive
modes of delivery. Delivery of genes expressing secretory
therapeutic proteins or peptides may even have ap-plication
to a number of nonpulmonary diseases. After the cloning of
the cystic fibrosis gene, there was great interest in the
delivery of genes directly to the lung surfaces via inhalation
and most early efforts focused on the use of nonviral vectors,
particularly cationic lipids. Early on, nebulization shear
forces, inefficient penetration of mucous barriers and inhibitory
effects of surfactant and other lung specific features generally
resulted in a lack of therapeutic effect. But in recent years,
a number of other nonviral and even viral vectors have been
delivered successfully in this manner. Poly-ethyleneimine
(PEI)-based formulations have proven stable during nebulization
and result in transfection of a very large proportion of epithelial
cells throughout the airways (though the level of transgene
expression per cell may be relatively low), as well as significant,
though lower levels of transfection throughout the lung parenchyma.
Most importantly, therapeutic responses have been obtained
in several animal lung tumor models when PEI-based complexes
of p53 and IL-12 genes were delivered by aerosol. This approach
may also prove useful as a means of localized genetic immuniza-tion.
In addition, inhalation delivery of some formulations seems
to be associated with surprisingly low toxicity and has resulted
in little or no immunostimulatory response to the unmethylated
CpG sequences in bacterially-produced plasmid DNA, which has
presented a challenge to repeated gene therapy via many other
modes of delivery.
[Back to top]
Minimally Invasive Cutaneous Delivery of Macromolecules
and Plasmid DNA Via Microneedles
Sion A. Coulman, David Barrow, Alexander Anstey, Chris
Gateley, Anthony Morrissey, Nicolle Wilke, Chris Allender,
Keith Brain and James C. Birchall
The stratum corneum (SC) represents a significant barrier
to the delivery of gene therapy formulations. In order to
realise the potential of therapeutic cutaneous gene transfer,
delivery strategies are required to overcome this exclusion
effect. This study investigates the ability of microfabricated
silicon microneedle arrays to create micron-sized channels
through the SC of ex vivo human skin and the resulting
ability of the conduits to facilitate localised delivery of
charged macromolecules and plasmid DNA (pDNA). Microscopic
studies of microneedle-treated human epidermal membrane revealed
the presence of microconduits (10-20μm
diameter). The delivery of a macromolecule, β-galactosidase,
and of a ‘non-viral gene vector mimicking’ charged
fluorescent nanoparticle to the viable epidermis of microneedle-treated
tissue was demonstrated using light and fluorescent microscopy.
Track etched permeation profiles, generated using ‘Franz-type’
diffusion cell methodology and a model synthetic mem-brane
showed that >50% of a colloidal particle suspension permeated
through membrane pores in ~2 hours. On the basis of these
results, it is probable that microneedle treatment of the
skin surface would facilitate the cutaneous delivery of lipid:polycation:pDNA
(LPD) gene vectors, and other related vectors, to the viable
epidermis.
Preliminary gene expression studies confirmed that naked
pDNA can be expressed in excised human skin following mi-croneedle
disruption of the SC barrier. The presence of a limited number
of microchannels, positive for gene expression, indicates
that further studies to optimise the microneedle device morphology,
its method of application and the pDNA formulation are warranted
to facilitate more reproducible cutaneous gene delivery.
[Back to top]
Sequence and Time Dependence of Transfection Efficiency
of Electrically-Assisted Gene Delivery to Tumors in Mice
Maja Cemazar, Darja Pavlin, Simona Kranjc, Alenka Grosel,
Suzana Mesojednik and Gregor Sersa
Electrically-assisted gene delivery is a non-viral gene
delivery technique, using application of square wave electric
pulses to facilitate uptake of plasmid DNA into the cells.
Feasibility and effectiveness of this method in vivo
was already demonstrated, elaborating on pulse parameters
and plasmid construction. However, there were no studies performed
on sequencing and timing of plasmid DNA injection into the
tumors and application of electric pulses. For this purpose
we measured luciferase expression in two tumor models (LPB
fibrosarcoma, B16F1 melanoma) after elec-trically-assisted
gene delivery at varying time intervals between the pCMV-Luc
plasmid injection and electroporation. Expression of
luciferase was determined by measurement of its activity using
luminometer.
The results demonstrated that pCMV-Luc plasmid has
to be injected before the application of electric pulses,
since no measurable expression was detected in the tumors
when pCMV-Luc plasmid was injected after electroporation
of tu-mors. In both tumor models the highest transfection
efficiency was obtained when pCMV-Luc plasmid was
injected not less than 5 minutes but also not more than 30
minutes before the application of electric pulses. The results
also demon-strated variability in the transfection efficiency
depending on the tumor model. High expression was obtained
in B16F1 tumor model (~5500 pg luc/mg tumor) and lower in
LPB fibrosarcoma (~200 pg luc/mg tumor).
In conclusion, our results demonstrate that regardless of
the susceptibility of the tumors to electrically-assisted
gene de-livery, the best timing for pCMV-Luc plasmid is
between 30 to 5 minutes prior to the application of electric
pulses to the tumors.
[Back to top]
Topical DermaVir Vaccine Targeting Dendritic Cells
Julianna Lisziewicz, Laurene Kelly and Franco Lori
DermaVir employs a topical, non-invasive method for vaccine
delivery to dendritic cells. The vaccine product contains
plasmid DNA as the active ingredient, encoding authentically
expressed retroviral genes with appropriate safety modifications.
The non-viral delivery system packages the DNA within pathogen-like
nanoparticles and studies indicate that vaccine antigens are
taken up by epidermal Langerhans cells, the precursors of
dendritic cells. DermaVir loaded dendritic cells reach the
draining lymph node target but not the bloodstream nor indiscriminately
other organ systems. Safety data from DermaVir immunized infected
macaques indicate improved survival, absence of apparent toxicities
other than transient erythema and lack of recombination between
the vaccine DNA and the infectious viral DNA in-tegrated in
the host genome. DermaVir represents a potential new approach
for the treatment of HIV infection to be util-ized either
in conjunction with antiretroviral therapy or during structured
treatment interruption.
[Back to top]
Gene Delivery into Human Skin In Vitro Using
Biphasic Lipid Vesicle
Marianna Foldvari, Praveen Kumar, Martin King, Ravinder
Batta, Deborah Michel, Ildiko Badea and Mary Wloch
Topical gene delivery to the skin shows great potential
for painless, non-invasive administration of novel vaccines
and therapeutic agents. The challenge is to develop a pharmaceutically
acceptable system that can deliver suitable amounts of plasmid
DNA to produce the desired level of response. The purpose
of this study was to quantitatively assess DNA delivery by
a novel lipid-based biphasic delivery system into the viable
layers of excised human skin. Biphasic lipid vesicle formulations,
incorporating plasmid DNA were evaluated in vitro
in flow-through diffusion cells. Fifty mg DNA formulation
containing 10 μg
DNA was applied to full-thickness human breast skin for 24
hours. Residual formulation was removed and the skin was washed
with PBS, then tape-stripped, followed by DNase treatment
to remove surface bound DNA. Skin samples were homogenised
and digested overnight with Proteinase K. The resulting supernatant
was used as a template for quantitative PCR. Three formulations
yielded a significant degree of dermal absorption compared
to the controls. Formulation 26-3-2-DNA indicated that approximately
1x109 copies of plasmid were absorbed per cm2
skin. Other formulations resulted in 5 x 106 copies/cm2
skin (17C3-1-DNA) and 5 x 108 copies/cm2
skin (26-3-1-DNA). Biphasic vesicles delivered significant
quantities of plasmid DNA into the ‘viable’ layers
of human skin in vitro. The successful delivery of
this large (~ 4, 400 kDa) charged molecule through intact
stratum corneum represents a major advance in transdermal
macromolecule delivery.
[Back to top]
Thermotropic and Spectroscopic Behavior of Skin: Relationship
with Percutaneous Permeation Enhancement
Kumar Babita, Vinod Kumar, Vikas Rana, Subheet Jain and
Ashok Kumar Tiwary
Stratum corneum (SC) is comprised of lipids, protein and
low molecular weight water-soluble components. Changes in
these skin micro constituents can be understood by instrumental
methods like differential scanning calorimetry (DSC) and attenuated
total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy.
The former provides information about changes in thermotropic
behavior of SC lipids and proteins, whereas the latter provides
data about alterations at molecular and conformational level.
Most of the DSC thermograms of intact mammalian SC show two
reversible and two irreversible transitions in the temperature
range of 25-125 ºC. The reversible endotherms are ascribed
to lipid melting transitions, whereas the irreversible endotherms
are ascribed to protein denaturation. Similarly, the FTIR
spectral bands of SC occurring between 2920-2850 cm-1
and between 1650-1550 cm-1 have been suggested
to arise from lipid and protein molecular vibrations, respectively.
Treatment of skin with solvents or permeation enhancers alters
the composition of lipids or their molecular arrangement in
the skin microenvironment, which leads to changes in permeability
of drug molecules. Furthermore, inhibition of lipid synthesis
in epidermis with concomitant decrease in en-thalpy of lipid
endothermic transitions and reduction in height and area of
asymmetric and symmetric C-H stretching peaks have been found
to be directly correlated with enhanced permeation of drugs.
In addition, method of skin prepara-tion, type of skin, types
of enhancer etc. also influence both the nature and intensity
of responses recorded in spectro-graphs and thermograms. Therefore,
the modification in spectrographs and thermograms of skin
samples treated with various enhancers, vehicles etc. are
expected to provide better insight into their mechanism of
action on the skin. This review article shall critically evaluate
the thermotropic and infrared spectroscopic data of SC/epidermis
after various treatments.
[Back to top]
Polylactide-Co-Glycolide Microparticles with Surface
Adsorbed Antigens as Vaccine Delivery Systems
Manmohan Singh, Jina Kazzaz, Mildred Ugozzoli, Padma Malyala,
James Chesko and Derek T. O’Hagan
Several groups have shown that vaccine antigens can be encapsulated
within polymeric microparticles and can serve as potent antigen
delivery systems. We have recently shown that an alternative
approach involving charged polylactide co-glycolide (PLG)
microparticles with surface adsorbed antigen(s) can also be
used to deliver antigen into antigen presenting cell (APC).
We have described the preparation of cationic and anionic
PLG microparticles which have been used to adsorb a variety
of agents, which include plasmid DNA, recombinant proteins
and adjuvant active oligonucleotides. These PLG microparticles
were prepared using a w/o/w solvent evaporation process in
the presence of the anionic surfactants, including DSS (dioctyl
sodium sulfosuccinate) or cationic surfactants, including
CTAB (hexade-cyl trimethyl ammonium bromide). Antigen binding
to the charged PLG microparticles was influenced by several
factors including electrostatic and hydrophobic interactions.
These microparticle based formulations resulted in the induction
of significantly enhanced immune responses in comparison to
alum. The surface adsorbed microparticle formulation offers
an alternative and novel way of delivering antigens in a vaccine
formulation.
[Back to top]
Particulate and Vesicular Drug Carriers in the Management
of Tuberculosis
Rachna Rastogi, Yasmin Sultana, Asgar Ali and Mohammed
Aqil
Although oral drug therapy for tuberculosis exists and is
widely followed, its major drawbacks are lack of patient compliance
and development of adverse effects like hepatotoxicity on
long term use. Absence of new therapeutic agents and the above
mentioned demerits have led to search for alternative methods
for delivery of antitubercular agents. Colloidal drug carriers,
a popularly utilized delivery system has been deeply explored
for the cause. The article discusses the advances in the management
of tuberculosis by the use of particulate and vesicular drug
carriers by par-enteral, inhalational and oral routes. Use
of this delivery strategy has led to massive reduction in
the dosage resulting in toxicity alleviation. As a number
of studies have already been undertaken in experimental models,
it will be a promising tool in the prevention of relapse and
successful treatment of tuberculosis in patients.
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