Inhaled therapy with conventional or unformulated ATDs
Many patients continue to remain sputum smear-positive for Mycobacteriumtuberculosis despite ongoing chemotherapy, which is mainly
attributableto (other than drug resistance) extensive cavitary
lesions wherethe antimycobacterial drugs fail to reach when
administeredorally.
Sacks et al.
selected such patients of pulmonaryTB who were sputum smear-positive
after at least 2 months ofconventional treatment. The patients were
treated with gentamicinor kanamycin via nebulization as adjunctive
therapy while theconventional drugs were maintained in parallel.
The
frequencyof nebulization was thrice daily whereas the duration was
dictatedby practical considerations and smear conversion times whichranged from 9 to 122 days. It was observed that 86% (6 out of
7) of the patients with drug-susceptible TB and 58% (7 out of12) of
the patients with drug-resistant TB underwent smear conversionduring
the study period, suggesting that residual aminoglycosidesin sputum
expectorated from pulmonary cavities could inhibitintracavitary
bacillary growth and prevent transmission, thoughnot necessarily
affecting the bacteria inside the macrophages.Nevertheless, the
study did document the supportive role ofinhaled aminoglycosides in
patients with refractory TB.
Aerosol administration of interferon gamma (IFN- ),
a key cytokinein the immunological response against mycobacteria,
has alsobeen attempted. The initial studies were inconclusive as thepatients receiving adjunctive aerosol IFN-
became smear-negativeafter 1 month but continued to be
culture-positive and the smearresponse was not sustained.
However, when the aerosolizedIFN-
therapy was continued for 6 months (thrice weekly), mostof the
patients showed a definite radiological improvement anda reduction
in the size of the cavitary lesions.
It appearsthat merely aerosolizing an antimycobacterial compound may
beinadequate; for efficient bacterial killing, drugs need to beformulated into suitable delivery systems thereby ensuring theirrapid uptake into macrophages which harbour the tubercle bacilli.The dictum holds true for the majority of intracellular infections,and liposomes as well as micro/nanoparticles have emerged as
useful drug carriers () in this context.,
Hence,it is not surprising that these carriers have established
theirpotential for antitubercular inhaled therapy ().
Pulmonary delivery of liposome-encapsulated ATDs
Liposome-encapsulated drugs are especially effective against
intracellular pathogens and their demonstrated advantages include:
(i) the ability to formulate biologically active molecules;
(ii) the ability to encapsulate hydrophilic compounds; (iii)
reduction in toxicity of the active agent; (iv) increased therapeutic
index; (v) increased stability of labile drugs; (vi) improved
pharmacokinetics; (vii) increased delivery to target tissues;and
(viii) the feasibility of nebulization. Liposomes are morepopular as
intravenous ATD carriers.
However, keeping inmind that liposomes have been successfully
nebulized to treatintracellular pulmonary infections,
conventional (phosphatidylcholine/cholesterol)liposomes
encapsulating rifampicin and isoniazid were preparedin our
laboratory for nebulization. The loading of rifampicinwas better
compared with isoniazid.
The inhalable distearoylphosphatidylcholine/cholesterolliposomes
encapsulating ATDs, as prepared by Justo & Moraes,
also showed a satisfactory drug loading for isoniazid as well
as pyrazinamide. In their case, however, the encapsulation of
rifampicin, streptomycin and ethionamide was low. Aerodynamic
characterization of our formulation showed 94% of generatedaerosol
to be respirable, with an MMAD of 0.96 �0.06�m.
A single nebulization of liposomal ATDs to guineapigs could maintain
therapeutic drug concentrations in the plasmafor 48 h whereas free/unencapsulated
drugs were cleared by 24h. Liposomal drugs were present in the lungs
and more importantlyin the alveolar macrophages till day 5 post-nebulization,
suggestingthat liposome-based controlled ATD release may obviate the
needfor daily drug dosing. Our findings and predictions are
supportedby the results of Kurunov et al.,
who reported an equivalenttherapeutic efficacy of twice weekly
nebulized liposomal rifampicinand daily conventional rifampicin in a
murine TB model. Theauthors suggested that the liposomal formulation
helps in thepersistence of rifampicin in the lung tissue.
The specific targeting of liposomes towards the alveolar macrophagescan be achieved by coating the liposomes with alveolar macrophage-specificligands such as O-stearyl amylopectin (O-SAP) and maleylatedbovine serum albumin (MBSA). The therapeutic efficacy of O-SAP-coatedliposomal ATDs was recently reported, however, the intravenous
route was employed for liposomal administration.
Vyas etal.
prepared O-SAP- and MBSA-appended inhalable liposomes
entrapping rifampicin. In vivo studies in albino rats demonstrateda higher pulmonary delivery and better localization of ligand-appendedliposomes to alveolar macrophages compared with conventional
liposomes or free rifampicin, from 30 min to 24 h post-nebulization.
Subsequently, the alveolar macrophages were isolated, spreadas a
monolayer and infected with Mycobacterium smegmatis. The
percentage viability of the bacilli was significantly reducedto
10.9% in the case of MBSA- and 7.1% in the case of O-SAP-coatedliposomes, compared with 69% and 31% for control macrophages
and conventional liposome-treated macrophages, respectively.The
results were based on a single nebulization of liposomalrifampicin
and the authors speculated that an ideal situationof 0% viability
may be obtained by repeated dosing. It is thereforeclear that
nebulization of liposomal ATDs, coupled to the useof alveolar
macrophage-specific ligands, may improve the chemotherapyof
pulmonary TB especially in view of the fact that liposomesare known
to be safe when administered via the respiratory route.
However, with the use of biodegradable polymers in the arenaof
drug delivery, more emphasis began to be laid on the useof polymeric
systems for antitubercular inhaled therapy.
Pulmonary delivery of microparticle-encapsulated ATDs
The use of polymeric microparticles to deliver ATDs by different
routes (injectable, oral and aerosol) has been reported by several
investigators.
Because of its biodegradability and biocompatibility,poly (lactide-co-glycolide)
(PLG; a synthetic polymer) has beena popular choice as a drug
carrier.
By employing solventevaporation as well as spray drying methods, PLG
microparticlesencapsulating rifampicin were prepared.
The former techniqueresulted in spherical particles with 20% drug
loading and 3.45�m volume median diameter whereas the latter
techniqueproduced shrivelled particles with 30% drug loading and
2.76�m diameter. The microspheres were administered via insufflationor nebulization to guinea pigs, 24 h before aerosol infection
with M. tuberculosis H37Rv. The model was adopted as a
post-treatmentscreening method for antimicrobial efficacy.
The assessmentof colony forming units (cfu) 28 days post-infection
showeda dose�effect relationship, i.e. lower cfu with higherdoses of microspheres. The cfu count was significantly reduced
compared with free rifampicin. With a similar experimental approach,
the authors next evaluated the effect of repeated dosing ofthe
microspheres. At 10 days post-infection, half of the treatmentgroup
received a second dose of the microspheres. There wasa significant
reduction in cfu in lungs (but not in spleens)in the case of animals
receiving a single dose of the formulation,whereas two doses
resulted in a significant decrease in cfuin lungs as well as in
spleens.
It was realized that besidesthe methodology involved in
microparticle preparation, the surfacecharacteristics of dry powders
also play a key role in predictingparticle dispersion and pulmonary
deposition.
Although the results with rifampicin-loaded microspheres provedto
be encouraging, it was necessary to incorporate other ATDsbecause
the disease requires multidrug therapy for its cure.Hence, other
investigators encapsulated isoniazid with rifampicinin polylactide
microparticles for dry powder inhalation to rats.
Drug concentrations inside the alveolar macrophages were found
to be higher than that resulting from systemic delivery of free
drugs, an indication of the rapid phagocytic uptake and cytosolic
localization of the drug-loaded microparticles. The authorsdiscussed
that since alveolar macrophages migrate to secondarylymphoid organs,
loading these cells with microparticles mightlead to transport of
drugs to those very sites where macrophagesmigrate (mimicking the
course of spread of mycobacteria). Thatis to say, pulmonary delivery
of microparticle-encapsulatedATDs has the potential to reach
extrapulmonary sites of infectionas well. Unfortunately,
chemotherapeutic studies were not carriedout by the authors.
The rising incidence of multidrug-resistant TB (MDR-TB) is a
matter of great concern because the treatment involves the useof
second-line ATDs, which are more costly and toxic comparedwith the
first-line drugs used to treat drug-susceptible TB.Furthermore, the
treatment schedule is more prolonged with agreater risk of patient
non-compliance.
Some of the second-linedrugs, e.g. para-aminosalicylic acid
(PAS), need to be administeredin very large amounts (up to 12 g
daily), which is inconvenientto the patient. In order to reduce the
drug dosage, investigatorshave formulated an inhalable
microparticulate system for PAS,based on
dipalmitoylglycero-3-phosphocholine.
The microparticleswere produced by spray drying, possessed a 95%
drug loadingand were administered to rats via insufflation. The drug
wasmaintained at therapeutic concentrations in the lung tissuefor at least 3 h (the authors did not monitor the drug levels
further) following a single dose of just 5 mg of the dried formulation.Accelerated stability studies indicated that the formulation
was stable for up to 4 weeks and the authors suggested thatthe
technology could be extended to include other drugs suchas
rifampicin, aminoglycosides as well as fluoroquinolones.
Despite the satisfactory results obtained with microparticles,the
quest for better drug delivery systems ushered in the eraof
nanoparticles. The design and development of polymeric nanoparticles
for experimental antitubercular inhaled therapy have been therecent
focus of interest in our laboratory.
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