Pharmacokinetics, metabolism, and partial biodistribution of "pincer therapeutic" nitazoxanide in mice following pulmonary delivery of inhalable particles

Article abstract of DOI:10.1021/acs.molpharmaceut.6b01089

(Graph Presented) Nitazoxanide (NTZ) induces autophagy in mammalian cells and also has mycobactericidal activity, displaying a two-pronged therapeutic effect, on the host as well as the pathogen. The pharmacokinetics and biodistribution of inhaled NTZ wer

Full text of DOI:10.1021/acs.molpharmaceut.6b01089

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Article
Pharmacokinetics, Metabolism and Partial Biodistribution
of ‘Pincer Therapeutic’ Nitazoxanide in Mice
Following Pulmonary Delivery of Inhalable Particl
Anuradha Gupta, Sachin L. Tulsankar, Rabi S Bhatta, and Amit Misra
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01089 • Publication Date (Web): 06 Mar 2017
Downloaded from http://pubs.acs.org on March 7, 2017
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Pharmacokinetics, Metabolism and Partial Biodistribution of ‘Pincer Therapeutic’
Nitazoxanide in Mice Following Pulmonary Delivery of Inhalable Particles
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Anuradha Gupta, Sachin L Tulsankar, Rabi S Bhatta, Amit Misra*
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CSIRꢀCentral Drug Research Institute, Sector 10A, Janakipuram Extension, Mohiuddinpur,
Lucknow 226031, India
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*Correspondence: Telephone: +(91) ꢀ(522)ꢀ277ꢀ2460; Fax: +(91) ꢀ(522)ꢀ277ꢀ1941
Eꢀmail: amit_misra@cdri.res.in
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Short Title: Nitazoxanide Inhalation
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Abstract
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Purpose: Nitazoxanide (NTZ) induces autophagy in mammalian cells and also has
mycobactericidal activity, displaying a twoꢀpronged therapeutic effectꢀꢀ on the host as well as the
pathogen. The pharmacokinetics and biodistribution of inhaled NTZ were investigated. Methods:
Particles containing NTZ in a matrix of PLGA were prepared by spray drying. HPLC and LCꢀ
MS/MS methods were developed and validated. Particles were administered as inhalations to
mice. Drug concentrations in plasma and tissues were estimated at different time points. Results:
Drug loading (~36%), entrapment efficiency (>90%) and the conversion of NTZ into metabolites
in plasma and lung homogenates were assessed satisfactorily by HPLC. NTZ pharmacokinetics
and biodistribution following intravenous administration or inhalation were established by LCꢀ
MS. NTZ converted in to tizoxanide (99% in 30 min) and other metabolites. Pulmonary delivery
of NTZ entrapped in particles increased the halfꢀlife of the drug by factors of 3, 12 and 200 in the
plasma, lung tissue and alveolar macrophages respectively. Conclusions: Targeted delivery and
prolonged lung retention along with dose sparing of the kidneys was observed upon pulmonary
delivery as compared to intravenous administration.
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Keywords: Dry powder inhalation; Tizoxanide; HPLC; LCꢀMS; oneꢀcompartment.
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Abbreviations: DPI, Dry Powder Inhalation; ACN, Acetonitrile; TBAHS, Tetra Butyl
Ammonium Hydrogen Sulfate; LCMS, Liquid Chromatography Mass Spectrometry; NMR,
Nuclear Magnetic Resonance; NTZ, Nitazoxanide; TZX, Tizoxanide; TG, Tizoxanide
Glucuronide; SA, Salicylic acid; BA, Benzoic acid.
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1. Introduction
The lengthy and arduous multipleꢀdrug regimen for treatment of tuberculosis (TB) is primarily
focused towards the objective of killing the pathogen. Hostꢀdirected therapies are increasingly
being proposed with a view to eliciting host defense responses for the same purpose.1
Microautophagy or autophagy is a host response that can potentially result in killing of
intracellular Mycobacterium tuberculosis (Mtb),2 but equally importantly, has demonstrated preꢀ
clinical efficacy in healing the diseased lung.3, 4
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Targeted drug delivery to deep lungs by means of a Dry Powder Inhalation (DPI) is an efficient
way of maintaining high intracellular drug concentration reducing systemic drug exposure to
hostꢀdirected or ‘pincer’ therapeutic agents.3 The use of biodegradable polymers to prepare
inhalable particles also helps in controlling the drug release profile and generating desired
pharmacokinetics.5
NTZ is a nitrothiazolyl salicylamide having close resemblance to metronidazole. It is a light
yellow crystalline powder, practically insoluble in water, slightly soluble in acetone, ethyl
acetate, ethanol, and freely soluble in Nꢀmethyl 2ꢀpyrrolidone and dimethyl formamide. It is a
broad spectrum antiparasitic and antiprotozoal agent.6, 7 NTZ is a nonꢀcompetitive inhibitor of
pyruvate ferredoxin oxidoreductase (PFOR), an enzyme required for anaerobic energy
metabolism in prokaryotes8 and inhibits the mTORC1 complex in the host cell to induce
autophagy.9 It is effective against clinical isolates of Mtb, however, primarily by changing
bacterial membrane potential and disrupting pH homeostasis.10 In view of the dual action of
NTZ, i.e., on both the pathogen and the host, we term this agent as a “pincer therapeutic.”3 NTZ
is a prodrug, rapidly hydrolysed nonꢀenzymatically or by plasma esterases with a t of ~6 min11
½
to tizoxanide (TZX) or desacetyl nitazoxanide [(2ꢀhydroxyꢀNꢀ(5ꢀnitroꢀ2ꢀthiazolyl) benzamide)].
TZX is further glucuronidated to form tizoxanide glucuronide (TG). Therefore, the US FDA
recommends the quantification of both NTZ and TZX for pharmacokinetic and bioequivalence
studies. NTZ is absorbed from the gastrointestinal tract. One third of the oral dose is excreted in
urine and two thirds in faeces. TZX is found in urine, bile and faeces.11, 12 The halfꢀlife of TZX
in plasma is approximately 1.5 hrs and it is >99% bound to plasma proteins.13, 14
Various pharmacokinetic and biodistribution studies, stability studies and forced degradation
studies (photodegradation, hydrolysis, oxidation, acid and alkali induced degradation) on NTZ
have been reported,15 using spectrophotometric,16 RPꢀHPLC,17ꢀ19 and LCꢀMS methods. There is
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no report on pharmacokinetics and biodistribution analysis of NTZ in mice. Since the mouse is
an animal model of choice in translation of drug discovery and delivery system leads in the area
of TB, the present report describes analytical methods and results for pharmacokinetic and
biopharmaceutic analysis of NTZ and its metabolites.
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Material and methods
Materials
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Nitazoxanide (NTZ, CASꢀ55981ꢀ09ꢀ4, purity 98%) was purchased from Hangzhou Terphenyl
LCD Co., Ltd, Hangzhou, China. The primary metabolite/degradation product of NTZ, i.e.,
tizoxanide (TZX) was synthesized by refluxing NTZ with 1M HCL. TZX glucuronide (TG)
sodium salt was purchased from (CAS 221287ꢀ83ꢀ8) Santa Cruz Biotechnology, USA. Salicylic
acid (SA) and Benzoic acid (BA) was purchased from Merck, Mumbai India. HPLC grade
acetonitrile (ACN), analytical grade tetrabutylammonium hydrogen sulfate (TBAHS) and
ammonium acetate were purchased from Spectrochem Pvt. Ltd., Mumbai, India. All other
reagents and chemicals were obtained from Merck, Mumbai, India. LCꢀMS grade acetonitrile
and methanol were purchased from Merck Millipore. Milli Q water was obtained from a Milli Q
water system from Merck Millipore.
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Methods
Synthesis and structure confirmation of TZX
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TZX was prepared by acidic hydrolysis of NTZ. NTZ (250mg) was taken in a 250 ml round
bottom flask and refluxed with 100 ml of 1M HCl at 100°C for 24 hr. The product was filtered
and reꢀcrystallized three times with chloroform. The structure of TZX was confirmed (Table S1)
by UVꢀvisible spectroscopy (UVꢀ1700 Pharma Spec, Shimadzu), Mass spectroscopy [(JMSꢀ
T100LC, Accu TOF (DARTMS)] and 1H and 13C NMR spectroscopy (Bruker DRXꢀ300; and
JEOL AL 300 FTꢀNMR).
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HPLC method development and validation
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The HPLC system (Shimadzu Corporation, Kyoto, Japan) consisted of dual piston reciprocating
pumps (LCꢀ10 AT Vp and LCꢀ20 AT) with a system controller (SCLꢀ10A Vp) and PDA detector
(SPDꢀM10A Vp). Data collection and analysis were performed using Class VP software. A
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LiChrospher 100 C18 column (RPꢀ18e, 5µm, 250mm×4mm, Merck) and ACN: 0.005M TBAHS
in triple distilled water (TDW) in the ratio of 50:50 was used as a mobile phase at a flow rate of
1ml/min to achieve separation at room temperature. The mobile phase was filtered through a
0.22µm membrane filter. Detection was carried out using a photodiode array (PDA) detector at
240 nm.
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Standard solutions of NTZ and TZX were prepared by dissolving accurately weighed 10 mg of
NTZ or TZX in 100 ml of ACN. The RPꢀHPLC method was validated as per International
Conference on Harmonization (ICH) guidelines20 in terms of linearity, accuracy, intraꢀday and
interꢀday precision and robustness of method. Limit of detection (LOD) and Limit of quantitation
(LOQ) for both NTZ and TZX was calculated from the values of standard deviation of response
(SD) and slopes (S) of the calibration curves.
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Estimation of NTZ and TZX in bulk and formulation
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Particles of volumeꢀmean diameter 3.8±0.3µm, aerodynamic diameter 2.2±0.2µm and NTZ
content of 36.3±1.4% w/w in a matrix of poly(lacticꢀcoꢀglycolic acid) (PLGA) were prepared by
spray drying.3 Briefly, both the drug and polymer were dissolved in acetone and spray dried
using inlet temperature 65ºC, feed rate 2ml/min, air flow rate 108 Nl/min. Drug loading and
entrapment efficiency was calculated by estimating drug by HPLC after dissolving the particles
in dichloromethane and extracting the drug using methanol as reported earlier.3
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NTZ degradation kinetics in plasma and lung homogenate ex vivo
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Rat blood plasma and lung homogenate were used to study the metabolic profile of NTZ. NTZ
converts to TZX nonꢀenzymatically. Thus, the source of the biomatrix is unlikely to contribute to
the rate of NTZ hydrolysis. However, NTZ is also metabolized by blood plasma esterases. To
our knowledge, there are no significant differences between activities of esterases present in rat
and mouse blood plasma, although both species differ significantly from humans.21 Since normal
rat plasma is easily obtained in abundance without killing the animal, we chose to employ this
resource as a surrogate.Blood (5 ml) was withdrawn from the tail vein and collected in
Vacutainer® tubes coated with heparin. The blood sample was centrifuged at 1000 rpm for 15
min at 4°C to obtain plasma. Aliquots (100 µl) of plasma and lung homogenate were spiked with
4µg/ml NTZ for different time periodsꢀ 5min, 15min, 30min, 1hr, 1.5hr, 2hr, 3hr, 6hr, 24hr, 48hr
and 72 hr. After incubation, 900µl of ACN was added to precipitate proteins and extract the drug
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and metabolites. The sample was vortexꢀmixed for 10min and centrifuged at 10,000rpm for
15min at 4°C. The supernatant was separated in a clean glass tube and vacuum dried. The sample
was reconstituted in 1 ml of ACN and injected for chromatographic analysis using HPLC. Blank
plasma and lung homogenate was also processed as above and the ACN extract injected to check
interference. The peak area was measured for calculation of NTZ and TZX concentrations at
different time points, with respect to the calibration curve. Degradation kinetics was fitted to
determine the decay rate constant.
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LC-MS method development and identification of metabolites
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The LCꢀMS system consisted of a Waters Milford USA (quaternary pump ꢀ600) equipped with
Waters 717 auto sampler and Waters in line AF degasser. The analytes were
chromatographically separated on a Waters SymmetryꢀShield C18 (5ꢁm, 4.6 × 150 mm) column,
using acetonitrile: 10mM ammonium acetate (pH 3), (80:20, v/v) as the mobile phase. All
separations were performed in binary mode at a flow rate of 0.7 ml/min and the injection volume
was kept at 20 ꢁl. Rinsing mode was set to before and after injection aspiration to ensure no
carry over effect using acetonitrile: water (50:50) as a rinsing solution.
Bioanalysis of analytes was performed using API 3200 mass spectrometer (Applied Biosystems,
Canada) coupled with an electro spray ionization (ESI) source using negative multiple reaction
monitoring (MRM) mode. Topiramate was used as internal standard (IS). The optimized mass
parameters for identification of major and minor metabolites is given in Table 1. Spectra of blank
ACN as well as blank plasma and tissue homogenate were recorded to identify any interferences
in detection due to biological sample.
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Table 1: LC-MS parameters for identification and quantitation of NTZ and its metabolites
Mass parameters
Source Parameters
LCꢀMS/MS System
Ion source
Values
API 3200
ESI
Ion spray voltage
Polarity (mode)
GSꢀ1
ꢀ4500
Negative
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GSꢀ2
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Curtain gas
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CAD gas
Temp.
3
250˚C
No
Splitter (Yes/No)
Probe
Xꢀaxis
Yꢀaxis
0.5 cm
0.5 cm
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Compound Parameters
Analyte
MRM(Q1/Q3)* DP* CE*
CXP* EP* Molecular
Dwell
(msec)
200
Weight
ꢀ10 339.36
ꢀ10 307.28
ꢀ10 265.24
ꢀ10 463.02
Topiramate
338.16/77.96
ꢀ90
ꢀ21
ꢀ30
ꢀ29
ꢀ70
ꢀ18
ꢀ21
ꢀ24
ꢀ1
ꢀ1
ꢀ1
ꢀ1
Nitazoxanide 306.1/186.1
200
Tizoxanide
Tizoxanide
glucuronide
263.8/217
439.9/264
200
200
Salicylic acid 136.9/93.10
ꢀ32
ꢀ29
ꢀ25
ꢀ18
ꢀ1
ꢀ1
ꢀ10 138.12
ꢀ10 122.12
200
200
Benzoic acid 121.1/76.9
Abbreviation: MRM (Q1/Q3) (Multiple Reaction Monitoring (Precursor ion/ Product ion); DP (Declustering potential); CE
(Collision Energy); CXP (Collision cell exit potential); EP (Entrance potential).
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Pharmacokinetics and biodistribution in mice
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NTZ pharmacokinetics and biodistribution were investigated with the approval of the IAEC
(approval number IAEC/2014/149/DATED 03.12.2014). Healthy male Swiss mice aged 8ꢀ10
weeks and weighing 22ꢀ25g were used. Animals were bred and housed ethically in the National
Laboratory Animal Facility located in our Institution in conformity with the US NIH’s Guide for
the Care and Use of Laboratory Animals.
NTZ was dissolved in DMSO and diluted with PBS. NTZ solution was filter sterilized and 806
µg/kg was administered intravenously to mice through the tail vein. For delivering inhalations,
an inꢀhouse nose only apparatus was used.22 This apparatus is constructed of a 15 ml centrifuge
tube with a hole just large enough to accommodate the nares of the test animal, and another hole
in the apex that allows flexible tubing to be admitted into the interior of the tube. When a rubber
bulb at the other end of the tubing is actuated, turbulent air enters the tube and aerosolizes
powder placed in the cap of the tube. Powder weighing approximately 12.5 mg was placed in the
apparatus. Mice were manually restrained with nostrils inserted in the delivery port. The powder
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was fluidized with turbulent air admitted by squeezing a rubber bulb once per second for 30
seconds. Under these conditions, the inhaled dose of NTZ was 18.42 ± 1.36 µg / mouse or
750µg/Kg.3
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Following intravenous and inhalation administration, sets of four animals were anaesthetized and
blood samples were withdrawn from hepatic portal vein for complete exsanguination at 4 min, 8
min, 12 min, 16 min, 30 min, 1h, 3h, 6h, 12h, 24h, and 48h. Bronchioꢀalveolar lavage (BAL)
was conducted to recover lung macrophages and viscera were collected and weighed fresh. NTZ,
TZX, TG, BA and SA concentrations was determined by LCꢀMS following extraction of drug
from plasma, BAL cell lysate and organ homogenate samples with acetonitrile. A oneꢀ
compartment PK model was fitted to data on concentrations in tissue homogenates and blood
plasma, using PK Solver as an addꢀin to Microsoft Excel® and PK parameters was calculated.
Results and Discussion
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HPLC method development and validation
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The HPLC method was suitable for detection and quantitation of NTZ and TZX in formulations
and ex vivo samples. HPLC conditions were optimized to obtain good peak shapes, baseline
resolution and purity indices of ≥0.9999. The method was linear in the concentration range of 1ꢀ
20µg/ml with a correlation coefficient of 0.99. The values of LOD and LOQ were 5.49 ng/mL
and 16.62 ng/mL for NTZ, and 5.43ng/ml and 16.45 ng/ml for TZX. Accuracy of analyte
recovery was ~ 99% (Table S2). To determine the intraꢀday and interꢀday precision of the
method, % CV was calculated and was within acceptable range as shown in Table S3. The
robustness of the method was checked by deliberately changing the mobile phase composition
and flow rates. Changes in retention time, peak shape, tailing and effect on separation were
observed and are shown in Table S4. Using the analytical HPLC method, it was established that
spray drying yielded particles with a practical drug content of 36.36 ± 1.39 % and the process
had an entrapment efficiency of 90.9 ± 3.48 %.
NTZ degradation kinetics in plasma and lung homogenate ex vivo
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Almost all NTZ (99%) spiked into plasma samples was observed to be metabolized to TZX
within 30 minutes. TZX itself is not a stable metabolite, as indicated by timeꢀdependent
reduction in TZX peak area in plasma and lung homogenate samples. NTZ converted in to TZX
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very rapidly and TZX decayed exponentially with the values of exponential decay rate constant
of 24.79 hꢀ1 and 82.42 hꢀ1 in plasma and lung homogenate respectively (Figure 1).
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B
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-1
-2
0
-1
-2
0 12 24 36 48 60 72
Time (hrs)
0 12 24 36 48 60 72
Time (hrs)
C
D
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0 12 24 36 48 60 72
Time (hrs)
0 12 24 36 48 60 72
Time (hrs)
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Figure 1: Degradation of NTZ (filled symbols) in blood plasma (A) and lung homogenate (C)
was rapid while TZX (open circles) decayed exponentially at 37°C. Mean ± SD of three replicate
experiments is shown. The exponential decay rate constant of TZX in calculated by fitting the
natural log of TZX concentrations to time was 24.79 hꢀ1(R2= 0.993) in plasma (B), while in lung
homogenate (D), the decay constant was obtained from fitting natural log of TZX concentrations
to time (y = A1e (ꢀk/t) + y0) was 82.42 hꢀ1 (R2= 0.987).
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LC-MS method development and identification of metabolites
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The sensitivity of the HPLC method was not sufficient for biopharmaceutics, so an LCꢀMS
method was developed for identification and quantitation of NTZ, TZX and minor metabolites.
The retention times of topiramate (IS), NTZ, TZX, Tizoxanide glucuronide (TG), Salicylic acid
(SA), and Benzoic acid (BA) in LCꢀMS were 2.51, 2.78, 2.77, 2.09, 2.27 and 2.51 min
respectively. The respective chromatograms are shown in Figure 2. The metabolites of NTZ are
summarized in Figure 3. Calibration curves were plotted over 0.97ꢀ1000ng/ml concentration
ranges for each analyte in every organ used for determining drug concentration. The value of the
correlation coefficient was 0.99 in each case. The value of LOQ and presence of metabolites is
shown in Table 2. For determining pharmacokinetics and biodistribution, the sum total of NTZ
and TZX was considered.
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210
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Figure 2: LCꢀMS chromatograms of (A) Topiramate, (B) NTZ, (C) TZX, (D) TZXꢀglucuronide,
(E) Salicylic acid, and (F) Benzoic acid.
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Figure 3: Structure and metabolic conversion of NTZ into its metabolites
Table 2: Parent compound and metabolites that were either “found” or “not found” in
plasma, cells or tissue homogenates at 72 hours after IV or inhalation dosing, and their
limits of quantitation
Intravenous
Organs/
LOQ (ng/ml)
Plasma
Lung
BAL cells
Liver
Spleen
Kidney
Heart
Inhalation
Organs/
LOQ (ng/ml)
Plasma
NTZ
TZX
TG
SA
BA
Not found
Not found
Not found
Not found
Not found
Not found
Not found
Found (0.97) Found (0.97) Found (7.85) Not found
Found (0.97) Found (0.97) Not found
Not found
Not found
Found (0.97) Not found
Found (0.97) Not found
Not found
Found (7.85) Not found
Found (31.2)
Found (0.97) Found (0.97) Found (15.6) Found (31.2)
Found (0.97) Found (0.97) Not found
Found (0.97) Found (3.9)
TZX TG
Not found
Not found
NTZ
SA
BA
Not found
Found (0.97) Found (0.97) Found (7.85) Not found
Lung
BAL cells
Liver
Found (0.97) Found (0.97) Found (0.97) Found (15.6) Not found
Found (0.97) Found (0.97) Found (0.97) Found (7.85) Found (3.9)
Not found
Not found
Found (0.97) Found (1.95) Found (7.85) Not found
Found (0.97) Found (0.97) Found (15.6) Found (31.2)
Spleen
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Kidney
Heart
Not found
Not found
Found (0.97) Found (0.97) Found (15.6) Found (31.2)
Found (0.97) Found (3.9)
Found (7.85) Found (15.6)
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Pharmacokinetic parameters were calculated by applying non compartmental, one compartment
and two compartment models. Based on the value of R2 and AIC, a one compartment model was
best fitted to data and PK parameters were calculated (Table 3). The concentrationꢀtime graphs
highlight the differences in concentrations over time as shown in Figure 4. NTZ was retained in
the lungs and alveolar macrophages following inhalation and intracellular drug concentration
were maintained for up to 48 h. This observation suggested that efficacy studies should examine
an alternateꢀday dosing regimen.23 NTZ was not detected in plasma after i.v. dosing in mice
despite the reported t of 6 min in humans following perꢀoral administration.11 TZX was
½
detected up to 6 h. TZX glucuronide and minor metabolites benzoic acid and salicylic acid were
detected at later time points but their amount was not taken into consideration for calculation due
to variability. NTZ and TZX were both found in BAL cells, plasma and lungs after inhalation
dosing. TZX was detected for up to 48 h demonstrating that NTZ particles were able to maintain
drug concentrations for a prolonged period of time. PK parameters indicated that the t of NTZ
½
increased from 0.68 h to 2.28 h in plasma, 0.19 h to 11.6 h in lungs and 0.10 h to 21.94 h in
alveolar macrophages. Clearance was reduced from 7.81 to 0.04 (ꢁg/kg) /(ng/ml)/h following
intravenous and inhalation dosing.
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During peerꢀreview, it was pointed out that relatively higher concentrations were observed in the
heart, liver and spleen following pulmonary delivery as compared to IV injection. A possible
explanation for these observations may be that particles deposited in the lungs traffic to these
organs. Since the particles are fairly large (volumeꢀmean diameter 3.8±0.3µm), it is extremely
unlikely that they would enter blood circulation by crossing epithelial barriers. It is far more
likely that macrophages on the lung and airway surface pick up deposited particles and move to
the bloodstream and then to visceral organs. This speculation is supported by the observations
that macrophageꢀrich organs (liver, spleen) also show high drug concentrations, whereas the
kidneys (which macrophages infiltrate only in disease or upon injury) show low concentrations.
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A
B
3600
3200
2800
2400
2000
1600
1200
800
320
280
240
200
160
120
80
400
40
0
0
10
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0.1
1
1
1
1
10
10
10
10
100
0.1
1
10
10
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10
100
100
100
100
14000 D
2400
2000
1600
1200
800
400
0
C
12000
10000
8000
6000
4000
2000
0
0.1
100
0.1
1
F
E
100
80
60
40
20
0
2000
1600
1200
800
400
0
0.1
100
100
0.1
1
640 G
H
1800
1600
1400
1200
1000
800
600
400
200
0
560
480
400
320
240
160
80
0
0.1
0.1
1
Time (h)
245
246
247
248
249
250
251
Figure 4: Pharmacokinetics and partial bio distribution of NTZ + TZX. Drug concentrations time graph
resulting from IV injection (blue empty circles) or inhaled (red empty circles) in mice (N=3 per time
point). (A) Plasma, (B) heart, (C) lungs, (D) cell lysate, (E) BAL fluid, (F) liver, (G) spleen (H) kidney.
Data are shown as means ± standard deviation. Respective predicted concentrations obtained after one
compartment modelling are shown by scatter points. Data from three animals per timeꢀpoint ± SD is
plotted.
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Table 3: Pharmacokinetic parameters calculated using a one-compartment model to data from three animals per time-point
Intravenous
6
7
8
9
Parameter/
Organs
C0
ng/ml
ka
1/h
k10
1/h
t1/2ka
h
t1/2
h
V
CL
(µg/kg)
AUC 0-t
ng/ml*h
AUMC
ng/ml*h^2
MRT
h
(µg/kg)
/(ng/ml)
/(ng/ml)/h
0.28±0.05
24.51±7.18
7.81±3.31
17.59±3.15
1.72±1.17
6.10±2.44
2.92±0.29
51.03±7.95
Plasma
Cell lysate
Lungs
Heart
Liver
Spleen
Kidney
BAL Fluid
3206.79±407.67
235.15±58.32
874.26±650.09
188.43±6.62
3401.19±2097.8
527.55±421.55
2080.22±1368.5
26.13±7.28
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1.14±0.4
6.81±0.4
8.81±6.3
4.11±0.7
9.26±7.7
5.18±4.3
7.82±5.3
1.61±0.3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.68±0.22 0.26±0.03
0.10±0.01 3.60±1.04
0.19±0.23 1.63±1.55
0.17±0.03 4.28±0.15
0.54±0.85 0.39±0.37
0.14±0.12 1.41±0.32
0.18±0.20 0.71±0.73
0.44±0.07 32.57±9.4
3007.3±620.1
34.6±9.0
114.9±42.5
46.8±8.7
952.9±1066.8
55.5±54.1
277.6±28.1
16.03±2.2
3139.9±1666.4
5.1±1.4
38.6±53.8
11.9±4.2
1617.45±2758.7
31.3±26.9
76.9±91.6
10.0±1.4
0.98±0.32
0.15±0.01
0.27±0.33
0.25±0.04
0.78±1.23
0.20±0.17
0.26±0.29
0.63±0.10
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Inhalation
Parameter/
Organs
A
ng/ml
ka
1/h
k10
1/h
t1/2ka
h
t1/2k10
h
V/F
(µg/kg)
/(ng/ml)
CL/F
(µg/kg)
/(ng/ml)/h
0.45±0.33
AUC 0-t
ng/ml*h
AUMC
ng/ml*h^2
MRT
h
Plasma
Cell lysate
Lungs
814.54±138.20
7484.62±921.47
1377.95±442.56
209.55±38.88
1443.34±143.15
483.39±36.31
1304.39±130.90
78.39±27.67
182.22±10.33
164.99±116.22
113.27±181.34
273.32±17.07
233.59±70.80
271.53±24.34
1739.7±2079.8
168.78±100.4
0.48±0.4
0.04±0.0
0.07±0.0
0.84±1.3
0.15±0.1
0.48±0.4
4.46±0.8
0.83±1.3
0.00±0 2.28±1.73 1.01±0.19
.0
0.01±0 21.94±10. 0.11±0.01
.0
0.06±0 11.16±3.5 0.64±0.25
.1
0.00±0 5.27±4.41 3.94±0.70
.0
0.00±0 5.84±3.37 0.56±0.05
.0
0.00±0 2.23±1.76 1.68±0.13
.0
0.00±0 0.16±0.03 0.63±0.07
.0
0.01±0 5.13±5.11 11.14±3.1
.0
2454.6±147.9
4
172595.6±566 8346942.8±5904 31.66±14.6
98.6 206.9
19455.9±3378 331950.4±10351 16.18±5.1
10483.81±11698 3.29±2.49
.25
0.00±0.00
0.04±0.01
2.78±4.13
0.08±0.04
0.78±0.55
2.76±0.24
6.67±9.15
17
9
.9
0.1
Heart
1435.0±1220.
7
11584.5±5709 118849.2±11900 8.44±4.86
16342.53±16120 7.61±6.37
.5
Liver
.8
3.7
Spleen
Kidney
BAL fluid
1491.9±1067.
8
293.4±25.3
6601.7±8339.1
3.22±2.53
0.23±0.04
7.40±7.37
68.5±18.5
401.0±344.8
5600.9±7398.2
Where C0, A: Drug concentration at time zero, ka: First order absorption rate constant, k10: Elimination rate constant (firstꢀorder) from the central (1)
compartment,
t
1/2ka: Absorption halfꢀlife, t1/2: Halfꢀlife of drug in plasma, t1/2k10: Elimination halfꢀlife, V: Volume of distribution, CL: Total body clearance, AUC0-t: Area
under the curve from time zero to t, AUMC: Total area under the first moment curve, MRT: Mean residence time, F: Fraction of administered dose which
reaches the systemic circulation unchanged.
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The high payload DPI formulation of NTZ, intended to target alveolar macrophages following
aerosol delivery to the lungs, maintained a high intracellular drug concentration for a longer
duration than intravenous injection. The HPLC method developed and validated was sufficient
for the purpose of determining drug content in the formulation, to estimate drug loading and
entrapment efficiency. For determining drug and metabolite concentrations in animal tissue, a
more sensitive LCꢀMS method was required. Using this method, it was established that NTZ is
converted into TZX which then gives rise to TG, SA and BA as terminal metabolites. NTZ
particles served the purpose of deep lung targeting and increasing the halfꢀlife of drug as evident
from secondary PK parameters.
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Acknowledgements
The authors acknowledge CSIRꢀCDRI Sophisticated Analytical Instrument Facility (SAIF) for
mass and NMR spectroscopy. AG received a Senior Research Fellowship from CSIR. Study
funded by DBT grant PR10468/MED/29/815/2013 and CSIR grants BSC0112 and ESC0103.
This is CDRI Communication No. 159/2014/AM.
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