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Molecular Pharmaceutics 2012, 9(4), 1011−1016

Partial biodistribution and pharmacokinetics of isoniazid and rifabutin following pulmonary delivery of inhalable

microparticles to Rhesus macaques

Rahul Kumar Verma, Jatinder Kaur Mukker, Ravi Shankar Prasad Singh, Kaushlendra Kumar Priya Ranjan Prasad Verma† and Amit Misra.*

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, 226001, and Birla Institute of Technology, Mesra, Ranchi 835215 India. Telephone: 91-522-261-2411 Ext 4302; Fax: 91-522-

2623405.

[email protected] RECEIVED DATE

TITLE RUNNING HEAD: Inhalable Microparticles Biodistribution and PK.

TABLE OF CONTENTS GRAPHIC.

Abstract

Dry powder inhalations (DPI) of microparticles containing isoniazid (INH) and rifabutin (RFB) are under preclinical development for use in pulmonary tuberculosis. Microparticles containing 0.25, 2.5 or 25 mg of each drug were administered daily for 90 days to rhesus macaques (n=4/group). Single inhalations or intravenous (i.v.) doses were administered to separate groups. Drugs in serum, alveolar macrophages and organ homogenates were assayed by HPLC. RFB/INH in lungs (101.10  12.90 / 101.07  8.09 µg/g of tissue) were twice that of the liver concentrations (60.22  04.97 / 52.08  4.62 µg/g) and four times that of the kidneys (22.89  05.22 / 30.25  3.71 µg/g). Pharmacokinetic parameters indicated operation of flip-flop kinetics. Thus, elimination half-life (t½) of RFB and INH was calculated as 8.01  0.5 and 2.49  0.23 hr, respectively, upon i.v. administration, and as 13.8  0.8 and 10.43  0.77 hr following a single inhalation; or 13.36  3.51and 10.13  3.01 at presumed steady state (day 60 of dosing). Targeted and sustained drug delivery to non-human primate lungs and alveolar macrophages was demonstrated. Flip-flop serum pharmacokinetics were observed and non-linearity in some pharmacokinetic parameters at logarithmic dose increments was indicated. The results suggest that human patients would benefit through improvement in biodistribution following DPI.

KEYWORDS: biodegradable microparticles, dry powder inhalation, alveolar macrophages, tissue distribution, pharmacokinetic parameters, monkeys

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Introduction:

Pulmonary drug delivery systems have been proposed as dry powder inhalation (DPI) formulations targeting alveolar macrophages that harbor Mycobacterium tuberculosis and congeners, the causative agents of tuberculosis (TB).^1-4 Inhaled microparticles (MP) containing anti-TB agents demonstrate significantly higher efficacy than oral doses of the same drug against experimental TB in mice and guinea pigs.^5-7 It is proposed that controlled release of rifabutin (RFB) and isoniazid (INH) incorporated in the MP not only targets a bolus of the two drugs to the cytosol, but also creates pharmacokinetic synergy within the macrophage cytosol. Synergy may emerge due to rapid release of INH from phagocytosed MP, concomitantly with slow release of RFB. INH possesses the highest early bactericidal activity (EBA) among known anti-TB drugs, while RFB exercises a sustained kill effect ⁸. However, it is not as if pulmonary delivery of MP containing anti-TB agents restricts biodistribution of drugs to the alveolar macrophages alone. In mice, inhaled MP deliver long-lasting drug concentrations to lung macrophages and lung tissue, and appreciable proportions of the delivered drugs distribute to the blood and visceral organs after DPI.⁸

Experiments reported here were carried out to investigate: (a) the biodistribution of RFB and INH resulting from oro-pharyngeal DPI administration of MP to an appropriate non-human primate model;

(b); the extent of drug distribution to non-target tissues like blood, liver and kidneys; and (c) whether conclusions may be drawn in respect of a preferred dose level and dosing interval that would be necessary and sufficient to deliver and sustain high intracellular concentrations in lung macrophages, but allow minimal ‘leakage’ of the drugs to non-target sites.

The relevance of the above objectives to preclinical development of inhalable MP is as follows.

Species differences in pharmacokinetics and biodistribution of drugs following pulmonary delivery are recognized.⁹. Administration of inhalable MP to animals (including humans) through the oropharynx is likely to generate a different pattern of biodistribution and pharmacokinetics compared to analogous experiments on animals such as mice and guinea pigs that breathe exclusively through the nose.^{3, 8, 10} Rhesus macaques breathe through both nose and mouth, and are a more appropriate model of DPI delivery to human patients of TB.^{11, 12} Since TB is a chronic disease, steady-state kinetics and biodistribution are more relevant to clinical outcome. The major aim of the present study was, therefore, to evaluate drug biodistribution following repeated inhalations, in organs where efficacy and toxicity is most profoundly manifested, in a non-human primate model. Thus, the target site was the lung macrophage and, to a lesser extent, lung tissue, whereas the blood and peripheral organs were non-target sites. Drug concentrations were investigated in lung and airway macrophages, the lung lumen as represented by broncho-alveolar lavage fluid (BALF), and in lung homogenates. Strong concerns have been expressed regarding the value of estimating antibiotic concentrations in tissue homogenates,¹³. This is because differences between intra- and extra-cellular concentrations that may be significant for bactericidal activity are not reflected in homogenate concentrations. This criticism may be mitigated in situations where (a) control of rates of availability of the antibiotic to the target site is exercised by the formulation rather than biological barriers between blood and tissue compartments, (b) antibiotic therapy is prolonged (as in TB) so that drug accumulation overrides most time-lag concerns, and (c) where steady-state tissue samples at different dose levels have been examined, as in the present case.

The use of a controlled-release formulation for antibiotic delivery could impose a rate-limiting step and time delays in distribution across body compartments.⁴ Figure 1 shows a schematic diagram depicting the expected pharmacokinetic processes following inhalation of microparticles.

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Figure 1: Diagrammatic representation of putative compartments of drug distribution following DPI.

MP= microparticles, M = macrophages.

The present report demonstrates that inhalation efficiently targets microparticle-incorporated drugs to the alveolar macrophage and lung tissue, sparing the blood, liver and kidneys from exposure to the highly toxic anti-TB drugs.

Experimental Section

MP containing 1 part RFB, 1 part INH and two parts poly (D,L-lactic acid) of intrinsic viscosity 0.8 and free drugs and their analytical standards were prepared at Lupin Laboratories Research Park, Pune, India. The preparation and characterization of these MP has been reported earlier.³ Briefly, a solution comprising poly(lactic acid) and RFB in a ratio of 2:1 in dichloromethane was mixed with a solution of INH in methanol, and spray-dried using a Buchi 190 spray-dryer. The spray dried product had a mass median aerodynamic diameter of 3.57 µm; within geometric standard deviation of 1.41 µm; and was blended 80:20 with Inhalac-230®. Dichloromethane, n-pentane, acetonitrile, methanol other reagents and solvents used in the experiments were of HPLC grade and were obtained from Merck, India. Triple distilled water was used throughout the study.

The animal study procedures were approved by the Institutional Animal Ethics Committee of the Central Drug Research Institute, Lucknow, India and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Government of India). Twenty four rhesus macaques (Macaca mulatta) of either sex with average weight 5.7 kg were used from the rehabilitation colony of the Central Drug Research Institute. These animals had been used as untreated controls in unrelated studies in the past. Animals were housed, fed and handled ethically as per the guidelines of the CPCSEA.

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Dosing and sampling

Four animals each were randomly assigned to six experimental groups. Each group was treated as summarized in Table-1. Inhalation dosing was carried out by interfacing the aerosolization apparatus reported earlier¹⁴ with an infant inhalation mask as shown in Figure 2. Dose delivery from the device was calibrated as described earlier. Briefly, charges of 500 ±0.76, 60 ±0.33 and 10 ±0.36 mg of the DPI formulation were required for delivering 100 ±6.0, 10 ±2.6 and 1 ±0.39 mg MP by actuating the apparatus 30, 30 and 15 times respectively, once per second. These correspond to dose levels of 0.25, 2.5 and 25 mg of each drug per animal.

Table 1: Experiment groups and sampling

Frequency and Route

Groups Administration Dose of INH and RFB (mg)

Animal ID

Samp- les

Repeated dose Inhalation (5 days/week; 90 days)

I Untreated Controls 0 1-4

-0.25, 1, 2, 4, 8, 12, 24

II 100 mg Blank MP day^-1 0 5-8

III 1 mg drug MP day^-1 0.25 9-12

IV 10 mg drug MP day^-1 2.5 13-16

V 100 mg drug MP day^-1 25 17-20

Single dose inhalations*

VI-A 1 mg drug MP 0.25 21-24

VI-B 10 mg drug MP 2.5 21-24

VI-C 100 mg drug MP 25 21-24

Single dose intravenous*

VI-D 0.1 ml DMSO-PBS 0.25 21-24

VI-E 0.1 ml DMSO-PBS 2.5 21-24

VI-F 1 ml DMSO-PBS 25 21-24

*Washout of 7 days between doses

For i.v. dosing, RFB (5, 50, and 500 mg respectively) was dissolved in 1, 2, and 6 % dimethyl sulfoxide (DMSO). Similarly, 5, 50, and 500 mg of INH were dissolved in phosphate-buffered saline (PBS). The two solutions were mixed and volume made up to 5 ml with normal saline. The final concentration of DMSO at the highest dose level was 6 % v/v. Solutions were sterile-filtered just before injection into the cubital or great saphenous vein.

Tissue samples were collected at the time of terminal sacrifice after 90 days of dosing. Bronchioalveolar lavage was conducted before harvesting tissues, as follows. The thoracic cavity was opened and lungs intact with trachea were excised. The trachea was cannulated and lungs were lavaged 3-4 times with chilled PBS containing 0.5 mM EDTA. Bronchio-alveolar lavaged fluid (BALF) recovered in sequential lavages from individual animals was pooled to obtain a single sample per animal, centrifuged and macrophages obtained were counted and kept at -20C for further analysis. Samples of remaining lung

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tissue, liver and kidneys were blotted on sheets of blotting paper to constant weight, minced using a scalpel blade, and homogenised in known volumes of PBS using a tissue homogenizer (Ultra-Turrax, Ika, Germany). Homogenates were stored at -20°C till bioanalysis. Blood samples of about 1 ml volume were drawn with a 23 Gauge needle and syringe at time-points shown in Table 1, from either of these veins, avoiding phlebotomy at the same site for at least 4 hrs. Samples used to determine steady-state pharmacokinetics were collected on day 60 of dosing. Serum was separated from blood samples by centrifugation.

Figure 2: Hand-held apparatus for administration of inhalations to monkeys.

Sample processing

INH was extracted from cell lysates, serum and tissues using the method described by Hutchings et al.¹⁵ with some modifications. Briefly, 200 μl of NaCl (20%) was added to 500 μl of serum or 1ml cell lysate or tissue homogenate. INH was extracted using 3ml of chloroform: butanol (70:30v/v) by vortexing for 1min and centrifugation at 4000×g for 10 min and the supernatant was collected. This process was repeated three times and supernatants pooled. The drug was recovered from the organic phase with 0.5 ml phosphoric acid (30mM) by vortexing for 1min. The aqueous phase containing the drug was separated by centrifugation (4000×g, 10 min) and 200μl was decanted. The sample was neutralized with 4 μl of KOH (4 M) just prior to the injection on to the HPLC column.

RFB was extracted from 500 μl of serum or 1 ml of cell lysate or tissue homogenate using an equal volume of a solvent system: dichloromethane:n-Pentane (1:1 v/v) by vortexing.¹⁶ The organic layer was separated by centrifugation (3000×g, 10 min) and the extraction repeated three times. Butylated hydroxy toluene (BHT, 0.1%w/v in acetonitrile) was added to the samples prior to extraction to minimize drug degradation. The organic layers from three extractions were pooled in a glass test tube and vacuum- dried (Heto-Holten, Denmark). The sample was reconstituted in 500μl of methanol and filtered through a 0.22μ filter before injecting into the HPLC column.

HPLC assay

The drugs were analyzed using a Shimadzu Class VP HPLC system comprising a dual-piston reciprocating pump (LC-10Avp) with system controller (SCL-10Avp), a syringe loading injector and UV-visible dual wavelength detector (SPD-10AVvp) with a Luna C18 column (5μm,4.6×250 mm,

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Phenomenex). Isocratic elution was employed with different solvent systems for analysis of INH and RFB. INH was analyzed using a mobile phase comprising 3% acetonitrile in triethylamine acetate buffer at pH 6.0, at flow rate of 1 ml/min with the detector wavelength at 262 nm. The drug eluted at ~6 min.

RFB was eluted using a mobile phase consisting of acetonitrile and 0.05 M potassium dihydrogen phosphate (55:45 v/v), pH-4.10 at flow rate of 1ml/min with the detection wavelength at 275nm. RFB eluted at ~5.5 min. Standard curves were made by spiking known amounts of drugs to serum and tissue homogenates. The lower limits of detection and quantitation (LOD and LLOQ) of RFB were 10 and 20 ng/ml, while for INH these were 60 and 200 ng/ml.

Data analysis

Arithmetic means and standard deviations of results from 4 animals per test group were calculated using Microsoft Excel. Straight lines were fit to the Natural logarithms (ln) of serum concentrations observed upon i.v. administration to individual animals. The intercept was Natural antilog-transformed to obtain concentration at time 0 (C0), and the inverse of the slope was used as the estimate of the elimination rate constant (kel). Acceptance criteria for these parameters were adjusted regression coefficients > 0.95 and coefficients of variation between replicates lower than 25%. Doses were normalized to animal body weight (mg/kg). Non-compartment analysis (NCA) was carried out on all datasets using Win Nonlin ver 5.3 (Pharsight, USA). Pharmacokinetic parameters were calculated using software-generated bounds, estimated by curve stripping. Results are reported only in the case of the 25- mg dose level wherein curve fitting converged for all animals in the group with the above acceptance criteria, and parameters matched with those calculated manually using the open, one-compartment model following i.v. administration as above.

Results and Discussion

Biodistribution

Drugs were extracted from alveolar macrophages and BALF which indicates drug deposition in the lung lumen at the time of terminal sacrifice (day 90). Necropsies of lungs, liver and kidneys were harvested and analysed for drug content. The DPI resulted in substantial deposition in the airways and lungs, as well as uptake of drugs into alveolar macrophages (Figure 3). Drug concentrations decreased in the order: lungs>liver>kidneys. Very low concentrations of both drugs were observed in tissues from animals administered 1 mg of MP (0.25 mg of each drug) /day, often below quantitation limits. Dose- dependent tissue concentrations were observed at the two higher doses. Levels of RFB/INH in the lungs were much higher (101.10  12.90 / 101.07  8.09 µg/g of tissue at 25 mg, and 14.32  3.53 / 13.28  2.37 µg/g at 2.5 mg doses, respectively) in comparison to accumulation in other organs such as liver (60.22  04.97 / 52.08  4.62 µg/g of tissue at 25 mg and 3.67  0.95 / 3.38  0.72 µg/g at 2.5 mg) and kidneys (22.89  05.22 / 30.25  3.71 µg/g of tissue at 25 mg and 0.56  0.41 / 0.15  0.13 µg/g at 2.5 mg doses). Drug levels in the BALF, representing an approximation of drug amounts theoretically available for absorption were 25.7  1.8 / 21.7  1.8; 6.2 ± 1.4 / 8.5  1.6 and 2.2 ± 0.1 / 3.2 ± 0.5 µg/ml at the three doses of RFB / INH, in descending order.

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Figure 3: Biodistribution of INH and RFB in lung and airway macrophages (cells), bronchioalveolar lavage fluid (BALF) and tissue homogenates of lung, liver and kidneys of animals receiving inhalable microparticles on day 90 of repeated administration. Columns represent group means, and error bars the S.D. (n=4).

Serum Pharmacokinetics of Single i.v. Doses

The serum concentration-time profiles of the two drugs in monkeys receiving 0.25, 2.5 or 25 mg of each drug by a single i.v. injection are depicted in Figure 4A and 4B. Administration of 0.25 mg of the two drugs, especially INH, resulted in very low serum concentrations after 4 hr. Pharmacokinetic parameters of the two drugs following i.v. injection at the 25 mg dose level are listed in Table 2. Serum t½ of RFB may be calculated as 8.01  0.5 hr and that of INH as 2.49  0.23 hr. The acylator status of the monkeys, although potentially capable of significant impact on pharmacokinetics, was not documented. The area under the concentration-time curve (AUC) of RFB was about two times larger than that of INH, but the index AUC above minimum inhibitory concentration (MIC) was comparable for both drugs.

Single inhalation

Non-compartment analysis of serum concentration-time data yielded secondary pharmacokinetic parameters in respect of the 25 mg dose level are listed in Table 2. Data resulting from lower doses was not adequate for accurate estimation of pharmacokinetic parameters.

Plots representing data obtained after a single inhalation of 0.25, 2.5 and 25 mg each of RFB and INH as MP are shown in Figure 4C and 4D. Scatter points represent values observed in individual animals, while solid lines indicate a point-to-point fit to group means of the data at each dose level.

Serum levels obtained after pulmonary delivery indicated that MP sustained drug release after delivery to the lungs, the oropharynx and possibly, the gastro-intestinal tract if a part of the dose was swallowed (Figure 1); imposing a flip-flop on the serum pharmacokinetics. Thus, t½ Vz, AUC and AUC/MIC increased in comparison to i.v. administration. These changes were more prominent in the case of INH.

Repeated inhalation: steady-state

Pharmacokinetic parameters were derived by NCA of the concentration-time profiles following 60 days of repeated inhalation dosing (Table 2). The time after previous dose () was 24 h. The values of t½ were

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similar to those observed after a single inhalation. Figure 4E and 4F show the serum concentration-time data. The results indicate extension of tmax and enhancement of AUC at steady state, and a corresponding reduction in Cl. These observations suggest that 25 and 2.5 mg doses resulted in drug accumulation during the 24-hr dosing interval.

Figure 4: Mean serum concentration versus time profiles of RFB (A, C, E) and INH (B, D) at dose levels of 0.25 (green lines), 2.5 (red lines) and 25 mg (blue lines) of each drug, in monkeys administered a single i.v. injection (A, B), a single inhalation of MP containing (C, D) and 60 consecutive doses by inhalation (E, F). Lines represent a point-to-point fit. Scatter points corresponding to data from individual animals at each dose-level are plotted. Points corresponding to line colour represent group means (n=4) and error bars show standard deviation (SD).

Discussion

Concentrations of RFB and INH in lung macrophages, lumen and tissue following DPI compared favourably with levels observed in the liver and kidneys with regard to demonstration of targeting

A B

C D

E F

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(Figure 3). Although intracellular concentrations in lung macrophages were below limits of quantitation at the lowest dose level (0.25 mg), drug levels in the lung lumen (BALF) and lung tissue homogenates suggest that proportionate levels might prevail within lung macrophages of animals receiving the lowest dose. Therefore, it was concluded that dose levels between 0.25 and 2.5 mg would be appropriate for humans. Further, drug concentrations in the liver homogenates were undetectable at the lowest dose, and were 3.7 to 60.2 µg/g of tissue at higher doses (Figure 3). Similarly, concentrations in the kidney homogenates ranged between 0.15 and 23 µg/g of tissue. INH hepatotoxicity, as indicated by induction of apoptotic cell death of cultured hepatoma lines, is observed only above INH concentrations greater than 26 µM (3.6 µg/ml).¹⁷ Thus, at dose levels between 0.25 and 2.5 mg, the contribution of drugs present in inhaled MP to adverse effects arising out of anti-TB chemotherapy is likely to be minimal.

Table 2. Mean (n=4) pharmacokinetic parameters of RFB and INH at 25 mg per animal ± standard deviations.

Dr ug

C0/C

max

SD t½(

z)

S

D Vz SD Clobs SD AUC

∞

SD AUC/M

IC^a SD

(µg/ml) hr (ml/kg) (ml/hr/kg) (hr. µg/ml) (hr) Intra-

venous RF

B 4.90 0.0 7

8.0 1

0.5 0

1173.

1

86.

5

101.

68 8.02 56.62 3.7

9 566.21 37.8 6 IN

H 6.88 0.1 6

2.4 9

0.2

3 837.0 80.

6

235.

15

38.9

1 24.75 2.8

5 494.98 56.9 7 Single

Inhalat ion

RF

B 3.16 0.2 5

13.

80 0.8 3

1648.

8

232 .4

107.

9

16.2

5 70.94 13.

18 709.45

131.

75 IN

H 3.12 0.1 5

10.

44 0.7 7

1593.

0

205 .8

143.

3

20.7

6 54.78 8.2

5 1095.68 165.

09 Steady

State

RF

B 7.07 0.5 4

13.

36 3.5 1

1703.

8

213 .4

57.2 7

18.7

6 100.41 9.7

9 1004.10 97.8 7 IN

H 5.49 0.9 8

10.

13 3.0 1

1587.

8

205 .9

108.

58

12.5

1 122.35 10.

42 2446.96 208.

47

Our previous reports demonstrated targeted and controlled drug delivery to the alveolar macrophage upon administration to rodents^{2, 3, 18} and suggested that the pattern of release of the two agents created a pharmacokinetic synergy.2, 3, 8, 18, 19 Results reported here indicate that even at steady state, INH from inhaled particles reaches systemic circulation faster (observed tmax= 4 hr) than RFB (observed tmax= 8 hr) and is eliminated at comparable rates (t½ = 10 ± 3. and 13.4 ± 3.5 hr respectively). This observation suggests that the flip-flop imposed by sustained release serves to maintain drug levels of RFB for longer periods than would be expected from the t½ observed following i.v. administration (Table 2). Thus, RFB may be expected to exercise a more sustained bactericidal effect following inhalation of microparticles while INH exercises its early bactericidal activity.

Since the focus of the present work was to target inhaled particles to lung macrophages, systemic drug delivery following pulmonary administration is, from this perspective, an incidental outcome.

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Nonetheless, documentation of these observations serves the purpose of highlighting the possibility of significant systemic bioavailability following DPI of MP containing RFB and INH. These observations further indicate the desirability of keeping the inhaled dose low (0.25-2.5 mg) if the objective of pulmonary delivery of particles is to merely provide an adjunct that targets alveolar macrophages.

In spite of large difference in pharmacokinetic parameters between mice⁸ and monkeys, pharmacokinetic curves in serum were similar when compared directly with either a single i.v. bolus or a single inhalation, and relative proportions of the drug in alveolar macrophages and serum were similar in the two species.

The minimum inhibitory concentration (MIC) of INH is 0.05 μg/ml²⁰ and that of RFB is 0.1 μg/ml, which represents the median MIC against 30 strains of M. tuberculosis.²¹ Using these values, the pharmacokinetic / pharmacodynamic (PK/PD) index represented by the area under the curve above the MIC (AUC/MIC) may be calculated, as shown in the Table 2. The index for RFB was comparable between single i.v. and inhalation doses, but large increments were observed at steady state in the serum, a peripheral, non-target site with respect to the formulation under test. The PK/PD index of INH nearly doubled on administration of inhalations. It appears worthwhile to investigate whether a reduction in the concomitant oral dose could be proposed when the DPI is tested for efficacy as adjunct therapy.

In reporting biodistribution, cytosolic concentrations are plotted here in terms of μg/ml of cell lysate (Fig 3). We submit that amounts per cell or million cells might be more meaningful units to assess cytosolic drug concentrations, but difficult to compare with data on lung homogenates. Converting the μg/ml values plotted in Fig 3 to μg per million cells, we obtain values of 0.69 ± 0.13 (INH) and 0.83 ± 0.14 (RFB) at the 25 mg dose level and 0.15 ± 0.04 and 0.15 ± 0.05 respectively at the 2.5 mg level.

Thus, it is concluded that intracellular concentrations, relevant to bactericidal activity against intracellular bacilli are overestimated if lung homogenates alone are assayed. Finally, biodistribution data also confirms that a dose interval of 24 h is sufficient to deplete drugs from lung macrophages as well as other tissue, but trace amounts remain in the lung lumen at even the lowest dose.

The predictive power of the present studies in conjunction with the mouse studies reported earlier for suggesting important human pharmacokinetic parameters can only be established after proposed experiments on humans conclude.

Acknowledgments: Mr. Dinesh Kumar, Mr. Vijay Kumar and Mr. Mohammad Saleem rendered invaluable help in animal handling. Funded by CSIR NMITLI Grant 5/258/4/2002 and NWP0035, this is CDRI Communication Number 7687.

Supporting Information: This information is available free of charge via the Internet at http://pubs.acs.org/.

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