e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

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In vivo lung deposition and sub-acute inhalationtoxicity studies of nano-sized alendronate sodiumas an antidote for inhaled toxic substances inSprague Dawley rats

Shaheen Sultanaa,1, Rashid Alib,2, Sushama Talegaonkara,3,Farhan Jalees Ahmada,4, Gaurav Mittalb,5, Aseem Bhatnagarb,∗

a Jamia Hamdard, Faculty of Pharmacy, Department of Pharmaceutics, Delhi 110062, Indiab Institute of Nuclear Medicine and Allied Sciences, Department of Nuclear Medicine, DRDO, Brig. S K MazumdarMarg, Delhi 110054, India

a r t i c l e i n f o

Article history:

Received 19 March 2013

Received in revised form

17 May 2013

Accepted 31 May 2013

Available online 7 June 2013

Keywords:

Alendronate sodium

Technetium-99m

Biodistribution

Gamma scintigraphy

Sub-acute inhalation toxicity

Histopathology

a b s t r a c t

Introduction: Alendronate sodium is a bisphosphonate agent used for the treatment of osteo-

porosis and other bone diseases. It has a strong chelating property to bind or, to some

extent, counteract the effects of substances, such as magnesium, calcium citrate, ferrous

fumarate, carbonyl iron, as well as the zinc gluconate, sulfate and acetate salts. The objec-

tive of the present study was to evaluate lung deposition and sub-acute inhalation toxicity

of the alendronate sodium respiratory formulation.

Methods: Particle dimension of aerosols of alendronate was measured using a particle size

analyzer. Alendronate was radiolabeled using Technetium-99m for in vitro and in vivo

biodistribution studies. Alendronate at doses, 0.5%, 1.0%, and 1.5% in ethanol-saline respira-

tory formulation was inhaled twice a day up to 5 weeks for inhalation toxicity investigations.

Hematological, biochemical and lung toxicity biomarkers in bronchoalveolar lavage (BAL)

fluid were determined at the end of the experiment. Histopathological analysis of lung

tissues was carried out to observe any microscopic changes

Results: Particle size analysis revealed the size within 300–500 nm. Anderson cascade

impactor results showed that the particles exhibited higher respirable fraction (55.52%)

with MMAD of 4.66 �m. Hematology, serum biochemistry and lung toxicity biomarkers

in BAL fluid performed in the sub-acute toxicity studies indicated no adverse effects of

alendronate sodium inhalation except for a significant increase in cholesterol levels and

marginal increase in BAL fluid protein. At autopsy, no histopathological changes in major

organs were observed.

∗ Corresponding author. Tel.: +91 981144102.E-mail addresses: shaheen634@yahoo.co.uk (S. Sultana), rashidinmas@gmail.com (R. Ali), stalegaonkar@gmail.com

(S. Talegaonkar), farhanja 2000@yahoo.com (F.J. Ahmad), gauravmittal23@gmail.com (G. Mittal), dr.aseembhatnagar@gmail.com,assem bhatnagar@indiatimes.com (A. Bhatnagar).

1 Tel.: +91 9456433520.2 Tel.: +91 9910016875.3 Tel.: +91 9818453518; fax: +91 11 26059663.4 Tel.: +91 9810720387; fax: +91 11 26059663.5 Tel.: +91 9891560750.

1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.etap.2013.05.016

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647 637

Conclusions: The lung deposition and safety evaluation data observed from these studies

suggested that aerosolized nanosized alendronate sodium by the inhalation route could

be a new and promising route of administration as an antidote to radioactive substances

through an increase in the bioavailability of the drug as well as a decrease in side effects on

systemic delivery.

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. Introduction

adiation poisoning, also called radiation sickness, is a formf damage to organ or tissue due to excessive exposure to ion-

zing radiation. The term is generally used to refer to acuteroblems caused by a large dosage of radiation in a shorteriod, though this also has occurred with long term expo-ure or chronic effects. Radiation exposure can increase therobability of attracting some other diseases, mainly can-er and genetic damage (www.cancer.org). During exposure,adioactive material is inhaled either as aerosol or colloidalarticles and absorbed therein. Some of the material maye swallowed as a food contaminant or by ciliary movementhrough the respiratory tract where they can stay indefi-itely (Marcus, 2006). Conventional treatment of the radiationoxicity includes decorporation of radionuclide by certainrugs which binds the toxic materials in body and make itnavailable for absorption. The treatments available are notery effective and rapid and require decorporation therapy toeduce the incidence of cancer and early death due to systemicissue damage. It is necessary to develop a novel decorpo-ation and radio-protective formulation which may be moreffective than the existing ones and must be cost-effectiveo ensure availability at mass scale. As significant amountsf radioactivity directly reaches the lungs, a delivery system

s required that traps or neutralizes toxic inhaled substancest an entry point only (lungs directly). In principle, inhala-ion delivery of therapeutics offers an attractive, non-invasivelternative to injections and other modes of administrationChoi et al., 2001). Moreover, lungs have a large surface area, areelatively tolerant of foreign substances and much more per-

eable than gastrointestinal and nasal mucosa or skin, whichontributes to rapid bioavailability of the radio protective for-ulation (Choi et al., 2001).Alendronate sodium is a bisphosphonate class drug used

or the treatment of osteoporosis and other bone diseasesPerez-Lopez, 2004; Russell, 2007). The pharmacologicalction of sodium alendronate is based on its affinity for theydroxyapatite crystals of bone and selective inhibition ofsteoclastic bone resorption during the remodeling cycle (Lin,996; Russell et al., 1999). It is reported as a strong chelatinggent and may be added to bind or, to some extent, counterhe effects of substances, such as magnesium, calciumitrate, ferrous fumarate, zinc gluconate, zinc sulfate, zinccetate and carbonyl iron (www.healthcare.com). Infact,ts chelation property for uranium has been well estab-ished in vivo (Houpert et al., 2004). Oral administration of

odium alendronate has been reported but it is associatedith gastrointestinal intolerance and oral bioavailability of

odium alendronate is very low, around 1.7% (Lin et al., 1992;raham and Malaty, 1999). Studies on alternative routes of

© 2013 Elsevier B.V. All rights reserved.

administration i.e., intravenous, intramuscular, subcutaneousand nasal have been conducted to overcome the adverse gas-trointestinal effects and the poor bioavailability of sodiumalendronate. However, it presents the risk of nephrotoxicity,local tissue damage and irritation at the site of injection(Monkkonen et al., 1990; Lin et al., 1992; Karie et al., 2006).

The pulmonary route of administration has gained atten-tion for targeted drug delivery during the treatment of localand systemic diseases for to lung due to its large absorp-tive surface area, thin pulmonary epithelium and rich bloodsupply (Tsapis et al., 2003; von Wichert and Seifart, 2005;Patton and Byron, 2007). The performance of an inhal-able system mainly depends on the physical characteristicsof particles such as mass median aerodynamic diameter(MMAD) which in turn depends upon shape, size and den-sity of aerosolized particles (Khilnani and Banga, 2008). LowerMMAD (1–5 �m) facilitates retention in lower respiratoryregion (Suarez and Hickey, 2000; Telko and Hickey, 2005).Hence, better targeting and deposition of antidotes in alve-olar spaces necessitate delivery of nanosized particles. Suchparticles will result in fast action and deep penetrate into thelungs. There are two principal means for pulmonary deliverywhich include the use of liquid nebulizers and dry powderinhalers (DPI). Nebulizers are gaining attention because ofpotential advantages such as stability, small droplets size, easyto formulate, low cost and better targeting efficiency (Hess,2008).

The present work is focused on the development of aninhalable respiratory formulation of alendronate sodium forthe treatment of uranium/radioactive toxicity, and investiga-tion of in vitro and in vivo deposition into lung via inhalation.The work also covers wide scope as the same formulationif sufficiently targeted to the lungs is expected to enhancethe oral bioavailability of bisphosphonates and in vivo safetyto assess any toxicity during sub-acute inhalation expo-sure.

2. Materials and methods

2.1. Chemicals and reagents

Sodium alendronate was received as a gift sample fromDr. Reddy’s laboratory (Hyderabad, India). Thiobarbituric acid(TBA), trichloroacetic acid (TCA), hydrogen peroxide (H2O2)and bovine serum albumin (BSA) was procured from SigmaChemical Company, St. Louis, MO, USA. Ethanol (absolute

ethanol, 99.9% pure) was purchased from Changshu YangyuanChemical, China. All other routine chemicals used in thisinvestigation were of research grade and were purchased fromMerck India Ltd., Mumbai.

d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

Fig. 1 – Schematic representation of radiolabeling

638 e n v i r o n m e n t a l t o x i c o l o g y a n

2.2. Experimental animals

Specific pathogen free male Sprague Dawley rats weighing200–220 g obtained from the Central Animal House Facility ofthe institute were used for the study. All animal experimentswere approved by the Institutional Animal Ethical Commit-tee duly constituted for the purpose and confirmed to generalnational guidelines on the care and use of laboratory animals.The animals were housed in polypropylene cages and werekept in a room maintained at 25 ± 2 ◦C with a 12 h light/darkcycle. They were allowed to acclimatize for one week beforethe experiments and were given free access to standard labo-ratory animal feed (Golden Feed Laboratory, Delhi, India) andwater ad libitum.

3. Formulation preparation andcharacterization

Respiratory fluid contained alendronate sodium in 30%ethanol and 70% saline solution. Ethanol was added to pro-duce sub micronic aerosol particles and to increase rate of drugoutput. The particle size distribution of generated aerosolswas studied by analyzer after nebulization of the solutionby Ultra compressor (Alpha Neb Plus, Germany) attached tospacer. Aerosols were generated by the nebulizers by using drycompressed air at 40 lb/in2 with a flow rate of 4 or 6 l/min. Par-ticle size was measured after 2 min of equilibrium. The datawere interpreted for 30 s to achieve stable values. All sampleswere analyzed in triplicate.

3.1. Aerosol generation and delivery

A whole body exposure chamber for aerosol inhalation wasused in conjunction with a large spacer (‘Anukool’) indige-nously developed by DRDO, India. The spacer attached tocommercially available nebulization assembly (Acorn II; Mar-quest Medical, Englewood, CO) produced aerosols of sodiumalendronate nebulization formulation. The aerosol particlesize was measured with the help of particle size analyzer(Model 310A, LASAIR II, USA).

3.2. Radiolabeling protocol

A 50 �l stannous chloride solution (1 mg/ml) was added to5 mg of alendronate. Finally, an appropriate amount of 99mTc-Pertechnetate was added and incubated for 20 min (Fig. 1).Percentage radiolabeling efficiency was determined by instantthin layer chromatography (ITLC) using acetone as a mobilephase.

3.3. Estimation of respiratory fraction

Respiratory fraction (Rf) of nebulized formulation of alen-dronate was determined using Anderson Cascade Impactor(ACI; Copley Scientific, Nottingham, United Kingdom). Three

milliliter of optimized formulation containing 1 mg alen-dronate was nebulized by jet nebulizer through spacerconnected to mouth piece (initiation port) of Andersoncascade impactor (ACI). The wash solutions from various

procedure for alendronate sodium.

stages such as initiation port, pre separator deposit, andimpactor stages were collected and quantified for drug con-tent by high-performance liquid chromatography (Shimazdu)(Sultana et al., 2010). The amount deposited at the variousstages was expressed as percentage alendronate per actua-tion. Respiratory fraction (Rf) was calculated as the ratio ofpercentage of total drug deposited in the lower stages of theACI (stage 2–8) to total theoretical dose. Rf describe the per-centage of aerosolized deposited deeply to the lungs. Theexperiments were repeated in triplicate.

3.4. In vivo biodistribution studies

All animal experiments conducted were approved by theSocial Justice and Empowerment Committee for the pur-pose of control and supervision on experiments on animals.Male Sprague Dawley rats, weighing 200–220 g were selectedfor the study. A 3 ml solution containing 1 mg alendronateand 1–2 mCi was nebulized to rats. Inhalation chamber foradministration of nebulized alendronate in animals for biodis-tribution studies are shown in Fig. 2. The rats were sacrificed atdifferent time intervals (0.5 h, 2 h, 4 h and 24 h) and blood wasobtained by cardiac puncture. Subsequently, tissues (heart,lung, liver, spleen, kidney, stomach, intestine) were dissected,washed with normal saline, made free from adhering tissues,and weighed, and then their radioactivity was measured ina shielded well-type gamma scintillation counter (Capintec,USA).

3.5. Subacute inhalation toxicity studies

Twenty-four male Sprague Dawley rats were randomly dividedinto four groups of six rats each. The groups were designatedas group I, II, III and IV. The animals of group I served as controland received no treatment. Group II, III, and IV animal receivedaerosols of sodium alendronate respiratory formulation inconcentration of 0.5, 1.0 and 1.5% respectively twice a day for20 min up to 5 weeks through whole body inhalation assemblyfollowing Schedule-‘Y’, guidelines of Indian Council of Med-ical Research (ICMR), Delhi, India. All animals were observed

twice daily for any morbidity and mortality (Ji et al., 2007). Oninitiation of dose administration, clinical examinations wereperformed daily prior to and approximately 1 h followingdose administration and detailed physical examinations were

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647 639

Fig. 2 – Schematic representation of; (a) nebulizer assembly and (b) distribution of radioactive count in different regions ofn

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erformed weekly. All the animals were sacrificed and dis-ected after 24 h of last exposure. Tissue samples of differentital organs (lungs, liver, kidney, stomach, heart and spleen)ere collected and preserved in 10% buffered formalin solu-

ion for histopathology analysis to observe any microscopichanges.

. Observations

.1. Gross pathology

orphological parameters of toxicity, i.e., mucous membranenasal secretion), eye irritation, tear secretion, excessive blin-ing, salivation, cyanosis, lethargy, pyloerection (ruffled fur),aralysis, skin irritation, edema, erythema, respiratory rate,leeping habits as well directed and non-directed movementsithin the cage were observed throughout the experiment.

.2. Body weight, food and water intake

hanges in body weight gain, food and water intake wereecorded and local injuries were studied during treatmentf animals. The changes in body weight of the animal wereecorded weekly.

.3. Hematological analysis

he animals were fasted overnight prior to necropsy and bloodollection. Blood collection was done by cardiac puncture, athe time of necropsy into EDTA containing vials for immedi-te analysis of hematological parameters. The values of redlood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb),ematocrit (HCT), mean corpuscular volume (MCV), mean cellemoglobin (MCH) and platelets counts were determined bytandard clinical procedures using an automatic hematolog-cal analyzer (Beckman-Coulter LH-500, Diagnostic Systems)nd compared with controls.

.4. Serum biochemistry analysis

lood samples for biochemical investigations were collected

n plain tubes and centrifuged at 4000 rpm at 4 ◦C for 10 min tobtain the serum. Analysis of biochemical parameters; aspar-ate aminotransferase (AST), alanine aminotransferase (ALT)lkaline phosphatase (ALP), cholesterol, triglycerides and

glucose was determined with a reagent kit of Randox labora-tory Ltd., UK using an automated analyzer (Hitachi 912 Model).

4.5. Analysis of lung toxicity biomarkers inbronchoalveolar lavage (BAL) fluid

Bronchoalveolar lavage (BAL) fluid was collected as per previ-ously reported method (Reynolds, 1987; Qamar and Sultana,2010; Ali et al., 2012). Briefly, rats were anesthetized andthen euthanized by exsanguinations. The trachea and lungswere exposed by thoracotomy after which a cannula wasinserted into the trachea and ligated using a thread. Twenty-five ml/kg body weight of warm phosphate-buffered saline(0.15 M NaCl–50 mM phosphate, pH 7.4; 37 ◦C) was instilled intolungs via a syringe fitted with tracheal cannula. Phosphate-buffered saline was allowed to stay in lungs for 30 s thenretrieved and re-instilled with the help of syringe. The pro-cess was repeated three times with the same solution. Volumeof recovered BAL fluid from each rat was 22.47 ± 0.38 ml/kgbody weight. BAL fluid was centrifuged (300 g, 10 min) to col-lect a cellular supernatant which was stored at −20 ◦C. Thesupernatant was used for the estimation of total proteinconcentration and other enzymatic parameters. All determi-nations were made in triplicate, and the average value wascalculated.

4.6. Total cell counts

The cell pellet obtained after centrifugation of BAL fluid waswashed three times with phosphate buffer saline immedi-ately after the BAL fluid collection. The cell pellet was resuspended and diluted to the appropriate amount using phos-phate buffer saline solution. Total cell counts were measuredusing an aliquot of BAL fluid by the method as reported ear-lier (Sole et al., 1996) using hematological automatic analyzer(Beckman-Coulter LH-500, Diagnostic Systems).

4.7. Total protein

Total protein in BAL fluid was analyzed by an earlier reportedmethod (Bradford, 1976) to evaluate lung vascular permeabil-

ity (Kasahara et al., 2008). Sample protein concentrations weredetermined from a standard curve using bovine serum albu-min (BSA) standards. The assay was based on the absorbanceshift from 465 to 595 nm that occurs when Coomassie

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640 e n v i r o n m e n t a l t o x i c o l o g y a n

Blue G-250 binds to proteins in an acidic solution. In brief,10 �l of samples was taken in which 90 �l distilled water wasadded. One milliliter of Bradford reagent was mixed to eachtube and vortexes gently for thorough mixing and incubatedat room temperature for 5 min. Samples were then read fortheir absorbance at 595 nm using micro plate reader (PerkinElmer, Pyris 6DSC).

4.8. Alkaline phosphatase

Alkaline phosphatase (ALP) is a lysosomal enzyme indica-tive of tissue damage. Activity of ALP was determined bythe method (Henderson et al., 1995), where ALP catalyzesthe reduction of p-nitrophenol phosphate to p-nitrophenoland phosphoric acid in the presence of magnesium ions andadenosine monophosphate. The rate of change in absorbanceat 400 nm is directly proportional to ALP activity in the sample.

4.9. Lactate dehydrogenase

Lactate dehydrogenase (LDH) is a soluble, cytosolic proteinpresent in blood. The concentration of LDH readily increasesin lung tissues after exposure of inhaled lung toxicants. Anincrease in the cytosolic enzyme LDH in BAL fluid is indicativeof cell damage and lysis. It is a very sensitive indicator for anylung injury. LDH catalyzes the oxidation of lactate to pyruvatewith simultaneous reduction of nicotinamide adenine dinu-cleotide (NAD). LDH activity was estimated by a previouslydescribed method (Kornberg, 1955). In brief, the reaction mix-ture consisted of 0.2 ml of sample, 0.1 ml of NADH (0.02 M),0.1 ml of sodium pyruvate (0.01 M), 1.1 ml of sodium phosphatebuffer (0.1 M, pH 7.4) and distilled water in a total volume of3 ml. Formation of reduced NAD (NADH) results in an increasein absorbance at 340 nm and enzyme activity was calculatedas nmol NADH oxidized/min/mg of protein of BAL fluid.

4.10. Superoxide dismutase

Superoxide dismutase (SOD) activity was evaluated toestimate endogenous defences against superoxide anions.Activity of the enzyme was measured spectrophotometricallyby monitoring the inhibition of pyrogallol auto-oxidation. Thesample (100 �l) was added to Tris–HCl buffer (pH 8.5) to makethe final volume 3 ml. Pyrogallol (25 �l) was added and changesin absorbance at 420 nm were recorded at 1 min interval for3 min. The increase in absorbance at 420 nm after addition ofpyrogallol was inhibited by the presence of SOD (Marklund andMarklund, 1974). The enzyme activity was calculated by usinga molar extinction coefficient of 4.02 × 103 M−1 cm−1.

4.11. Estimation of malondialdehyde

The assay for membrane lipid peroxidation was carried outusing a method (Wright et al., 1981) with slight modifications.The reaction mixture in a total volume of 3.0 ml contained1.0 ml BALF, 1.0 ml of TCA (10%) and 1.0 ml TBA (0.67%). All

the test tubes were placed in boiling water bath for a period of45 min. The tubes were shifted to ice bath and then centrifugedat 2500 rpm for 10 min. The amount of malondialdehyde (MDA)formed in each of the samples was assessed by measuring

a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

optical density of the supernatant at 532 nm. The results wereexpressed as the �mol MDA/ml BALF by using a molar extinc-tion coefficient of 1.56 × 105 M−1 cm−1.

4.12. Catalase

Catalase is an antioxidant enzyme like superoxide dismu-tase and glutathione peroxidase, which is produced naturallywithin the body. It is one of the most efficient anti-oxidantenzymes found in nearly all living organisms exposed to oxy-gen and catalyzes the decomposition of hydrogen peroxideto water and oxygen, using either an iron or manganesecofactor. Level of CAT can also be used as a biomarker to assessthe toxicity of inhaled toxicants pre and post-treatment. CATactivity was estimated following a previously reported method(Claiborne, 1985). Briefly 50 �l sample was added to cuvettecontaining 2.95 ml of 19 mM/l solution of H2O2 prepared inpotassium phosphate buffer (0.1 M, pH 7.4) in a total vol-ume of 3 ml. The change in absorbance was monitored at240 nm at 1 min interval for 3 min. Presence of catalase decom-poses H2O2 leading to a decrease in absorbance. Change inabsorbance was calculated as nmol H2O2 consumed per minper mg of protein.

4.13. Macroscopic examination and organ weights

A complete necropsy was conducted on all animals. Necrop-sies included, but were not limited to, examination of theexternal surface, all orifices, and the cranial, thoracic, abdom-inal, and pelvic cavities, including viscera. During necropsy,organ weights of vital organs (lungs, liver, heart, spleen andkidneys) were recorded and appropriate organ weight ratioswere calculated in relation to total body weight.

4.14. Histopathological analysis

The lungs tissues were excised for histopathological analysis.Tissues were processed by standard histopathological tech-niques. Histopathological changes in lungs and respiratoryroute were specifically observed. Tissues were fixed in 10%formalin and embedded in paraffin. 5 �m size sections werecut from the stomach and lung tissue of each group. Thesections were deparaffinized using xylene and ethanol. Theslides were washed with phosphate buffer saline (PBS) andpermeabilized with permeabilization solution (0.1 M citrate,0.1% Triton X-100). The deparaffinized sections were stainedwith hematoxylin and eosin. Tissue histology was evaluatedand microscopic changes were analyzed. Tissue sections wereobserved under a light microscope (Olympus BX 60), at a mag-nification of 10× and 40× and compared with those of controlanimals.

5. Statistical analysis

Numerical data are expressed as mean ± SD. Body weight,

change in body weight, food consumption, clinical pathologyand organ weight data were analyzed using Student t-testto determine inter group differences. Differences betweengroups were analyzed using analysis of variance (ANOVA)

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647 641

Fig. 3 – ACI study comparison for % deposition in each stagef

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Fig. 4 – Biodistribution studies of oral and inhaledalendronate sodium formulation in rats.

or nebulized alendronate using nebulizer at 60 l/min (n = 3).

ollowed by Dunnett’s multiple comparisons test. Minimumriterion for statistical significance was set at P < 0.05 for allomparisons. Clinical pathology values for white blood cellypes occurred at a low incidence (i.e., monocytes, eosinophils,nd basophils) were not subjected to statistical analysis.

. Results

.1. Formulation preparation and characterization

he size of aerosols for Sodium alendronate nebuliza-ion formulation delivered to experimental animals throughebulizer-spacer assembly was found to be in the range of00–500 nm.

.2. Respiratory fraction

istribution pattern of radiolabeled nebulization formulationf alendronate particles in various stages of cascade impactory gamma scintigraphy is shown in Fig. 3. More than 49.5%f particles were retained in nebulizer at the end of nebu-

ization process, while between 4 and 7% were found to beetained in the mouthpiece and spacer, respectively. The emit-ed dose was 50.45% where a sufficient amount reached to theower stages (stages 2–8, where stage 8 indicated filter) whenompared with Ip and Ps region (denote mouth, throat andropharynx) where 10% retention was observed. The MMADnd GSD calculated for the designed particles were 4.66 �m

nd 3.07 ± 0.1, respectively (Table 1). % Rf of nebulized drugas significantly high (55.52%) that ensured better aerosoliza-

ion and lung deposition behavior.

Table 1 – Andersen cascade impactor (ACI) results for thenebulized alendronate particles measured using an airflow rate of 60 l/min.

Particle size attributes ACI (inhalation data)

Emitted dose (%) 50.48 ± 0.5Respiratory fractiona (%) 55.52Mass median aerodynamic

diameter (�m)4.66

GSD 3.07 ± 0.1

a Respiratory fraction calculated as ratio of total drug deposited inthe lower stages of the ACI (stages 2–8) to total theoretical dose.

6.3. In vivo biodistribution studies

The respiratory formulation of alendronate in nebulizer wasinhaled by the rats until completely dried. For comparisonan oral solution was administered through cannula in rats.Biodistribution of inhaled alendronate particles and oral solu-tion in rats are demonstrated in Tables 2 and 3. A comparativesignificant biodistribution increase in lung deposition of neb-ulized alendronate was observed when compared with oralsolution (Fig. 4). A decrease in activity from 4.387% to 3.655%was observed after 4 h. Studies revealed that 1.87% activitywas still in the lungs of rats after a period of 24 h. The biodis-tribution of nebulized particles in other organs was observedto be negligible beside stomach which showed 3.18% in 30 min.The stomach showed a lower deposition of nebulized particleswhen compared with conventional oral drug delivery (wheremostly radiolabeled drug reside in stomach). Even the plasmastudies exhibited ∼9 times increase in absorption when theparticles delivered through pulmonary route when comparedwith oral route. Biodistribution studies revealed that consid-erable amount was reaching to the lungs and, therefore, themodel could be used for toxicity studies.

7. Sub-acute inhalation toxicity studies

7.1. Gross pathology

No mortality and morbidity or any sign of behavioral changesor toxicity were observed during the experimental procedureand period of the study in all the animal groups. The ani-mals were at normal nutritional status and healthy and nodifferences were noted with respect to control. Morphologicalcharacteristics (fur, skin, eyes, and nose) appeared normal. Notremors, convulsion, salivation, diarrhea, lethargy or unusualbehaviors such as self mutilation, walking backward and soforth were observed; gait and posture, reactivity to handlingor sensory stimuli, grip strength were all normal.

There were also no significant changes in pre- and post-treated weight of test groups II, III and IV as compared tocontrol (group I) (Fig. 5). In group I, rats gained weight through-

out the duration of treatment whereas weight loss in thelast week was observed in group IV animals receiving 1.5%alendronate respiratory solution. While animals of groups II

642 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

Table 2 – Biodistribution studies of oral alendronate sodium at various time intervals.a

Organs Percent deposition (%/g)

0.5 h 2 h 4 h 24 h

Lungs 0.006 ± 0.01 0.006 ± 0.02 0.005 ± 0.02 0.001 ± 0.03Liver 0.012 ± 0.02 0.017 ± 0.05 0.009 ± 0.05 0.004 ± 0.10Spleen 0.010 ± 0.04 0.005 ± 0.04 0.005 ± 0.06 0.003 ± 0.05Kidney 0.020 ± 0.10 0.035 ± 0.22 0.061 ± 0.01 0.021 ± 0.11Stomach 10.29 ± 0.45 9.883 ± 0.55 6.764 ± 0.12 1.45 ± 0.23Intestine 0.647 ± 0.21 0.531 ± 0.24 1.620 ± 0.25 0.65 ± 0.10Blood 0.03 ± 0.06 0.032 ± 0.02 0.019 ± 0.01 0.016 ± 0.03Heart 0.005 ± 0.14 0.004 ± 0.09 0.009 ± 0.05 0.001 ± 0.04

a Data are expressed as mean ± SD (n = 3).

Table 3 – Biodistribution studies of nebulized alendronate sodium at various time intervals.a

Organs % Deposition (%/g)

0.5 h 2 h 4 h 24 h

Lungs 4.385 ± 0.13 3.745 ± 0.39 3.655 ± 0.34 1.87 ± 0.15Liver 0.087 ± 0.006 0.099 ± 0.02 0.0825 ± 0.02 0.1226 ± 0.05Spleen 0.15 ± 0.04 0.076 ± 0.001 0.099 ± 0.03 0.0705 ± 0.02Kidney 0.26 ± 0.15 0.181 ± 0.08 0.1675 ± 0.01 0.182 ± 0.05Stomach 3.18 ± 2.20 2.62 ± 0.58 0.7085 ± 1.0 0.1075 ± 0.02Intestine 0.473 ± 0.075 0.553 ± 0.13 0.624 ± 0.19 0.126 ± 0.11Blood 0.267 ± 0.16 0.113 ± 0.01 0.101 ± 0.01 0.087 ± 0.03Heart 0.101 ± 0.15 0.099 ± 0.06 0.085 ± 0.01 0.076 ± 0.03

a Data are expressed as mean ± SD (n = 3).

and III animals gained weight in a similar fashion as of thegroup I.

7.2. Hematological analysis

There were no treatment-related changes in hematologicalparameters between control and treated groups, indicatingthat the nebulized alendronate sodium particles, in spite ofreaching into the blood in substantial amount were not toxic

to circulating red cells, nor interfered with their productionand that of platelets. The values of RBCs, WBCs, Hb, HCT, MCV,MCH and platelets were found to be within normal range in

Fig. 5 – Body weight gain pre and post-inhalation ofalendronate sodium respiratory formulation.

treated animal groups and there were no significant changes(P > 0.05) as compared to the control animals (Table 4).

7.3. Serum biochemistry analysis

The results of biochemical assessment of different treatedgroups of alendronate sodium are shown in (Table 5). Therewere no significant alterations in the levels of AST, ALTand ALP, which were the good indicator of liver functions,suggesting that sub-chronic administration of nanosized par-ticles unaltered hepatocytes and the normal metabolismof the animals. The parameters like glucose, protein andtriglyceride were within the normal values. The results werenon-significant when compared with control groups (P > 0.05).However, cholesterol level was significantly reduced (P < 0.001)in rats receiving 1.5% alendronate respiratory formulationindicating beneficial role of drug as antihyperlipidemic agenton lipid metabolism (Guney et al., 2008). Comparable ureavalue of all treated animals demonstrated normal functioningof the kidney.

7.4. Macroscopic examination and organ weights

No adverse aerosol inhalation-related macroscopic findings

were noted at the scheduled necropsies. The weights of vitalorgans (lung, liver, kidney, heart and spleen) were not sig-nificantly different in experimental animals as compared tocontrol (Table 6).

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647 643

Table 4 – Effect of inhalation of alendronate sodium respiratory formulation on hematological parameters.

Hematological parameters Groups

Group I Group II Group III Group IV

RBC (106/�l) 9.46 ± 0.46 9.24 ± 0.43 8.87 ± 0.46 9.11 ± 0.52WBC (106/�l) 9.97 ± 1.40 10.79 ± 1.33 10.36 ± 1.48 10.02 ± 1.62Hb (g/dl) 16.33 ± 1.53 16.73 ± 1.17 16.47 ± 1.66 16.53 ± 1.71HCT (%) 45.48 ± 2.86 43.47 ± 2.73 43.79 ± 3.28 44.66 ± 3.47MCV (fl) 56.99 ± 2.49 57.85 ± 2.37 57.42 ± 2.46 57.81 ± 2.78MCH (pg) 20.92 ± 1.76 20.29 ± 1.66 19.71 ± 1.86 20.36 ± 1.75MCHC (%) 36.72 ± 5.85 35.56 ± 6.41 34.20 ± 6.44 35.16 ± 6.97Platelets (103/�l) 359.67 ± 25.30 375.25 ± 28.05 389.67 ± 30.46 376.33 ± 32.57

Group I: control; group II: 0.5% alendronate sodium; group III: 1% alendronate sodium; group IV: 1.5% alendronate sodium respiratory formulationinhalation. RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean cell hemoglobin;MCHC, mean cell hemoglobin concentration. Data are represented as mean ± SD (n = 6). The differences among the treatment groups vs. controlare not statistically significant (P > 0.05).

Table 5 – Effect of inhalation of alendronate sodium respiratory formulation on serum biochemical parameters.

Serum biochemistry parameters Groups

Group I Group II Group III Group IV

AST (U/l) 62.47 ± 4.68 63.18 ± 5.26 64.40 ± 5.88 65.88 ± 5.77ALT (U/l) 137.37 ± 12.79 138.4 ± 12.50 140.87 ± 13.94 142.25 ± 14.57ALP (U/l) 259 ± 5.43 254 ± 2.56 251 ± 3.34 246 ± 1.78Urea (mg/dl) 16.54 ± 1.52 17.63 ± 1.68 19.12 ± 1.84 19.832 ± 1.86Uric acid (mg/dl) 2.43 ± 0.25 2.46 ± 0.24 2.48 ± 0.26 2.52 ± 0.28Creatinine (mg/dl) 0.50 ± 0.056 0.56 ± 0.055 0.55 ± 0.058 0.57 ± 0.060Glucose (mg/dl) 83.24 ± 8.12 81.11 ± 7.62 85.18 ± 8.24 85.21 ± 8.34Cholesterol (mg/dl) 73.14 ± 4.90 70.22 ± 5.53 69.82 ± 4.68 74.32 ± 5.79Triglyceride (mg/dl) 66.27 ± 5.65 64.20 ± 5.88 68.39 ± 6.17 70.50 ± 6.54

Group I: control; group II: 0.5% alendronate sodium; group III: 1% alendronate sodium; group IV: 1.5% alendronate sodium respiratory formulationinhalation. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase. Data are expressed as mean ± SD (n = 6).

stical

7

Toogir(is

The differences among the treatment groups vs. control are not stati

.5. Analysis of lung toxicity biomarkers in BAL fluid

otal cell counts, total protein, lactate dehydrogenase, super-xide dismutase, malonaldehyde and alkaline phosphatasebserved in BAL fluid of experimental animals of all the testroups showed no significant change with respect to controlndicating no adverse effect of inhalation of alendronate respi-atory formulation confirming its safety and no lung injury

Table 7). However, total protein concentration was marginallyncreased in group II and group IV treated with 0.5% and 1.5%odium alendronate respiratory formulation.

Table 6 – Effect of inhalation of alendronate sodium respiratory

Vital organs O

Group I Group II

Lungs 0.0070 ± 0.00087 0.0069 ± 0.000Liver 0.0396 ± 0.0047 0.0398 ± 0.004Kidney 0.0040 ± 0.00052 0.0038 ± 0.000Spleen 0.0025 ± 0.00030 0.0023 ± 0.000Stomach 0.0058 ± 0.00072 0.0059 ± 0.000

Group I: control; group II: 0.5% alendronate sodium; group III: 1% alendronatinhalation. Data are represented as mean ± SD (n = 6). The differences am(P > 0.05).

ly significant (P > 0.05).

7.6. Histopathological analysis

Histopathological assessment of lungs of treated animalsreveals that inhalation of alendronate sodium respiratoryfluid causes no significant inflammatory cell infiltration, nointerstitial and alveolar edema, vascular congestion and alve-olar collapse. The tissues were comparatively the same asthat in the control group (Fig. 6). Bronchial passages and

alveolar spaces are within the normal limit in all treatedgroups even at the highest concentration group (group IV).No polymorphonuclear leucocytes in terminal bronchiole

formulation on organ/body weight ratio of vital organs.

rgan/body weight ratio

Group III Group IV

86 0.0072 ± 0.00085 0.0071 ± 0.000866 0.040 ± 0.0045 0.0402 ± 0.005151 0.0038 ± 0.00054 0.0042 ± 0.0005331 0.0027 ± 0.00034 0.0026 ± 0.0003374 0.0058 ± 0.00073 0.0061 ± 0.00075

e sodium; group IV: 1.5% alendronate sodium respiratory formulationong the treatment groups vs. control are not statistically significant

644 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

Fig. 6 – Photomicrographs of lungs: (i) control (no treatment), (ii) 0.5%, (iii) 1.0%, and (iv) 1.5% alendronate sodium respiratoryformulation treatment: (A) lung micrographs at 10×; (B) lung micrographs at 40×.

Table 7 – Effect of inhalation of alendronate sodium respiratory formulation on BAL fluid parameters.

Lung toxicity biomarkers in BAL fluid Groups

Group I Group II Group III Group IV

Total cell counts 0.0254 ± 0.0052 0.0256 ± 0.0053 0.0258 ± 0.0052 0.062 ± 0.0056Total protein 16.42 ± 1.75 18.78 ± 1.88 17.54 ± 2.24 18.86 ± 1.96Lactate dehydrogenase 13.41 ± 1.59 14.29 ± 1.68 15.93 ± 1.78 15.73 ± 2.2Alkaine phosphatase 47.06 ± 4.74 49.08 ± 4.87 48.74 ± 4.79 48.40 ± 5.7Superoxide dismutase 12.42 ± 1.45 13.28 ± 1.50 13.46 ± 1.56 14.20 ± 1.54Malondialdehyde 4.16 ± 0.24 4.38 ± 0.27 4.44 ± 0.31 4.88 ± 0.34Catalase 15.25 ± 0.85 15.78 ± 0.88 14.66 ± 0.92 14.24 ± 0.94

Group I: control; group II: 0.5% alendronate sodium; group III: 1% alendronate sodium; group IV: 1.5% alendronate sodium respiratory formulationinhalation. Total cell counts: million cells/ml; total protein: mg/dl lactate dehydrogenase: unit/ml; alkaline phosphatase: unit/l; superoxidedismutase: unit/mg protein; malondialdehyde: �mol MDA/ml; catalase: �mol H2O2 consumed/min/mg protein. All the values are mean ± SD of

s. co

six rats in each group. The differences among the treatment groups v

were observed in any of the lungs of inhaled rats. Insteadthe groups showed dose dependent increase in polymor-

phonuclear leukocyte in alveolar spaces. Summary chartfor histological observations in lungs has been tabulated inTable 8.

Table 8 – Histopathological observations of lungs of animals tre

Parameters

Group I

Bronchial passages WNL

Alveolar spaces WNL

PMNs in alveolar spaces +

PMNs in terminal bronchioles Nil

Group I: control; group II: 0.5% alendronate sodium; group III: 1% alendronatinhalation. WNL, within normal limits; PMN, polymorphonuclear leukocyt

ntrol are not statistically significant (P > 0.05).

8. Discussion

In the present study respiratory formulation of alendronatesodium was investigated for lung deposition and toxicity

ated with alendronate sodium respiratory formulation.

Groups

Group II Group III Group IV

WNL WNL WNLWNL WNL WNL+ + ++Nil Nil Nil

e sodium; group IV: 1.5% alendronate sodium respiratory formulatione.

p h a r

eitorlplhhewaNwtsia

apdifittlatmRsf

atottbhail

owaeadadwvmai

e n v i r o n m e n t a l t o x i c o l o g y a n d

valuation. The formulation was meant as antidote fornhaled radioactive toxic substances fulfilling the manda-ory task of generating animal safety data prior to carryingut phase-1 human trials. During radioactive poisoning,adioactive/toxic material inhaled either as aerosol or col-oidal particles reaches into the lungs and absorbed thereinroducing rapid onset of toxicity. Once incorporated into the

ungs these are very difficult to remove and are extremelyarmful. The superior chelation ability of bisphosphonateas been reported in the literature (Xu et al., 2008; Doganat al., 2005). Since chelated metals will not be absorbed andill be excreted through the gastrointestinal tract (GIT),

lendronate may be used to reverse radioactive poisoning.anoformulation containing nebulized fluid of alendronatehen inhale, reaches into the pulmonary region, neutralize

oxic material in lungs and thus make it unavailable forystemic absorption. Such a targeting approach will resultn quick and deep penetration of antidote into the lungs inppreciable quantities, reduce dose and increase survivability.

In this study, ethanol (30%) and saline (70%) were selecteds nebulization fluid formulation. Ethanol was added toroduce submicronized aerosolized particles having bettereposition efficiency. When the alcohol is added as a mist

nstead of a liquid, the drug particles enters the bloodstreamaster and the effects are more immediate. In fact vapor-zed particles dried instantaneously leaving only drug intohe alveolar regions and thus reduced the chances of lungoxicity by alcohol. Previous studies suggested that beingow surface tension, alcohol nebulization increased compli-nce and decreased resistance in normal subjects. Being lessoxic, ethanol has been widely used as an excipient in several

arketed formulations (e.g., Tornalate, Azmacort, Decadronespihaler, and Bronkometer) (Choi et al., 2001). The particleize distribution was estimated in the range of 300–500 nm andound to be appropriate for inhalation delivery.

The study describes in vitro drug deposition of sodiumlendronate respiratory formulation in lungs. Percentage dis-ribution of alendronate nanosized particles in various stagesf cascade is represented in Fig. 4. ACI results demonstratedhat majority of particles deposited in 1, 3 and 4 stages. Emit-ed dose and MMAD of aerosolized particles was found toe 50.48% and 4.66 �m, respectively. The particles showed aigher retention in the lower stages and, hence, suitable forlveolar deposition. Rf fraction of nebulized drug was signif-cantly high (65%) that ensured the better aerosolization andung deposition behavior.

A whole body exposure chamber was used for inhalationf drug aerosols as it provided an exposure environment inhich the test subjects were unrestrained and could move

bout freely within cages. The system also provided efficientxposure to a large numbers of animals at the same time,nd was very useful for chronic exposure studies whereaily exposure duration might be as much as 24 h. Since thenimals left are unrestrained, the experimental procedureoes not result in any significant physical stress to the animal,hich may otherwise affect the experimental findings to a

ariable extent (Ali et al., 2012). However, the designed systemay lead to oral and dermal exposure of the test compound

nd thus reducing the inhalable fraction. Any such exposuren the present study with no adverse effects may be taken as

m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647 645

an added confirmation of the safety of alendronate aerosols.The in vivo biodistribution of nebulized alendronate in ratmodel showed that the drug significantly deposited in thelungs and retained for a considerable period of time.

The assessment of toxicity by inhalation route is sim-ilar to that used for toxicology studies conducted by anyother route of administration. Common parameters which arestudied included assessment of body weight, food consump-tion, clinical signs of toxicity, clinical pathology (hematology,clinical chemistry), gross necropsy (except nasal cavity), andhistopathology. In the present study there was no mortality,no adverse effects on any of the hematological and bio-chemical parameters were noticed following treatment withaerosols of alendronate respiratory fluid in any of the con-centration studied. The changes in body weight have beenused as an indicator of adverse effect of respiratory fluid (Teoet al., 2002). Since no remarkable changes were observed inanimal behavior, body weight and organ weight at all doselevels in treated rats as compared to control group, it couldbe inferred that sodium alendronate respiratory fluid wasnon-toxic at the doses administered. Data analysis of ani-mal’s blood parameters can be translated for risk evaluation inhumans, since changes in hematological and serum biochem-istry system have a higher predictive value for human toxicity.The hematopoietic system is considered as one of the mostsensitive targets of toxic compounds and denotes physiologi-cal and pathological status in man and animals (Diallo et al.,2010).

Transaminases (AST and ALT) and ALPs are generally usedas indices for liver and kidney damage respectively (Hayes,2007; Raza et al., 2002). No significant change was found inserum levels of AST, ALT, and ALP enzymes post sodiumalendronate inhalation. Sodium alendronate, therefore, didnot provoke any detrimental effect on liver and kidney. Theinhalation of alendronate significantly reduced the cholesterolvalues of the blood. This could be an interesting finding thatthe drug particles affected the cardiovascular system and canbe utilized in lowering lipid concentration.

The collection and analysis of BAL fluid has become anestablished technique to study cellular and soluble compo-nents of the lower respiratory tract (Costabel and Guzman,2001). The study of cells and proteinaceous substances inlung washings provides an insight into the effects of anyparticular inhalation agent on lung biochemistry. BAL fluidanalysis was therefore done to ascertain any alendronatesodium inhalation induced lung toxicity. BAL is a minimallyinvasive procedure that offers an opportunity to investigateintra-alveolar alterations associated with lung diseases(Reynolds, 2000) and a valuable tool for studying immune andinflammatory mechanisms in pulmonary disorders (Costabeland Guzman, 2001; Reynolds, 2008). There were also noindications of nonspecific immune responses, as indicated bynormal ALP and LDH activity in BAL fluid. These studies indi-cated that aerosol delivery of alendronate directly to rodentlungs resulted in no activation of the local cellular immunesystem. Total serum protein which are synthesized in the

liver, is used as an indicator of liver impairment, attributed tochanges in protein and free amino acid metabolism and theirsynthesis in the liver (Vasantharaja et al., 2012). It could alsobe due to the production of heat shock proteins or destructive

d p h

r

646 e n v i r o n m e n t a l t o x i c o l o g y a n

free radicals or could be a part of toxicant induced apoptosis(Sobha et al., 2007). Marginally increased in total proteinconcentration of BAL fluid might be due to slight inductionof edema in alveoli of lungs by inhaled submicronic drugparticles which has been associated with dehydration state(Halim et al., 2011). Several enzymes in blood serum have beenconsidered as a relevant oxidative stress indicators. It is alsoan indirect method to detect free radical production in livingcreatures (Jain et al., 2008). Therefore, activities of serum MDAand LDH have been commonly used in the detection of tissuedamage caused by oxidative stress. An increase of theseenzyme activities in the extracellular fluid or serum is a sen-sitive indicator of even minor cellular damage and indicatesstress-based tissue impairment. Treated groups did not showany significant alterations in MDA level and the results wereremained closed to control indicating no stress induced tissuedamage in any treated group (Table 7). Our data confirmsthat there are no changes in lung injury markers in BAL fluidpost-inhalation of submicron sized sodium alendronate.

The lack of toxicity of sodium alendronate on specificorgans was further confirmed by histopathological assess-ment. No morphological changes were observed in alveolarmacrophages. Histopathological examination of lung fromcontrol and treated animals showed normal architecture,suggesting no microscopic changes and morphological distur-bances at all dose levels. Sections of control lung tissues havethe appearance of fine lace because of thin-walled alveoli andterminal bronchioles showing normal histology and no accu-mulation of inflammatory cells in alveoli. We also investigatedthe tracheal ultra-structure in rats that received drug expo-sure. The exposed animals did not exhibit any cellular influxto the site of administration, and the alveolar macrophagesexhibited no altered morphology relative to controls. Infact thelungs of alendronate-treated rats did not develop any edemaor alveolitis although they showed concentration dependentperivascular inflammation. The adherence of polymorphonu-clear leukocytes (PMNLs) to the pulmonary blood vesselsrepresented acute inflammation. On the other hand thealveoli and bronchioles were normal and do not revealed anyhistological changes in any treated groups. Non-significantaccumulation of mild inflammatory cells in alveolar spaces,normal alveoli and bronchioles confirmed no significant lunginjuries.

9. Conclusions

The respirable formulation prepared and evaluated in thisstudy offers great promise as a formulation for the lungdelivery of sodium alendronate which exhibits good in vitroaerosolization in nanosized particles that penetrate deeplyinto the lung parenchyma and alveolar region. These nano-sized particles were further retained for a considerable periodin the lungs. The hematology, serum biochemistry and bron-choalveolar lavage studies showed that inhalation of sodiumalendronate respiratory formulation did not induce significant

increases of lung toxicity markers as compared with control.The study highlights the safety of the sodium alendronateformulation in nanosize formulation which can be used asan antidote for radioactive compounds. The inhalation of

a r m a c o l o g y 3 6 ( 2 0 1 3 ) 636–647

sodium alendronate respiratory formulation shows increasedlung bioavailability than oral administration which can mini-mize the site effects of the drug and increases the therapeuticefficacy. These results suggested that the pulmonary routefor delivery of sodium alendronate could be a new and safeadministration route for future therapy.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors are thankful to Institute of Nuclear Medicine andAllied Sciences (INMAS), Defence Research and DevelopmentOrganization, Ministry of Defence, Government of India forproviding the animals for in vivo studies to carry out this work.The authors are also thankful to University Grant Commissionfor awarding the financial support during this work.

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