Acute and Repeated Dose Toxicity Studies of Different β-Cyclodextrin-Based Nanosponge Formulations

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Acute and Repeated Dose Toxicity Studies of Different ␤-Cyclodextrin-Based Nanosponge Formulations PRAVIN SHENDE,1 YOGESH A. KULKARNI,2 R. S. GAUD,2 KIRAN DESHMUKH,3,4 ROBERTA CAVALLI,4 FRANCESCO TROTTA,1 FABRIZIO CALDERA1 1

Department of Chemistry, University of Torino, Italy SPP-School of Pharmacy and Technology Management, NMIMS University, Mumbai, Maharashtra, India 3 Faculty of Pharmaceutical Sciences, Jodhpur National University, Jodhpur, Rajasthan, India 4 Department of Science and Technology of Pharmaceutics, University of Torino, Italy 2

Received 6 December 2014; revised 11 February 2015; accepted 12 February 2015 Published online 9 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24416 ABSTRACT: Nanosponges (NS) show promising results in different fields such as medicine, agriculture, water purification, fire engineering and so on. The present study was designed to evaluate toxicity of different NS formulations (namely, S1–S6) synthesized with different cross-linking agents such as carbonyl diimidazole, pyromellitic dianhydride and hexamethylene diisocynate; and preparation methods in experimental animals. Acute and repeated dose toxicity studies of formulations were carried out as per OECD guidelines 423 and 407, respectively. For acute toxicity study, formulations were administered to female rats at doses of 300 and 2000 mg/kg orally. The general behaviour of the rats was continuously monitored for 1 h after dosing, periodically during the first 24 h and daily thereafter for a total of 14 days. On day 14, animals were fasted overnight, weighed, and sacrificed. After sacrification, animals were subjected to necropsy. For repeated dose toxicity study, rats of either sex were orally administered with formulations at the dose of 300 mg/kg per day for a period of 28 days. The maximally tolerated dose of all formulations was found to be 2000 mg/kg. Repeated administration of formulations for 28 days did not show any significant changes in haematological and biochemical parameters in experimental animals. These results indicate that C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association the formulations are safe, when tested in experimental animals.  J Pharm Sci 104:1856–1863, 2015 Keywords: cyclodextrin nanosponge; bulk condensation method; interfacial condensation method; acute toxicity; repeated dose toxicity; OECD guidelines; complexation; nanotechnology; pharmacokinetics; protein delivery; surface active

INTRODUCTION Cyclodextrin-based nanosponges (NS) are spherical, regular shape, cross-linked nanoparticles. NS emerge with promising results in different fields such as medicine as drug carrier, drug solubiliser, controlled-release matrix system; agriculture as longevity enhancing agent for fruits and flowers; water purification as adsorbing agent, fire engineering as smoke adsorbent and flame retardant and so on.1 NS are condensed, complexed and/or polymerised $-cyclodextrin ($-CD) with different cross-linkers such as hexamethylene diisocynate (HMDI),2 diphenyl carbonate,3 pyromellitic dianhydride (PMDA),4 carbonyl diimidazole (CDI)5 and so on. NS are mostly prepared by polymer condensation and interfacial condensation methods.2 NS, a nanoparticle-based drug delivery system have numerous applications in pharmaceuticals such as enhancing the dissolution rate, solubility and stability of drugs, to mask unpleasant flavours, to convert liquid substances to solids and to prolong the release of drug.5 NS showed superior complexing ability than natural cyclodextrins towards many molecules leads to increased encapsulation efficiency and stability. This behaviour has been exploited to improve the solubility and bioavailability of poorly water-soluble drugs. Particularly, paclitaxel and tamoxifen encapsulated in NS showed an in-

creased bioavaibility compared with the free drug after their oral administration to rats.6,7 To develop therapeutic formulations, safety requirements are mandatory. In acute previous toxicity study, the oral administration of PMDA cross-linked $-NS in rats at the dose of 2000 mg/kg does not show any signs of toxicity up to 14 days and no animals died. This indicates that $-NS was nontoxic in rat to an oral dose of 2000 mg/kg of body weight.4 The present study was designed to evaluate toxicity of different NS formulations (namely, S1– S6) with different cross-linking agents such as CDI, PMDA and HMDI; and preparation methods in experimental animals.

MATERIALS AND METHODS Materials $-Cyclodextrin was gifted from Roquette Italia SpA (Cassano Spinola, Italy). CDI, PMDA, HMDI and potassium hydroxide were obtained from Sigma–Aldrich (Munich, Germany). Dichloromethane, dimethyl formamide (DMF), acetone, dimethyl sulfoxide (DMSO), triethyl amine (TEA) and ethanol were purchased from Sigma–Aldrich (Munich, Germany). All other chemicals and reagents were of analytical grade. Methods

Correspondence to: Pravin Shende (Telephone: +011-0917550; Fax: +0116707855; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 1856–1863 (2015)  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

1856

Preparation of Cross-Linked NS The cross-linked NS were prepared in 1:8 molar ratio of $-CD and CDI or HMDI or PMDA (Table 1) by the methods

Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Table 1. Test Compounds Serial Number 1 2 3 4 5 6

Formulation Code

Test Compound

S1 S2 S3 S4 S5 S6

BNS–CDI (1:8)–CM BNS–CDI (1:8)–NM BNS–HMDI (1:8)–CM BNS–HMDI (1:8)–NM BNS–PMDA (1:8)–CM BNS–PMDA (1:8)–NM

as described below.

r (1) Bulk condensation method (CM) r (i) CDI cross-linked NS 17.42 g of anhydrous $-CD (15.35 mmol) was added to 100 mL DMF in round bottom flask to attain complete dissolution by monitoring clear solution. Then, 19.91 g of CDI (122.8 mmol) was added as a cross-linking agent and the solution allowed to react at 100°C. After the completion of condensation reaction, the solid block of cross-linked cyclodextrin was roughly ground in mill or unreacted reagents were completely removed by Soxhlet extraction with ethanol.8

r (ii) HMDI cross-linked NS 3.91 g of anhydrous $-CD (3.44 mmol) was added to 16 mL DMSO in round bottom flask to achieve complete dissolution by observing clear solution. Then, 4.64 g of HMDI (27.5 mmol) was added as a cross-linking agent and the solution allowed to react at 70°C for 4 h. After the completion of reaction, the solid block of cross-linked cyclodextrin was roughly ground in mill or unreacted reagents were completely removed by Soxhlet extraction with ethanol.

r (iii) PMDA cross-linked NS 11.35 g of anhydrous $-CD (10.00 mmol) and 17.45 g of PMDA (80.0 mmol) were dissolved in 100 mL of DMSO containing 2.7 mL TEA (19.4 mmol) and were allowed to react at room temperature for 3 h. Once the reaction was over, the solid obtained was ground in a mortar and Soxhlet extracted with acetone for 24 h.

r (2) Interfacial condensation method (NM) 1.135 g of $-CD (1.000 mmol) was completely dissolved in 20 mL of 0.1 M aqueous solution of potassium hydroxide under magnetic agitation or by means of sonicator. 1.297 g of CDI (8.00 mmol) or 1.346 g of HMDI (8.00 mmol) or 1.745 g of PMDA (8.00 mmol) was dissolved in methylene chloride to obtain an organic CDI or HMDI or PMDA solution. The alkaline $-CD solution was added to the organic CDI or HMDI or PMDA solution under continuous agitation. After 30 min of reaction, the precipitate was washed with deionized water and centrifuged at 3000 rpm for 10 min. The filtrate was collected and dried in vacuum dessicator to obtain the NS.9 The schematic representation of the structure of NS is shown in Figure 1. DOI 10.1002/jps.24416

1857

Suspensions of test compounds were prepared by using 1% sodium carboxy methyl cellulose (CMC) for oral administration. Particle Size The sizes of NS were measured by dynamic light scattering using a Brookhaven particle sizer (New York, USA) equipped with particle sizing software. NS were suitably diluted with distilled water for each measurement. The measurements were made at a fixed angle of 90° for all NS samples and 25°C. Experimental Animals Wistar albino rats were purchased from Bharat Serum (Thane, India). All animals were housed in animal facility with temperature 25 ± 2°C, relative humidity of 75 ± 5%, and a 12-h light– dark cycle. Standard basal diet (Nutrimix Laboratory Animal Feed, Maharashtra, India) and purified water were provided ad libitum to the animals. Rats were assigned to each dose group by stratified random sampling based on body weight. The animals were kept under laboratory conditions for an acclimatization period of 7 days before carrying out the experiments. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted as per the norms of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. Acute Toxicity Study The acute toxicity of samples was carried out in rats weighing 150–170 g, using the OECD guideline no. 423.10 The animals were divided into 13 groups containing three female rats in each. The NS samples were prepared in 0.1% sodium CMC in water before oral administration and were administered at doses of 300 and 2000 mg/kg body weight, after a short fasting period. The control group received 0.1% CMC solution. The general behaviour of the rats was continuously monitored for 1 h after dosing, periodically during the first 24 h (with special attention given during the first 4 h), and daily thereafter for a total of 14 days. Various cage side observations were performed, such as behaviour patterns, somatomotor activity, changes in eyes and mucous membranes, skin and fur and also respiratory, circulatory, autonomic and central nervous systems. Animals were strictly observed for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The body weight, food and water intake for the treatment and control group were recorded weekly. On day 14, animals were fasted overnight, weighed and sacrificed. After scarification, the animals were subjected to necropsy. Repeated Dose Toxicity Study Repeated dose toxicity study was carried out on Wistar rats of either sex (150–170 g) as per OECD guidelines 407. Animals were divided in six groups, each group containing five males and five females. Group I was control group that received vehicle. Group II, III, IV, V, VI and VII received test compounds S1, S2, S3, S4, S5 and S6 NS formulation, respectively, at dose 300 mg/kg. Test compounds were administered orally in morning using gavage for 28 days. All rats were observed daily for morbidity and mortality. Body weight, food intake and water intake were measured at 7-day interval starting from 0 day till 28 days. On 28th Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

1858

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Figure 1. Schematic representation of the structure of NS.

day, blood samples were collected in heparinized tubes by retro-orbital method. Biochemical parameters such as cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT) total protein and blood urea nitrogen were determined using autoanalyser (Erba Chem 7, Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

Mannheim, Germany). Haematological parameters such as total red blood corpuscles, haemoglobin, white blood corpuscles (WBC) and platelet count were determined using automatic haematological analyzer (Sysmex Corporation, Kobe, Japan). DOI 10.1002/jps.24416

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Histopathology Liver, kidney, stomach, intestine, spleen, pancreas, adrenal, lungs, heart, brain and gonads were fixed immediately in 10% formalin for routine histopathological examination. The tissues were embedded in paraffin and then sectioned, stained with hematoxylin and eosin and were examined under light microscope.

Statistical Analysis Statistical evaluation of difference between biochemical and haematological parameters of control and treated group was carried out by using GraphPad Prism ver. 5.00 for Windows. Comparison among different groups was performed by using analysis of variance test (ANOVA). All data are expressed as mean ± SEM; p values less than 0.05 were considered to be significant.

RESULTS

1859

polydispersity indices (data not shown). It suggests that particle size of NS depends on process of grinding as well as the preparation method. The particle sizes of NS were 450–550 nm by trituration and 40–60 nm by vibrational rod milling.4 Acute Toxicity Study After single oral administration of NS samples at 300 and 2000 mg/kg dose levels, animals in all treated groups did not show any lethal effects or mortality, throughout the test period (Table 3). The animals did not show any changes in the general appearance during the observation period. Morphological characteristics (fur, skin, eyes and nose) were unchanged. The treated animals did not show any tremors, convulsion, salivation, diarrhoea, lethargy or unusual behaviours such as selfmutilation, walking backward. There was no significant difference in body weights, food and water intake in the treatment groups when compared with control animals (data not shown). The animals in the treatment group showed no changes in organ gross anatomy observed during necropsy, when compared with control.

Particle Size

Repeated Dose Toxicity Study

The particle sizes of NS formulations were between 400 and 550 nm in CM and 50 and 200 nm in NM (Table 2) with low

Administration of test NS formulations for 28 days did not show any adverse symptoms of toxicity and mortality. Body Control S1 S2 S3 S4 S5 S6

Body weight (g)

220

200

180

28

21

14

7

a

0

160

Days Control S1 S2 S3 S4 S5 S6

Body weight (g)

220

200

180

b

28

21

14

7

0

160

Days

Figure 2. Effect on body weight change in male rats (a) and female rats (b) in 28-day repeated dose toxicity study. Data are expressed as mean ± SEM. DOI 10.1002/jps.24416

Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

1860

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Table 2. Particle Size of Different NS Formulations Serial Number

Formulation Code

Particle Size (nm)

S1 S2 S3 S4 S5 S6

400–500 50–100 400–500 100–200 450–550 100–200

1 2 3 4 5 6

weight of animals gradually increased in all groups. No significant difference was observed among control and treated groups (Fig. 2). Comparison between food and water intake of treated groups and that of control group did not show any significant change (Figs. 3 and 4). All biochemical parameters are summarised in Table 4. Comparison among control and treated group shows no statistical significance between any of the biochemical parameter. Table 5 summarises results of haematological evaluation. No statistical significance was observed among the haematological parameters of control and that of treatment groups.

Table 3. Sign of Toxicity and Mortality Results of Acute Toxicity Study of Samples in Rats Group

Dose (mg/kg)

Sign of Toxicity (ST/NB)a

Mortality (D/S)a

Control S1 S1 S2 S2 S3 S3 S4 S4 S5 S5 S6 S6

– 300 2000 300 2000 300 2000 300 2000 300 2000 300 2000

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

a

Values are expressed as animal number. ST, sign of toxicity; NB, normal behaviour; D, died; S, survived.

Control S1 S2 S3 S4 S5 S6

20

g/day/rat

18 16 14

28

21

14

7

0

12

a

Days Control S1 S2 S3 S4 S5 S6

20

g/day/rat

18 16 14

b

28

21

14

7

0

12

Days

Figure 3. Effect on food intake in male rats (a) and female rats (b) in 28-day repeated dose toxicity study. Data are expressed as mean ± SEM. Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

DOI 10.1002/jps.24416

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Control S1 S2 S3 S4 S5 S6

22 20

mL/day/rat

1861

18 16 14

28

21

14

7

0

12

a

Days

Control S1 S2 S3 S4 S5 S6

22

mL/day/rat

20 18 16 14

28

21

14

7

0

12

b

Days Figure 4. Effect on water intake in male rats (a) and female rats (b) in 28-day repeated dose toxicity study. Data are expressed as mean ± SEM.

No treatment-related gross or microscopic changes were noticed in internal organs during the pathological examination.

DISCUSSION Toxicological evaluation is an important step to determine safety of drugs and excipients; it also helps for selection of safe dose for their use in humans and animals. It is worth of note that the fate of the parent cyclodextrins in the gastrointestinal tract differs based on the resistance to hydrolysis and enzymatic degradation. The $-CDs are practically resistant to stomach acid or salivary and pancreatic amylases and they are extensively hydrolyzed in the colon.11 A human study with healthy volunteers and ileostomists has shown clear evidence that $-CDs is poorly digested in the human small intestine, but it is almost completely degraded by the colonic microflora.12 The enzymatic degradation of $-CDs in the human colon can form either glucose or maltodextrins.13 The LD50 of $-CD is 18.8 g/kg after oral administration to rats.14 Previous in vitro studies were carried out to evaluate the NS safety. NS did not show haemolytic activity or cytotoxicity tested on different cell lines.3,15 The in vitro stability of NS against chemical degradation was also evaluated. Briefly, under DOI 10.1002/jps.24416

incubation of carbonate NS in acidic conditions (0.1 N HCl), a limited release of cyclodextrin units was detected after 2 h because of the initial degradation of the NS structure, whereas a basic environment (NaOH 0.1 N) did not show to affect the NS stability. On the contrary, NS obtained by using PMDA as a cross-linker appear less stable in basic solutions, also if they maintained their structure for 24 h.16 As there was no reports on the toxicity of the NS after oral administration, dose of 300 mg/kg was selected as starting dose in acute toxicity study.10 Mortality was not detected in control and treated groups of all selected dose levels throughout the study period. The results of the present study indicated that the oral LD50 of NS formulations was observed to be greater than 2000 mg/kg body weight, which suggested no toxicity of the compounds with good safety. Thus, the non-observed adverse effect level of all the NS tested may be set at 2000 mg/kg. However, acute toxicity data are not sufficient for clinical use owing to cumulative toxic effects over repeated dose administration. Hence, repeated dose toxicity study of all NS was carried out. The changes in the body weight are indications of the toxic effects of drugs and chemicals.17 In the present study, no significant changes were observed after administration of all NS at selected dose, for 28 days. All cellular blood components are Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

1862

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Table 4. Effect of Test Compounds on Biochemical Parameters in Male Rats and Female Rats in 28-Day Repeated Dose Toxicity Study (Mean ± SEM) Parameters (Mean ± SEM) Group Male

Control S1 S2 S3 S4 S5 S6

Glucose 106.20 108.38 111.96 108.73 108.21 112.62 111.0

± ± ± ± ± ± ±

Total Proteins

4.08 4.16 1.06 3.04 4.41 3.96 3.06

5.14 5.27 5.43 5.66 5.16 5.77 5.26

± ± ± ± ± ± ±

0.14 0.20 0.14 0.25 0.18 0.30 0.16

Blood Urea Nitrogen 21.02 20.09 23.84 21.93 23.26 20.98 22.69

± ± ± ± ± ± ±

1.26 0.43 1.11 1.35 1.43 0.91 1.67

Creatinine 0.57 0.55 0.57 0.54 0.58 0.58 0.58

± ± ± ± ± ± ±

0.03 0.05 0.04 0.02 0.05 0.04 0.04

Parameters (Mean ± SEM) Group Female

Control S1 S2 S3 S4 S5 S6

Glucose 107.30 106.84 108.56 111.71 110.16 108.45 106.6

± ± ± ± ± ± ±

Total Proteins

3.21 3.27 3.10 1.90 3.73 4.10 4.20

5.45 5.5 5.33 5.67 5.64 5.45 5.59

± ± ± ± ± ± ±

0.17 0.29 0.24 0.14 0.19 0.28 0.08

Blood Urea Nitrogen 22.00 20.90 21.76 22.94 22.25 20.10 20.86

± ± ± ± ± ± ±

1.64 0.96 0.93 0.72 1.19 1.13 0.92

Creatinine 0.53 0.58 0.53 0.59 0.55 0.58 0.54

± ± ± ± ± ± ±

0.03 0.05 0.03 0.04 0.05 0.05 0.03

Parameters (Mean ± SEM) Group Male

Control S1 S2 S3 S4 S5 S6

ALT 59.00 57.77 61.03 58.76 61.11 58.92 60.27

± ± ± ± ± ± ±

2.89 2.48 2.87 3.32 2.66 3.22 2.95

AST 305.20 289.40 323.90 301.30 304.40 320.40 288.6

± ± ± ± ± ± ±

Cholesterol 15.45 10.96 17.63 16.84 8.68 9.93 10.9

88.55 84.57 91.62 88.48 92.65 89.96 88.90

± ± ± ± ± ± ±

2.95 2.36 2.60 4.13 3.48 4.16 3.35

Triglycerides 87.73 83.68 90.57 86.91 91.19 89.32 88.67

± ± ± ± ± ± ±

2.46 1.54 4.02 1.71 1.90 3.66 3.25

Parameters (Mean ± SEM) Group Female

Control S1 S2 S3 S4 S5 S6

ALT 57.5 61.82 60.11 59.37 60.09 58.54 59.86

± ± ± ± ± ± ±

2.35 1.43 2.24 1.75 3.35 2.43 2.20

AST 295.80 305.30 301.80 323.70 318.40 288.10 309.3

derived from hematopoietic stem cells. Many chemicals or drugs affect hematopoietic system, which is indicated by changes in haematological parameters. So, analysis of blood parameters is relevant to risk evaluation. When data are translated from animal studies, the changes in the haematological system have a higher predictive value for human toxicity.18 Administration of all compounds did not show any significant changes in the haematological parameters such as white blood cells, red blood cells and haemoglobin between control and treated groups after 28 days of administration. The liver toxicity is represented by significant changes in enzymes such as ALP, AST and ALT.19 Liver is the site of synthesis of cholesterol and proteins, so the change in the levels of the same also indicates liver toxicity. Liver also controls glucose synthesis as well as produces free glucose from hepatic glycogen stores, it is the major area of synthesis and clearance of cholesterol.20 These enzymes and biochemical parameters Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015

± ± ± ± ± ± ±

Cholesterol 9.97 19.58 13.09 10.99 12.04 14.82 10.52

92.47 89.51 86.87 89.71 95.49 86.14 86.70

± ± ± ± ± ± ±

2.29 3.23 2.85 3.09 3.57 4.01 3.15

Triglycerides 92.25 88.45 89.90 87.18 90.64 88.09 91.00

± ± ± ± ± ± ±

4.19 3.46 3.85 3.54 3.38 3.08 3.1

were not altered by administration of compounds after administration for 28 days, indicating that there was no severe liver damage. Kidney damage is represented by changes in biochemical parameters such as urea and creatinine.21 These parameters remained normal after administration of NS for 28 days. Thus, all NS formulations did not produce any hepato and renal toxicity. The histopathological examination of important organs such as liver, kidney, stomach and intestine indicated no damage; these observations further support the safety compounds in rats.

CONCLUSIONS All NS after oral administration to rats were found safe at selected doses in acute and repeated dose toxicity study. DOI 10.1002/jps.24416

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

1863

Table 5. Effect of Test Compounds on Haematological Parameters in Male Rats and Female Rats in 28-Day Repeated Dose Toxicity Study (Mean ± SEM) Parameters (Mean ± SEM) Group Male

Control S1 S2 S3 S4 S5 S6

Red Blood Corpuscles 8.53 ± 0.21 8.39 ± 0.11 9.07 ± 0.16 8.55 ± 0.20 9.03 ± 0.16 8.69 ± 0.13 8.67 ± 0.25

(109 /L)

Haemoglobin (g/dL)

White Blood Corpuscle (109 /L)

Platelets (109 /L)

13.37 ± 0.17 13.61 ± 0.22 13.64 ± 0.18 13.49 ± 0.21 13.40 ± 0.19 13.72 ± 0.19 13.81 ± 0.18

10.66 ± 0.68 10.17 ± 0.43 10.41 ± 0.48 10.21 ± 0.54 11.02 ± 0.53 11.12 ± 0.91 10.75 ± 0.87

1040.8 ± 32.58 1072.2 ± 36.37 1037.4 ± 32.88 1044.8 ± 40.95 1022.8 ± 16.41 1047.2 ± 34.37 1056.04 ± 22.05

Parameters (Mean ± SEM)

Group Female

Control S1 S2 S3 S4 S5 S6

6.33 ± 0.16 6.57 ± 0.18 6.49 ± 0.29 6.64 ± 0.19 6.37 ± 0.14 6.54 ± 0.19 6.40 ± 0.12

12.26 ± 0.16 12.20 ± 0.25 12.60 ± 0.22 12.09 ± 0.18 12.56 ± 0.19 11.98 ± 0.19 12.68 ± 0.16

Thus, the study provides first systematic report on toxicity data of these hyper cross-linked cyclodextrin polymers after oral administration, showing a good biocompatibility with a potential negligible degradation during the gastrointestinal transit. Despite that, further investigations are needed to evaluate the fate of NS in the GI tract. On the basis of the results, $-CD NS might be considered a new platform for drug delivery with the potential of improving oral therapeutic formulations.

REFERENCES 1. Trotta F. 2011. Cyclodextrin nanosponges and their applications. In Cyclodextrins in pharmaceutics, cosmetics, and biomedicine: Current and future industrial applications; Bilensoy E, Ed. New Jersey: John Wiley and Sons, pp 323–342. 2. Trotta F, Shende P, Biasizzo M. 2012. Method for preparing dextrin nanosponges. Patent WO 2012/147069 A1. 3. Swaminathan S, Pastero L, Serpe L, Trotta F, Vavia P, Aquilano D, Trotta M, Zara G, Cavalli R. 2010. Cyclodextrin-based nanosponges encapsulating camptothecin: Physicochemical characterization, stability and cytotoxicity. Eur J Pharm Biopharm 74:193–201. 4. Shende PK, Trotta F, Gaud RS, Deshmukh K, Cavalli R, Biasizzo M. 2012. Influence of different techniques on formulation and comparative characterization of inclusion complexes of ASA with $cyclodextrin and inclusion complexes of ASA with PMDA cross-linked $-cyclodextrin nanosponges. J Incl Phenom Macrocycl Chem 74:447– 454. 5. Shende P, Deshmukh K, Trotta F, Caldera F. 2013. Novel cyclodextrin nanosponges for delivery of calcium in hyperphosphatemia. Int J Pharm 456:95–100. 6. Ansari KA, Torne SJ, Vavia PR, Trotta F, Cavalli R. 2011. Paclitaxel loaded nanosponges: In-vitro characterization and cytotoxicity study on MCF-7 cell line culture. Curr Drug Deliv 8:194–202. 7. Torne S, Darandale S, Vavia P, Trotta F, Cavalli R. 2013. Cyclodextrin-based nanosponges: Effective nanocarrier for tamoxifen delivery. Pharm Dev Technol 8:619–625.

DOI 10.1002/jps.24416

11.06 ± 0.50 10.53 ± 0.75 11.08 ± 0.45 11.55 ± 0.78 10.78 ± 0.86 10.87 ± 0.65 10.45 ± 0.36

869.0 ± 27.30 863.8 ± 23.26 885.8 ± 20.07 860.6 ± 26.58 896.4 ± 19.00 884.2 ± 20.87 877.6 ± 23.65

8. Trotta F, Tumiatti W. 2003.Cross-linked polymers based on cyclodextrins for removing polluting agents. Patent WO 03/085002. 9. Shende P, Trotta F. 2014. New applications of cyclodextrin nanosponges. Saarbrucken, Germany: Lap Labmert Academic Publishing. 10. OECD. 2001. The OECD guidelines for testing of chemicals, 423. Acute oral toxicity test. Paris, France: Organization of Economic Cooperation Development. 11. Irie T, Uekama K. 1997. Pharmaceutical applications of cyclodextrins: Toxicological issues and safety evaluation. J Pharm Sci 86:147– 162. 12. Flouri´e B, Molis C, Achour L, Dupas H, Hatat C, Rambaud JC. 1993. Fate of $-cyclodextrin in the human intestine. J Nutr 123:676–680. 13. Antenucci R, Palmer J. 1984. Enzymatic degradation of "- and $cyclodextrins by bacteroides of the human colon. J Agric Food Chem 32:1316. 14. Rowe RC, Sheskey PJ, Oweb SC. Handbook of pharmaceutical excipients. 2006. Pharmaceutical Press. London, UK. 15. Cavalli R, Trotta F, Tumiatti W. 2006. Cyclodextrin-based nanosponges for drug delivery. J Incl Phenom Macrocycl Chem 56:209– 213. 16. Trotta F, Zanetti M, Cavalli R. 2012. Cyclodextrin-based nanosponges as drug carriers. Beilstein J Org Chem 8:2091–2099. 17. Hilaly JE, Israili ZH, Lyouss B. 2004. Acute and chronic toxicological studies of Ajuva iva in experimental animals. J Ethnopharmacol 91:43–50. 18. Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Rhiouani H, Elhilaly J, Israili Z, Lyoussi B. 2008. Acute and sub-chronic toxicity of an aqueous extract of the leaves of Herniariaglabra in rodents. J Ethnopharmacol 118:378–386. 19. Hayes AW. 1989. Guidelines for acute oral toxicity testing. In Principles and methods of toxicity. New York: Raven. 20. Kaplan A, Jack R, Opheim KE, Toivola B, Lyon AW. 1995. Clinical chemistry interpretation and techniques. Williams & Wilkins. Philadelphia, USA. 21. Kulkarni YA, Veeranjaneyulu A. 2012. Toxicological evaluation of the methanol extract of Gmelina arborea Roxb. Bark in mice and rats. Toxicol Int 19:125–131.

Shende et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1856–1863, 2015