The ARRIVE Guidelines Checklist
Animal Research: Reporting In Vivo Experiments
Item Recommendations Paragraph/
Section and Page Number Title 1. Preparation, characterization and evaluation of fenofibrate: benzoic acid
cocrystals with enhanced pharmaceutical properties
Title Page, Page no. 1 Abstract 2. Background: Cocrystallization process involved the understanding of
interaction at molecular level between two molecules in context to their crystal packing and designing of new solids having improved physicochemical as well as pharmaceutical properties. In the present research, an attempt to increase the aqueous solubility and dissolution rate of a poorly aqueous soluble drug fenofibrate (FB) by formulation and evaluation of its cocrystals with benzoic acid (BZ) as a coformer, was carried out.
Results: The drug and coformer were cocrystallized by using solvent drop grinding method. For prediction of cocrystals formation CSD (Cambridge Structure Database) software was utilized. DSC (Differential Scanning Calorimetry), FT-IR (Fourier Transformation Infra-red Spectroscopy) and XRD (X-Ray Diffraction) techniques were used for analysis of cocrystals.
Albino rats were procured from institution as per IAEC guidelines for in- vivo anti-hyperlipidemic studies. The in-vitro dissolution profile of cocrystals, pure drug, their physical mixture and marketed formulation was found to be 89%, 39%, 47% and 61% respectively. The in-vivo anti- hyperlipidemic studies revealed an enhanced anti-hyperlipidemic activity of cocrystals compared to pure drug. The FB: BZ cocrystals also compared to the pure drug showed better dissolution profile and improved in-vivo anti- hyperlipidemic activity in rats.
Conclusion: The study proved that cocrystals can promise to improve in- vitro dissolution rate of poorly aqueous soluble drugs which in turn can lead to better in-vivo activities.
Species and Strain: In-vivo Triton-induced Hyperlipidemic studies were conducted on 24 Albino rates divided in four groups.
Conclusion: The in-vivo anti-inflammatory studies exhibited improved anti- hyperlipidemic activity compared to pure drug.
Page no. 1
INTRODUCTION
Background 3. a. Fenofibrate: Benzoic acid cocrystals has not been investigated for Anti- hyperlipidemic activity yet. So, in this study, Fenofibrate: Benzoic acid cocrystals based anti- hyperlipidemic activity in Triton-induced Hyperlipidemic studies models was investigated. Relevant references have been given in the manuscript.
b. Animal is sensitive to the proposed activity testing. Handling, storage and care of proposed animal species is easy and comfortable
Paragraph 2, Section Methods, Section 2, Page no. 3, 7.
Table 2 Objective 4. The research work requires animals to observe pharmacological effect of test
compounds inside the body by evaluating biochemical and behavioural parameters.
Paragraph 2, Section Methods, Section 2, Page no. 3, 7.
Table 2 METHODS
Ethical Statement
5. The animal experiments were designed according to the approved animals ethical committee and related recommendations for performing experiments on animals. The study was approved by Institutional Animal Ethics Committee of Maharishi Dayanand University, Rohtak, India [ Reference no.
1767/GO/Re/S/14/CPCSEA; dated: 15/06/2019]. Owners of the animals have given a written consent to the authors to use the animals for experimental work. Informed consent was confirmed by the IRB. The authors do not claim to contain any content comprising of any study on human subject.
Methods, paragraph 2, Page no. 3 Declaration, Section : Ethics approval and consent to
participate Study design 6. Table 2 In-Vivo anti- hyperlipidaemic study
S.
No. Group Treatment Dose Route of Administration
1. I Triton 300 mg/kg Injected intraperitoneal
2. II FB:BZ cocrystals 25 mg/kg Oral Route
3. III FB:BZ cocrystals 50 mg/kg Oral Route
4. IV FB 50 mg/kg Oral Route
*Total number of rats = 24
Paragraph 3, Section 3.3 and Table 2, Page no. 7
Experimental procedures
7. Table 2 In-Vivo anti- hyperlipidaemic study S.
No.
Grou p
Treatment Dose Route of Administration
1. I Triton 300 mg/kg Injected intraperitoneal
2. II FB:BZ cocrystals 25 mg/kg Oral Route
3. III FB:BZ cocrystals 50 mg/kg Oral Route
4. IV FB 50 mg/kg Oral Route
*Total number of rats = 24
Place: The study was conducted in laboratory of institution under prescribed conditions as per IAEC guidelines.
Time of Day: 12th December, 2019 at 10 A.M (N/A) Anaesthetic agents: N/A
Surgical procedure: (N/A) Euthanasia method: (N/A)
Instrument Used: CardioChek Lipid Profile Analyzer Kit (Supplier: Galaxy Informatics India Karol Bagh, Delhi) Model no. PLM 01 PLUS
Paragraph 3, Section 3.3 and Table 2, Page no. 7
Experimental
animals 8. Animal Species: Albino rats Sex: Either sex
Age: Mean range: 2.833. Average age of 2 to 3 months.
Weight: Mean range: 184.166. Average Weight 180-200 gm
Paragraph 3, Section 3.3 and Table 2, Page no. 7 Housing and
husbandry
9. Housing: Air-conditioned housing facilities were provided for storing the animals.
Cages: 4 transparent polycarbonate unbreakable cages were used for storing the animals. 6 animals in each cage were stored.
Food: They were treated with hygienic food and fresh water twice daily. To ensure uniform hydration, the rats receive 5ml of water by stomach tube.
Bedding Material: Corn Bedding Breeding Programme: N/A Husbandry conditions: N/A Light/Dark Cycle: N/A
Welfare-related assessments and interventions: N/A
Paragraph 3, Section 3.3 and Table 2, Page no. 7
Sample size 10. Sample Size: For each experiment, a brief detail of the Sample Size includes in paragraph 3, section 3.3 and Table 2 in the manuscript.
Transportation: University provided air-conditioned transportation.
Number of independent replications: N/A
Paragraph 3, Section 3.3 and Table 2, Page no. 7 Allocating
animals to experimental groups
11. a. Allocating animals to experimental groups: Similar experiments have been conducted in the past and number of animals used were 6-7 in each group.
b. For each experiment, a brief detail of the Sample Size includes in paragraph 3, section 3.3 and Table 2 in the manuscript.
Paragraph 3, Section 3.3 and Table 2, Page no. 7
Experimental outcomes
12. In-vivo anti-hyperlipidaemic study of cocrystals exhibited effect in dose- dependent manner. There was no significant reduction in serum lipids profile found in case of group I or control group rats treated with saline. The results revealed that cocrystals with 50 mg/kg dose significantly reduced the serum lipids followed by standard anti-hyperlipidaemic drug, FB 50 mg/kg.
Paragraph 3, Section 3.3 and Table 3, Page no. 7
Statistical methods
13. N/A Paragraph 3,
Section 3.3 and Table 3, Page no. 7 RESULTS
Baseline data 14. N/A Paragraph 3, Section 3.3 and Table 3, Page no. 7 Numbers
analysed
15. a. 24 Albino Rats were used for study
S. No. Group Number of Animals
1. I (Control) 6
2. II 6
3. III 6
4. IV 6
b. N/A
Paragraph 3, Section 3.3 and Table 2, Page no. 7
Outcomes and
estimation 16. In-vivo anti-hyperlipidaemic study of cocrystals exhibited effect in dose- dependent manner. There was no significant reduction in serum lipids profile found in case of group I or control group rats treated with saline. The results revealed that cocrystals with 50 mg/kg dose significantly reduced the serum lipids followed by standard anti-hyperlipidaemic drug, FB 50 mg/kg.
Table 3 Serum lipidemic data*
Groups Dose TC TG HDL LDL VLDL
I Control
(Triton X100- 300
mg/kg)
173.05
± 3 .89
169.48±4.
46
29.47±4.5 3
109.33±2.89 28.86±3.49
II Cocrystals
(25 mg/kg) 159.67
±4.85 122.31±3.
82 27.80±0.5
9 102.10±2.31 26.73±1.41 III Cocrystals
(50 mg/kg) 89.23±
3.72 81.37±3.3
5 38.12±1.1
3 45.97±2.09 15.83±0.98
IV FB (50
mg/kg) 108.2±
2.55 98.55±2.0
3 33.21±1.9
8 58.31±3.61 20.76±2.39
*values are mean of 6 ± S.D, TC- Total cholesterol, TG- Triglycerides, HDL- High density Lipids, LDL- Low Density Lipids, VLDL- Very Low-Density Lipids.
*values are mean of 6 ± S.D
Paragraph 3, Section 3.3 and Table 3, Page no. 7
Adverse events
17. N/A Paragraph 3,
Section 3.3 and Table 2, Page no. 7 DISCUSSION
Interpretation/
scientific implications
18. N/A Paragraph 3,
Section 3.3 and Table 2, Page no. 7 Generalisabili
ty/
translation
19. N/A Paragraph 3,
Section 3.3 and Table 2,
Page no. 7
Funding 20. N/A Paragraph 3,
Section 3.3 and Table 2, Page no. 7
