EVALUATION OF THE EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF DIABETES-INDUCED ERECTILE DYSFUNCTION

                                                                STUDY PROTOCOL

EVALUATION OF THE EFFICACY OF TEST FORMULATIONS IN RAT MODEL OF DIABETES-INDUCED ERECTILE DYSFUNCTION

1.0  TEST SYSTEM DETAILS:

Species                   : Rattus norvegicus (Rat)

Strain                     : Sprague-Dawley

Age                        : 6-8 weeks

Sex                        : Male

No. of animals       : 10/Group

Total animals         : 60

2.0   ALLOCATION OF GROUPS:

Group No.	Group Description	A Disease Induction agent administered	Agent for inducing penile erection	Treatment administered	Dose Volume and Route
G1	Normal Control	Sodium citrate buffer, i.p.	Apomorphine – 100 µg/kg, s.c.	0.5% MC,  q.d.	5 ml/kg, p.o.
G2	Disease Control	Streptozotocin 55 mg/kg in sodium citrate buffer, i.p.		0.5% MC,  q.d.
G3	Reference Control			Tadalafil-0.5 mpk, q.d. in 0.5% MC
G4	Treated with low dose of TF			TF -X1 mg/kg, q.d.  in 0.5% MC
G5	Treated with an intermediate dose of TF			TF -X2 mg/kg,  q.d. in 0.5% MC
G6	Treated with a high dose  of  TF			TF –X3 mg/kg,  q.d.. in 0.5% MC

 Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; X1, X2, and X3 are defined as the incremental doses of the Test formulations. 

1.0  METHOD:

·         Healthy animals will be selected and randomized based on their body weight. Animals will be allocated into 6 different groups consisting of 10 animals in each group.

·         Animals of Group G1 will receive sodium citrate buffer whereas animals of Group G2 to G6 will receive 55 mg/kg Streptozotocin (STZ) in sodium citrate buffer solution by intraperitoneal route to induce experimental diabetes mellitus (DM).

·         Blood sample level will be measured pre- and post-induction at 72 h and 1 week-post Streptozotocin administration, using a Glucometer.

·         One week later, rats with more than 300 mg/dL of blood glucose will be regarded as DM model rats.

·         After STZ injection, the rats will be fed with a standard pelleted diet ad libitum for 8 weeks.

·         Blood glucose will be additionally estimated in all rats once a week to confirm the maintenance of hyperglycemia.  

·         Eight weeks later, DM rats will be weighed and placed in a quiet, dark observation box for 10 min. Then, apomorphine will be injected subcutaneously into the neck of the rats at a dose of 100 µg/kg. The state of the rats in the above environment will be monitored using a video camera, and the number of penile erections of the rats will be recorded for 30 min.

·         The enlargement or growth of the penis, with the end of the penis exposed will be counted as one time.

·         The DM rats that did not exhibit penile erection will be considered DIED rats and included in the model group.

·        Fasting blood glucose levels will be measured at weekly intervals. Body weight will be measured weekly.

·         Animals of group G1 will serve as normal control and will receive 0.5% MC, p.o.

·         Disease control (G2) animals will receive 0.5% MC, p.o.

·         Animals of group G3 will be treated with the reference drug, Tadalafil  0.5 mg/kg BW, p.o., q.d. for 8 weeks.

·         Animals of groups G4 to G6 will be treated with test formulation at different dose levels 

·         After 8 weeks of treatment, animals will be again administered apomorphine as described above and a number of penile erections will be observed and recorded.

·         Additionally, an oral glucose tolerance test (OGTT) will be performed in overnight fasted rats by measuring tail vein blood glucose level after 20% oral glucose administration (2g/kg) at 0, 30, 60, 90, and 120min.

·         On the last day, animals will be euthanized after overdosing on thiopentone. After suitable anesthesia but before the animal dies, blood will be collected from the retro-orbital plexus. After the death of the animals, penile cavernous tissue will be collected for histological analysis.

2.0  ENDPOINT PARAMETER(S):

·         Number of penile erections

·         Body weight

·         Fasting blood glucose level

·         Oral glucose tolerance test

·         Glycosylated hemoglobin (HbA1c)

·         Serum will be separated for the detection of insulin, glucagon, serum endothelial NOS (eNOS), follicle-stimulating hormone (FSH), and testosterone.

·         Histological Analysis of penile cavernous tissue of rats.

 

3.0  REFERENCE(S):

1.      Wang JS, Feng JL, Li X, Chen ZL, Bao BH, Deng S, Dai HH, Meng FC, Wang B, Li HS. Effect of leech-centipede medicine on improving erectile function in diabetes-induced erectile dysfunction rats via PDE5 signaling pathway-related molecules. Pharmaceutical Biology. 2021 Jan 1;59(1):167-74 

2.      Pu XY, Wang XH, Gao WC, Yang ZH, Li SL, Wang HP, Wu YL. 2008. Insulin-like growth factor-1 restores erectile function in aged rats: modulation the integrity of smooth muscle and nitric oxide-cyclic guanosine monophosphate signaling activity. J Sex Med. 5(6):1345–1354.

3.      Zhang XH, Morelli A, Luconi M, Vignozzi L, Filippi S, Marini M, Vannelli GB, Mancina R, Forti G, Maggi M. 2005. Testosterone regulates PDE5 expression and in vivo responsiveness to tadalafil in rat corpus cavernosum. Eur Urol. 47(3):409–416.

4.      Ma HF, Liu Y, Wang B, Dang J, Ma JX, Zhu YT, Li HS. 2017. Clinical randomized controlled study for the treatment of erectile dysfunction by Tong Luo Xi Feng Qi Wei soup. China J Human Sex. 26:78–81.    

5.  Source Research Needs YouTube Channel: Diabetes-Induced Erectile Dysfunction Video

END OF DOCUMENT

You may like to read these links:   

1. List of All SOPs and Documents for In-vivo Laboratory

2. List of All SOPs and Documents for Study Protocol

3. List of Pharmacological Study Models with details about disease Inducing agents, Standard or Reference drugs, and related parameters.

4. List of OECD Guidelines or Toxicological Studies

5. List of Pharmacopeial Tests Quality Control Tests or Batch Testing Parameters (In-vivo)

6. Animal  Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)

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