Download After myocardial infarction carvedilol improves insulin resistance

Title of the Topic:
“Drug -Drug interaction studies of carvedilol with pioglitazone in
Diabetic Rats”
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Due to the advancement in the drug science and developments in medicinal treatment; mortality has
declined even in developing countries. Many potentially fatal diseases can be completely cured by
modern treatments; however, the number of patients suffering from particular chronic diseases like
diabetes and some of the cardiovascular disease have been increased because of the modern drugs
are not able to completely cure chronic diseases but, to certain extent to prevent further
deterioration associated with them patients have to take drugs for extended periods of time and due
to that it may land up in deleterious effect of the particular drug.
Due to the ineffectiveness on chronic diseases as well as the potential side effects, patients are often
led to explore complementary or alternative medicine in particular1.
Drug-drug Interactions [DDI] are still observed frequently in current pharmacotherapy and the
prediction of DDIs is of clinical importance for selecting regimens and adjusting doses2.
However, mainstream medical treatment for chronic diseases is mainly based on modern
drugs.Many studies have been performed regarding prediction of DDIs using in vitro experimental
data and those studies revealed involvement of inhibition or induction of cytochrome P450 (CYP), a
family of oxidative drug metabolizing enzymes expressed in the liver and the intestine3,4,5,6. The
specific P450 enzyme(s) involved in the biotransformation of a drug can be the defining
characteristic of its pharmacokinetic behavior, and drug interactions can occur when there is
competition
between
two
or
more
drugs
for
oxidation
by
the
same
P450
enzyme7.Thiazolidinediones (TZDs) act as agonists of peroxisome proliferator-activated receptor g
(PPAR-γ), and are powerful insulin sensitizers that have been used as anti-diabetic agents.
Pioglitazone and rosiglitazone are currently approved worldwide for the treatment of hyperglycemia
in patients with type 2 diabetes8.
The effects of pioglitazone are known to be mediated through its binding to peroxisome proliferator
activated receptor (PPAR), a member of the nuclear hormone receptor superfamily9. Pioglitazone
has significant effect on the cytochrome family enzymes10.
A large clinical study has recently shown that pioglitazone reduces the composite of all-cause
mortality, non-fatal myocardial infarction, and stroke in patients with type 2 diabetes11. Carvedilol
is a nonselective beta-adrenoceptor and selective alpha1-adrenoceptor blocker. So far, it has been
widely used in the treatment of heart failure, hypertension with or without diabetes12.As β-blockers
have been always reluctant to be used on the treatment of diabetes, because they often brought
negative effects on the glucose and lipid metabolisms. But it has been reported carvedilol had no
negative effects including insulin sensitivity besides glucose and lipid metabolisms13 14.It has been
suggested that carvedilol may provide greater benefit than traditional β-blockers in chronic heart
failure because of its antioxidant actions that synergize with its nonspecific β- and α-blocking
effects15.
After myocardial infarction carvedilol improves insulin resistance compared to metoprolol16.
So the present study is to investigate the effect of carvedilol along with pioglitazone in diabetic
rats16.
6.2 Review of Literature:
Diabetes mellitus magnifies the risk of cardiovascular morbidity and mortality. Besides the wellrecognized micro-vascular complications of diabetes, such as nephropathy and retinopathy, there is
a growing epidemic of macro-vascular complications, including diseases of coronary arteries,
peripheral arteries, and carotid vessels,Diabetes mellitus has been identified as a primary risk factor
for cardiovascular diseases and alters vascular responsiveness to several vasoconstrictors and
vasodilators . Most of the complications in diabetes are due to increased serum glucose and
increased generation of oxygen-derived free radicals, which lead to endothelium dysfunction17,18.
It has been previously reported that pioglitazone reduces oxidative stress and this may also
contribute as one of its reasons for reduction in blood pressure. In the present study pioglitazone
administration significantly reduced oxidative stress19.
A recent meta-analysis revealed beneficial effects of pioglitazone on cardiovascular disease20.
Consistent with these clinical observations, a number of experimental studies
show that pioglitazone has beneficial effects on insulin resistance21, endothelial function22,
hypertension23, and atherosclerosis24.Several experimental studies show that treatment with
pioglitazone attenuates cardiac hypertrophy and fibrosis in rats with salt-sensitive hypertension25,26.
Carvedilol, 1-[carbazole-(4)-oxy]-3-[(2-methoxy-phenoxyethyl)-amino]-2-propanol is a vasodilator
β-adrenoceptor antagonist used for the treatment of hypertension27,28. Carvedilol has been shown to
have multiple functions such as neuro-protection29-31,myocardial32,33 and endothelial protection34,35.
Preventive effects of carvedilol against smooth muscle cell proliferation have been shown 36,37.It has
also been reported that treatment with carvedilol both in vitro and in vivo increased the resistance of
isolated LDL against oxidation induced by macrophage or copper38,39.
The cytochrome P450 (CYP) enzyme super family, one of the most important drug-metabolizing
enzyme systems in humans, is responsible for the oxidative metabolism of a large number of
endogenous compounds (e.g., steroids) and xenobiotics (e.g., drugs)40. A total of 270 CYP gene
families found in various organisms-have been described to date40.The eighteen gene families that
exist in humans encode fifty-seven individual CYP genes
40
. Despite the large number of CYP
genes and enzymes, it appears that only the CYP1, CYP2, and CYP3 families of enzymes have a
major role in drug metabolism. The remaining CYP families all have essential roles in intermediary
metabolism. For example, the CYP4 family is involved in the oxidation of fatty acids,
prostaglandins, and steroids40.Pharmacokinetic and clinical issues may also occur in individuals
who are deficient in a specific P450 enzyme. For example, 5–10% of Caucasians are deficient in
CYP2D6 and 3–5% of Caucasians and 20% of Far Eastern populations lack functional CYP2C1941.
Thus, it is useful to delineate the involvement of particular P450 enzymes in metabolic pathways of
new pharmaceuticals to explain and forecast interindividual pharmacokinetic variability and help
predict drug-drug interactions as the activity of CYP enzymes has been reported to vary up to 50fold between individuals for some index metabolic reactions42.
6.3 Objective of study:
The objective of the present study is to evaluate the pharmacokinetic and pharmacodynamic drugdrug interaction of pioglitazone and carvedilol.
SPECIFIC OBJECTIVES

To arrive the therapeutic doses for carvedilol and pioglitazone in rats based on human dose.

To explore drug metabolism and drug interactions by in-vitro methods.
a) CYP inhibition studies using rat liver microsomes
b) CYP induction studies using rat liver microsomes

LC-MS/MS Method development and validation of pioglitazone and carvedilol.

To define/describe effect of drug-drug interaction on PK and PD properties of the drug
molecules

To standardize methods for estimating plasma levels of carvedilol and pioglitazone by
suitable methods.
MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from laboratory based studies by using Sprague dawley rats of either sex
weighing between 200-250 gm maintained at room temperature having free access to food (std
pellet diet), tap water ad libitum. These studies will be carried out in plasma of pretreated animals.
7.2 Method of Collection of Data:
Chemicals and reagents will be procured from standard companies. Pure samples of carvedilol and
pioglitazone will be collected from the companies manufacturing these chemicals.
Experimental animals
Laboratory bred Sprague dawley rats weighing between 200-250 g will be housed at 25° ± 5°C in a
well-ventilated animal house under 12:12 hour light and dark cycle.
Institutional Animal Ethics Committee approval for the experimental protocol will be obtained;
animals will be maintained under standard conditions in an animal house approved by CPCSEA.
Determination of in-vitro drug metabolism and in-vivo pharmacokinetic parameters:
In-vitro assays

Metabolic stability study:
Brief procedure:
1. Preparation of control and test compound solutions from 10 mM stock solutions in
DMSO.
2. Preparation of microsomes suspension (0.5mg/ml).
3. Add 1 µL of test compound or control (1.1 mM) in the reaction mixture, label as
incubation mixture.
4. Take 180 µL of aliquot from incubation mixture and label as T0, T5, T15 and T30.
5. Pre-incubate all the tubes at 37  10 C for 5 min in shaking water bath.
6. After pre-incubation add 20 µL of NADPH (10 mM) in all tubes.
7. At the end of the incubation period (0, 5, 15 and 30 min) of respective tubes, Reaction
is terminated by adding ice cold acetonitrile containing internal standard.
8. Vortex gently and centrifuge at 4000rpm for 20 min at 40C and loaded to LC-MS
system.
9. The data will be represented as % of drug metabolized based on the AUC of 0 minute
and 30 minutes of the drug incubated with rat liver microsomes; simultaneously the invitro (T1/2) will also be calculated.

CYP inhibition study:
The concentration of substrate and inhibitor used for the assay will be standardized,
based on this the concentration of our compounds (carvedilol and pioglitazone) for
the assay will be optimized.
Brief procedure:
1. Preparation of microsomes suspension.
2. Preparation of stock solutions of known CYP substrates (midazolam, bufuralol).
3. Preparation of stock solutions of known CYP inhibitors (ketaconazole, qunidine).
4. Preparation of stock solutions of compounds (carvedilol and pioglitazone).
5. Spiked, a volume of microsomes suspension, CYP substrates, CYP inhibitors. Mixed gently
by inverting and equilibrate at 370C for 5 min.
6. Spike 0.9µl test compound stock (2 & 20µM) in test tube labeled T.
7. Spike 0.9µl test compound solvent in test tube labeled test control.
8. Spike 0.9µl positive control (37µM) in test tube labeled positive control.
9. Mix the incubation mixture.
10. The reaction is intiated by adding NADPH to all samples and incubates at 370C for 10min.
11. Reaction is terminated by adding ice cold acetonitrile containing internal standard.
12. Vortex gently and centrifuge at 4000rpm for 20 min at 40C and loaded to LC-MS system.
in-vivo pharmacokinetic study:
Experimental Procedure:
1. Preparation of cannula
2. Preparation of animal for surgery
3. Cannulation
4. Conducting PK study

The animals will be weighed, marked and grouped accordingly.

Pre-dose samples will be collected from the animals to their respective pre-labeled
tubes containing anticoagulant.

The formulations will be prepared 0.5 hr prior to dosing.

For Group-1 animals, the test compounds will be administered as an IV bolus
injection slowly through tail vein.

For Group-2 animals, the test compounds will be administered through oral gavage
using a 16-G stainless steel intubation cannula.

Following dosing the animals will be observed closely for any abnormal behavior.

Blood samples (~200 μL) will be collected serially from the tail vein into prelabeled tubes, containing anticoagulant.

Plasma will be harvested from the blood samples by centrifugation at 4000 rpm for
10 min at 4 ± 2ºC and stored at -80ºC/-20ºC until analysis.
The data will be represented as % of drug metabolized based on the concentration of
0 minute and 30 minutes of the drug incubated with rat liver microsomes; simultaneously the invitro (t1/2) will also be calculated.
In-vivo pharmacokinetic data will be represented from plasma concentration-time curve from which
the parameters like Cmax (maximum concentration), Tmax (maximum time), Kel (elimination rate
constant), t1/2 (half life), CL (clearance), Vd (apparent volume of distribution) and AUC (area under
time curve) will be determined using trapezoid rule.
Pharmacodynamic interaction
The effective dose of pioglitazone and carvedilol will be selected for this interactive study. The
Sprague Dawley rats of either sex will be divided into two sets[Normal and Diabetic groups]
consisting of six animals each:
The groups may as follows:
Group I : Normal animals+Pioglitazone [10 days treatment]
Group II : Normal animals+Carvedilol [10 days treatment]
Group III : Normal animals+Pioglitazone + Carvedilol [10 days treatment]
Group IV : Diabetic animals+Pioglitazone [10 days treatment]
Group V : Diabetic animals+Carvedilol [10 days treatment]
Group VI : Diabetic animals+Pioglitazone + Carvedilol [10 days treatment]
Immediately after drug administration on 10th day 0.25 ml of blood samples will be withdrawn over
24 hrs (0, 0.5, 1, 2, 4, 8, 16 and 24 hours) by puncturing retro orbital vein under anesthesia effect
and subjected to analysis. The hypovolemia is prevented by intra-peritoneal administration of 0.25
ml of normal saline immediately after each withdrawal of blood.
Experimental model
The animals of the above group will be made insulin resistance by the injection of long acting
human insulin at 0.5 U/kg into peritoneal cavity of rats was repeated every eight hour daily1. After
15 days of insulin treatment the animals, which shows blood glucose level above 300 mg/dl or more
will be selected for the study43.
7.3 Does the study require any investigation or interventions to be conducted on patients or
the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various
parameters using rats as experimental animal model.
7.4 Has ethical clearance been obtained from your institute
Ethical Committee approval letter is enclosed.
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