INJECTABLE ELIXIR

TREATMENT BY CHEATING THE

BODYAgapito, Bacunot, Ballesteros Ibana, Manuel, Mascarenas, Valdevieso

Cagayan State University- CMS

A. Overview: Metabolic Disorder• Type 2 Diabetes- “insulin resistance”

- accumulation of glucose in the blood rather than being used as energy

- carbohydrates glucose X enter cell (insulin)

- symptoms: frequent urination, thirst, weakness, drowsiness and blurred vision

PREVENTION: early detection and regular check-ups

Damages and Risk Factors• Small (micro) blood vessels - Vision problem, nerve damage, kidney

damage• Large blood vessels - Stroke, heart disease, poor circulation

RISK FACTORS: OVERWEIGHT/OBESITY (metabolic disorder topic 2); sedentary lifestyle/ lack of exercise, family history,

DRUGS: corticosteroids, thiazide diuretics, mental illness drugs, HIV treatment drugsKaty Perry

Tests• Fasting Blood Glucose test

• Hemoglobin A1c

• Oral glucose tolerance test

Goal of type 2 diabetes treatment

KEEP BLOOD SUGAR IN NORMAL RANGE

normal ranges:

90-130 mg/dL fasting blood glucose

HbA1c of 6.5-7%

2-hour post meal <180mg/dL

MAIN and established treatment of type 2 diabetes: Diet and exercise

• Improving diet

• Exercising regularly

- decrease resistance of cells to action of insulin

-easier for glucose to enter cell

- NO ASSOCIATED WEIGHT LOSS

• MEDICATIONS: most common: metformin, sulfonylureas (but HYPOGLYCEMIA)

Currently available Medications and Treatments (video)

• Overtime drugs cannot help anymore

• In cases of obesity (usually seen in type 2 diabetes), the drugs mentioned cannot totally help and healthy normal lifestyle cannot be fully restored, thus studies/ approaches would like to pave their way to cure both diabetes and obesity at the same in one drug

Journal Report (January 2015)We would like to acknowledge:

Dr. Brian Finan, Ph.D.Group Leader

Division of Drug Discovery

Institute for Diabetes and Obesity (IDO)

Helmholtz Zentrum Munich

Business Campus Garching-Hochbruck

Message for the group: “ Best of Luck”

Dr. Matthias H TschöpInstitute for Diabetes and Obesity, Helmholtz Zentrum München,

German Research Center for Environmental Health, Neuherberg, Germany

.Department of Medicine, Division of Metabolic Diseases,Technische Universität München, Munich, Germany.

Dr. Richard D DiMarchi, Ph.D Department of Chemistry, Indiana University, Bloomington, Indiana, USA. 4Marcadia

Message for the group:

” I am forwarding to you the requested information.

I wish you great success with your education.”

B. INTRODUCTION

• Researchers in Germany

Combines action of 3 GI hormones to:

(The objectives of their study)

a.Improve glucose sensitivity

b.Calorie burning

c. while reducing apetite

Motivations /Issues• Previous works

• Other drugs are also triagonist, but ARE NOT STABLE

• Hence, first study to come up with a stable engineered peptides for cure of diabetes while reducing

weight by about 30 %

Kim Kardashian

The study: preclinical

• Previous studies: engineered peptides affecting GLP-1 and GIP (released by body after you eat to stabilize sugar level) in cases of type II diabetes (where this hormones are disrupted hypoglycemia)

• This new study: added another hormone GLUCAGON to GLP-1 and GIP in a protein

-glucagon acts very different from the two other hormones

- glucagon raises blood sugar levels by CONVERTING FAT to SUGAR

(Nsikan Akpan)

* When the drug was formed ,the protein was able to bind to and activate the cell receptors for these three hormones in order to produce an anti-obesity response (Finan and colleagues)

Jennifer Lawrence

“ As hyperglycemia progresses in diabetes, the body shuts the hormone off in

PRESUMED attempt to restore sugar balance, but that also stymies fat burning. In normal people, glucagon lowers fat and can

trigger weight loss”

(Nsikan Akpan)

Emma Watson

Significance• Triple hormone effect in a single molecule=

metabolic control centers are influenced: pancreas, liver, fat depots and brain (Finan)

• Body weight was reduced by a third, on average, and the blood glucose levels are halved (in 3-weeks for mice)

• Fat mass reduced by about 30%, but healthy lean mass was left (Akpan)

• If this can get ready for human trials can revolutionize how to treat obesity

Katy Perry

WHY RATS?•rats, dogs, cats, pig, monkeys•It is hard to study humans

-> ethical issuesInterspecies similarities and differences (Inagi, 1998)

* But all animals SHOULD be treated properly, even though they are subjected to studies.

Methodology

PEPTIDE SYNTHESIS

TWO TYPES

1.) Boc-based neutralization peptide synthesis

2.) Fmoc-based neutalization peptide synthesis

BOC-BASED NEUTRALIZATION PEPTIDE SYNTHESIS

0.2 mmol 4 – methylbenzhydrylamine (MBHA) resin used on highly modified Applied Biosystems 430A peptide synthesizer

DEPBT/DIEA for coupling

TFA for deprotection of amino-terminal amines

Peptide resinsPeptide resins

Hydrofluoric acid/ p-cresol (10: 0.5 vol/vol) for 1 hr with agitation

Hydrofluoric acid removed in vacuo

Cleaved and deprotected peptide

Precipitated in diethyl ether

FMOC-BASED NEUTALIZATION PEPTIDE

SYNTHESIS 0.1 mmol Rink 4 – methylbenzhydralamine

resin (Novabiochem) used on Applied Biosystems 433A peptide synthesizer by standard Fmoc methods

DIC/Cl- HOBt for coupling

20% piperidine/ dimethylformamide (DMF) for deprotection of N-terminal amines

Completed peptide resintreated

TFA/TIS/Anisole (9: 0.5: 0.5 vol/vol/vol) for 2 hrs with agitation

Removed ether

Crude peptide dissolved in:Aqueous buffer

20% acetonitrile (ACN)1% acetic acid (AcOH)

Lyophilization

Electrospray Ionization (ESI) or Matrix-assisted laser desorption/ionization time-of-light (MALDI-TOF) mass spectrometry•Confirms peptide molecular weight

Analytical reversed-phase HPLC in 0.1 % TFA with an ACN gradient•Confirms character

CELL LINES All are confirmed to be mycoplasma free and

tested on monthly basis.

General experimental approaches for in vivo

pharmacological experiments

group size of eight is optimal for in vivo evaluation

Smaller group size for genetically modified animals due to insufficient numbers

LEAN AND OBESE MICE

•randomized into treatment groups based on body weight, body fat/lean mass and blood glucose levels

•Housed on a 12h/12h light-dark cycle at 22 C with free access to food and water⁰

DIABETIC RODENTS

•randomized into treatment groups based on body weight, body fat/lean mass and blood glucose levels

DIO MICE

• Fed with diabetogenic diet at two months of age

• Housed on a 12h/12h light-dark cycle at 22 C with free access to food and water⁰

• Maintained for a minimum of 16 weeks before initiation of pharmacological studies between 6-18 months old

Rodent pharmacological and metabolism studies.

Body composition measurements

• Subcutaneal injections during light phase

• Body weight• Food intake

• Body composition measurements (fat and lean mass)

• echoMRI

Blood parameters

• Plasma insulin• Plasma FGF21, adiponectin, GIP,

GLP-1, and glucagon • Plasma cholesterol, triglycerides, ALT,

and AST • Plasma free fatty acid

• 6h of fasting• 1.5g per kg for DIO 2g per kg for lean

mice• Glucometer (0 min and at 15, 30, 60,

and 120 min after injection. )• AccuCheck glucometer

• 6 h of fasting• 0.75 units of insulin per kg body weight• handheld glucometer• (0 min and at 15, 30, 60, and 120 min

after injection

Glucose tolerance test

Insulin tolerance test

Statistical analyses

Acute glucose tolerance test with GLP-1R , GIPR and

GcgR antagonist

• 6-h fasted male DIO mice (n = 8 per group; age 9 months

•Pre treted with anatgonist

• triagonist 1 nmol per kg body weight)

•glucose via intraperitoneal injections

•handheld glucometer (−30, −15, 0, 15, 30, 60, and 120 min after the glucose administration.)

• one way or two way ANOVA

RESULTS

A. Effect of triagonist vs other treatments on Body Weight

Triagonist>GLP-1/GIP coagonist>GLP-1>glucagon>GIP>vehicle

B. Effects of triagonist vs other treatments on cumulative food

intake

Triagonist=GLP-1/GIP coagonist>GLP-1>GIP>glucagon=vehicle

C. Effect of triagonist vs other treatments in the blood glucose

level

Triagonist>GLP-1/GIP coagonist>GLP-1=GIP>vehicle>glucagon

Discovery of a unimolecular, balanced, high-potency

GLP-1/GIP/glucagon selective triagonist

• Challenges:a. Maintaining the individual affinity of each

ligand to its receptor

b. Eliminating the structural element that convey selective preference for each individual receptor

c. Introducing GIP agonism without destroying GLP-1 and GcG receptors

TheTriagonist: its featuresA. Aminoisobutyric acid @ pos 2

• convey resistance to dipeptidyl peptidase IV–mediated degradation and inactivation

• contribute to mixed agonism @ GLP-1R and GIPR9 but is detrimental to glucagon activity

Glu16, Arg17, Gln20, Leu27 and Asp28 was included

B. Site-specifically lipidated Lys 10 with palmitic acid through y-carboxylase spacer

• Enhance time action• Acyl moeity promotes albumin binding while also

supporting mixed agonism

TheTriagonist: its features

C. C-terminal–extended residues from exendin-4

• A reptilian-derived GLP-1 paralog, which results in a single molecule of 39 residues

• shows superior solubility, potency and balance at each of the three receptors

Can it be EFFECTIVE to Human?

• Triagonist has a similar activity profile (based on cyclic AMP (cAMP) induction) across all three constituent receptors originating from mice, rats and cynomolgus monkeys

is suitable for in vivo

pharmacological and characterization studies across different species in preclinical studies

The triagonist: dilemma

• Possibility that the peptide may bind to additional targets screened at over 70 different receptor targets

in high-throughput competitive binding assays

displayed no cross-reactive binding to any of the other screened receptors

highly specific for GLP-1R, GIPR and GcgR

Unimolecular triagonist possesses activity attributed to each targeted

receptor1. In mouse pancreatic beta cells (express

GLP-1R)• increased cAMP production confirming full GLP-1R

activity

2. In mouse adipocytes (express GIPR)• cAMP production was induced

3. In rat hepatocytes (express GcgR)• increased cAMP production

Triagonist activity at each cognate receptor

1. To confirm presence of GLP-1 activity• Mice pretreated with validated GLP-1R antagonist• Pretreatment ameliorated the improved glucose

tolerance observed with the triagonist alone

2. To confirm presence of GIP activity• Mice pretreated with validated GIP antagonist• antagonist blunted the improvement observed

with the triagonist alone

3. To confirm presence of GcgR activity• streptozotocin-treated mice pretreated with a

GcgR antagonist• inhibited the acute, transient hyperglycemic effect

otherwise observed with the triagonist alone

Metabolic benefits of Triagonist• It was the only compound to markedly improve glucose

tolerance and reduce food intake without inducing hypoglycemia

• Glucagon component contributes to the substantial weight-lowering efficacy (decreased BW by 26.6%) at low doses

• Generate the loss of fat without lean mass without influence in gastric emptying

• Insulin sensitivity• Increase plasma concentration of fibroblast growth factor• Decrease plasma concentration of cholesterol to a great

extent• Pronounced effect in lowering hepatic lipid content

Safeness of chronic treatment of triagonist

• Induced a dose dependent decrease in blood glucose without an observable hypoglycemia at any doses

• Did not reduce lean mass only fat mass

• Do not cause irreversible damage to cells

• The superior body weight loss induced may limit the dose escalation in translational and toxicological studies

Metabolic Efficacy of GLP-1 component of the Triagonist

EFFECTS ON BODY WEIGHT CHANGEHigh-fat diet-fed (HFD) Glp1r-1- Mice vs.

Wild-type HFD mice

The contribution of GLP-1 activity to the full efficacy of the triagonist underscore the necessity of integrated GLP-1 activity to minimize the diabetogenic risk of chronic and maximal GcgR agonism

Contribution of the GIP component of the triagonist

EFFECTS ON BODY WEIGHT CHANGEHigh-fat diet-fed (HFD) Glp1r-1- Mice vs.

HFD Gipr-1-1 mice

RESULT

The integrated GIP activity within the triagonist contributes to the glucose-lowering effect and helps buffer against the hyperglycaemic effect of glucagon activity, albeit to a lesser degree than GLP-1

Improved energy metabolism benefits of tri-agonism depend on GcgR signaling

EFFECTS ON BODY WEIGHT CHANGEHigh-fat diet-fed (HFD) Glp1r-1- Mice vs.

Wild-type HFD mice

RESULT

The triagonist decreased body weight by 7.7% and lowered fat mass in wild-type mice, yet had no measurable effect on body weight loss in HFD Ggcgr-1- mice.

Likewise, the triagonist lost its anorectic efficacy and lowering effect on fasted blood glucose, and failed to improve glucose tolerance in these HFD Gcgr−/−, which may be partially attributed to the existing hypoglycemia and inherent protection from glucose intolerance of Gcgr−/− mice27

GcgR Signaling component of the triagonist DOES NOT exacerbate pre-existing

hyperglycemia

Effects of Triagonist in an Insulin-Resistant Obese Mice

Prevented excessive weight gain Cumulative food intake was not altered Preserved proper islet architecture

Effects of Triagonist in Diabetic Fatty Rats Using Dose-dependent approach

Improved body weight

Fasting blood glucose with a rapid onset and sustained efficacy

Improved glucose tolerance and hemoglobin A1C

Preserved proper Islet cytoarchitecture

Optimal Metabolic Benefits of Triple Agonism predominantly Depend on Fine-

Tuning the Glucagon Component

GLUCAGON ACTIVITY

Modification of the Third Amino acid – GLP-1 and GIP were insensitive to changes

Substitution of Methionine Sulfoxide(MET (O)3) resulted in analog with 5% of initial glucagon character, and glutamic substitution resulted in negligible glucagon activity

Fine-Tuning of glucagon: Effect on metabolic and glycemic efficacies

The maximal dose of the glucagon analog alone increased

blood glucose, but this rise was prevented when supplemented with

the dual incretin coagonist, even at a relative ratio as little as one-

third to one-tenth of the glucagon dose

Extreme Glucagon Dosing

Tested the capacity of dual incretin coagonism to prevent the glucagon-mediated rise in blood glucose. Co-administration of escalated doses of Acylated Glucagon analog with a low dose the GLP-1 and GIP co-agonist

RESULT

Dual Incretin components counterbalance the potential diabetogenic liabilty of excessive GcgR agonism which is something that seemed more fragile when using just GLP-1 to buffer glucagon action.

Discussion

• Here the study explored the chemical capability of combining agonism at the glucagon, GLP-1 and GIP receptors into a single molecule, as well as the synergistic efficacy of this concerted triple agonism to reverse perturbed metabolism in rodent models of obesity and diabetes. As a first-degree proof of principle, it was simulated in vivo triple agonism by in situ co-administration of a glucagon analog as a second molecule with the validated GLP-1/GIP coagonist. This adjunctive triple agonism amplified the metabolic efficacy of the dual incretin in obese mice, thus establishing the pharmacological foundation to pursue the uncertain creation of a unimolecular triagonist with glucagon, GLP-1 and GIP activities.

• The design of the triagonist was inspired by previous observations in the discovery process of the mixed coagonists, as well as by the established sequence differences among the three endogenous hormones. The goal was to maintain high activity across all three receptors yet eliminate the inherent high selectivity resident in the native hormones.

. •

• this single-peptide triagonist is a hybridized peptide, not simply a conjugate multimer of the native ligands, and it features a single receptor-binding face. This conveys concerted yet independent and promiscuous agonism at each constituent receptor without cross-reactivity at other related G protein–coupled receptors, essentially serving as a master key to unlock signaling at each individual receptor.

• It was demonstrate here that this unimolecular triagonist potently reverses diet-induced obesity and prevents diabetes progression in rodent models to a greater extent than reciprocal coagonism at the individual receptors.

• Through a series of imbalanced triagonists with a step-wise selective blunting of glucagon activity, the study also demonstrated that glucagon activity that is aligned with GLP-1 and GIP activity is necessary for the maximal weight loss induced by the triagonist, but dampening the glucagon activity can promote better glycemic outcomes, albeit with less weight loss.

• Notably, the glucagon activity can be selectively fine-tuned with minimal structural or chemical change (a single amino acid change at position 3), providing the opportunity for a more personalized medicinal approach to obesity therapy that reflects the heterogeneous nature of the human condition, such as for Prader-Willi syndrome, neurodegenerative diseases or nondiabetes liver diseases associated with excessive fat deposition.

• Ultimately, the triagonist represents a sizable step forward beyond previous attempts of coagonism and reflects the growing notion that single-molecule polytherapies are emerging as the gold standard for obesity and diabetes medicines.

SUMMARY

Current/Existing Medication for Diabetes- Individually boost the levels of these hormones (GLP-1, GIP and Glucagon),

These drugs- limited effect on obesity and diabetes

• Dr. Matthias Tschop- Endocrinologist

- protein that takes structural pieces of GLP-1, GIP and Glucagon

-merge them into a single molecule

• The peptide- rationally designed– Fully potent– Balanced– Triple agonist

Results of the study- “Unparalled”

-compared to earlier test

-3 hormones alone

-together as co-agonist

Lab Mice

3 Weeks

-Body Weight reduced by 1/3

-Blood glucose levels reduced by 1/2

3 hormone- SPECIFICALLY and EQUALLY target three receptors

-GLP-1

-GIP

-Glucagon

-NO cross-reactive binding to any other receptors (over 70 screened recptors).

Benefits and Safety of the Triagonist

•Improve glucose tolerance and reduce food intake without Hypoglycemia

•Decrease body weight by 26.6%- Low dose

•Loss of Fat without reduction of Lean Mass

•No observable hypoglycemic effect @ any dose

•No irreversible damage to cells

•Superior body weight loss- limit dose escalation in translational and toxicological studies

This Study- VERY SIGNIFICANT

Revolutionise Obesity Treatment (diabetes type 2 treatment as well)

Loosing WeightObese People Healthy People=

Effectiveness to Humans

-similar activity profile (cAMP induction)

-3 receptors from mice, rats and cyronolgous monkey

-suitable for in vivo pharmacological and characterization studies across different species in pre-clinical studies

What’s next?Pre-Clinical TrialsClinical TrialsMarket

U.S. Food and Drugs Authority, 2014

THANK YOU FOR LISTENING!?

Dr. Brian Finan, Ph.D.

Dr. Matthias H Tschöp,Ph.D

Dr. Richard D DiMarchi, Ph.D

•Inagi, Katsuhide. 1998. An Anatomical Study of the rat Larynx: establishing the Rat Model for Neuromuscular Function.Sage Journals. Retrieved from http://oto. sagepub. com/content /118 /1/74.short (July 27, 2013)•Science Alert•U.S. Food and Drugs Authority. 2014. How Drugs are Developed and Approved. http://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/•Youtube: https://www.youtube.com/watch?v=Imc2spQR3dy

REFERENCES:

•Bayat, et al. (2013). The effects of education based on extended health belief model in type 2 diabetic patients: a randomized controlled trial. Journal of Diabetes and Metabolic Disorders. doi: 10.1186/2251-6581-12-45 (March 12, 2015).

•Day, Caroline. (2001). The rising tide of type 2 diabetes. Sage Journal: The British journal of Diabetes and Cardiovascular disease. doi: 10.1177/14746514010010010601 (March 12, 2015).

•Finan, et al. (2015). A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nature Medicine. doi:10.1038/nm.3761

•Hall, John. (2011). Guyton and Hall textbook of Medical Physiology.12 th ed. Elsevier. Philadelphia.

•Murray, et al. (2009). Harper’s Illustrated Biochemistry. 28th ed. Mc Graw Hill.China

•Pratley, Richard. (2013). The early treatment of type 2 diabetes. The American Journal of Medicine. doi: http://dx.doi.org/10.1016/j.amjmed.2013.06.007 (March 14, 2015).

•Crew, B. (2014). New anti-obesity can cut body weight by 30%. Science Alert. http://www.sciencealert.com/new-anti-obesity-drug-can-cut-body-weight-by-30 (Januray 8, 2015)

•Zhou, et al. (2013). Lifetime direct medical cost of treating type 2 diabetes and diabetic complications. American Journal of Preventive Medicine. http://dx.doi.org/10.1016/j.amepre.2013.04.017 (March 14, 2015).

•Video source:

•Understanding Type 2 Diabetes. (2014). Prime Media Inc. Retrieved from: https://www.youtube.com/watch?v=Imc2spQR3dY or www.AnimatedDiabetesPationt.com