Transnasal Delivery of a Gonadotropin-releasing Hormone Analog In Miniature

Swine

Mark Santos (Microbiology Major)Department of Biochemistry and Molecular Biology

Robert Gyurik, Zhengmao Li, Carl Reppucci, Scott Kavanaugh, Roger Wells and Stacia A. Sower

OutlineBackground

My Study

Results

Conclusions

Hypothalamus and Pituitary Gland

Human Brain

Vertebrate 1 2 3 4 5 6 7 8 9 10Mammal pGlu His Trp Ser Tyr Gly Leu Arg Pro GlyNH2

Guinea Pig pGlu Tyr Trp Ser Tyr Gly Val Arg Pro GlyNH2Chicken - I pGlu His Trp Ser Tyr Gly Leu Gln Pro GlyNH2

Rana pGlu His Trp Ser Tyr Gly Leu Trp Pro GlyNH2Seabream pGlu His Trp Ser Tyr Gly Leu Ser Pro GlyNH2

Salmon pGlu His Trp Ser Tyr Gly Trp Leu Pro GlyNH2Medaka pGlu His Trp Ser Phe Gly Leu Ser Pro GlyNH2Catfish pGlu His Trp Ser His Gly Leu Asn Pro GlyNH2Herring pGlu His Trp Ser His Gly Leu Ser Pro GlyNH2

Chicken -II pGlu His Trp Ser His Gly Trp Tyr Pro GlyNH2Dogfish pGlu His Trp Ser His Gly Trp Leu Pro GlyNH2

Lamprey - III pGlu His Trp Ser His Asp Trp Lys Pro GlyNH2Lamprey - I pGlu His Tyr Ser Leu Glu Trp Lys Pro GlyNH2Whitefish pGlu His Trp Ser Tyr Gly Met Asn Pro GlyNH2

Vertebrate Family of Peptides

Mihael Freamat

What is Gonadotropin-Releasing Hormone (GnRH)?

Strategy for Developing GnRH Analogs

pGlu1 - His2 - Trp3 - Ser4 - Tyr5 - Gly6 - Leu7 - Arg8 - Pro7 - Gly10NH2

Agonist(Stimulator)

Antagonist(Inhibitor)

D-Amino Acid(Especially Bulky Aromatics)

D-Amino Acid(Especially Basics)

D-Ala

EthylamideD-Amino Acid

(Especially Aromatics) NαMe-Leu

Identification of novel GnRHs and Receptors can contribute to the development of novel GnRH therapeutics.

How Is GnRH Used Medically10,000 Analogs Developed

http://perso.wanadoo.fr/pharma.rocheville/contraception_nouveautes.htm

Westside Pregnancy Resource Center

FertilizationContraceptionCancer Therapy

http://www.news-medical.net/images/breast%20cancer%20cell.jpg

Breast cancer cell

Delivery System of Insulin-2005

Drug Delivery Technology

Why Miniature Swine?Swine have been shown to have similar mucosal membrane properties

to humans making them an ideal test subject (Bollen et al., 2000).

Pig & Human Nose

Iowa State University

OutlineBackground

My Study

Results

Conclusions

ObjectiveTo determine if the GnRH analog DAla6Pro9NEt can successfully be

delivered via a transnasal application.

GnRH Complex

Biological Effect

Measure GnRH & LH

MethodsCatheter Implantation to 4 Miniature Swine

Plasma Collection for GnRH Analysis

Serum Collection for LH Analysis

GnRH Extracted From Plasma

Samples Sent to Dr. Richard Barb

Samples Assayed Using a Specific

Radioimmunoassay

Samples Assayed Using a Specific

Radioimmunoassay

Data Analyzed

Dr. Roger Wells and Van Gould

This study was completed using 4 miniature swine aged for 4 months. They were cared for according to IACUC requirements.

Animals

The TreatmentsPig 1: 0% CPE 0% GnRH Analog (Control)

Pig 2: 2% CPE 1% GnRH Analog

Pig 3: 0% CPE 1% GnRH Analog

Pig 4: 0% CPE 2% GnRH Analog

Transnasal Drug Delivery

Sampling Times

Week 1 Days 1+2

Week 2 Days 1+2

Week 1 Days 3+4

Week 2 Days 3+4

0 Hours30 Min60 Min90 Min

180 Min24 Hours

0 Hours30 Min60 Min90 Min

180 Min360 Min

24 Hours

Blood Collection

Tubes for Blood Collection

Steps for GnRH Analysis

• Centrifuge Blood• Extract

• Centrifuge• Filter

• Sep Pak• RIA

• Analysis

Centrifuge Blood

GnRH ExtractionUsing a Polytron

Centrifugation

Filtration

Separation by Sep Pak

Radioimmunoassay

Analysis

OutlineBackground

My Study

Results

Conclusions

Week 1 Days 1+2

Week 2 Days 1+2

Week 1 Days 3+4

Week 2 Days 3+4

0 Hours30 Min60 Min90 Min

180 Min24 Hours

0 Hours30 Min60 Min90 Min

180 Min360 Min

24 Hours

Sampling Times

GnRH - Week 1 Days 1 + 2

These results show a significant increase in GnRH concentration in the blood using the Bentley Formulation with GnRH

BB B B

BB

J

J

J

J

J

J

H

H

H

H

H

H

FF

F

FF

F

0 150 300 450 600 750 900 1050 1200 13500

200

400

600

800

1000

1200

1400

GnR

H p

g/0.

1ml

Sampling Time (Minutes)

B ControlJ 2%CPE 1%GnRHH 0%CPE 1%GnRHF 0%CPE 2%GnRH

Week 1 Days 1+2

GnRH - Week 1 Days 3+4

These results show a significant increase in GnRH concentration in the blood using the Bentley Formulation with GnRH

BB

BB

B B

BJ

J

J

J

J

J JH

H

H

H HH

HF

F

F

F

FF F

0 150 300 450 600 750 900 1050 1200 13500

50

100

150

200

250

GnR

H p

g/0.

1ml

Sampling Time (Minutes)

B ControlJ 2%CPE 1%GnRHH 0%CPE 1%GnRHF 0%CPE 2%GnRH

Week 1 Days 3+4

GnRH - Week 2 Days 1+2

These results show a significant increase in GnRH concentration in the blood using the Bentley Formulation with GnRH

B B B B B BJ

J

J

J

JJH

H

H

H

H H

F F FF

F F0 150 300 450 600 750 900 1050 1200 1350

0

20

40

60

80

100

120

GnR

H (P

g/0.

1ml)

Time (Minutes)

B 0% CPE 0% GnRHJ 2% CPE 1% GnRHH 0% CPE 1% GnRHF 0% CPE 2% GnRH

Week 2 Days 1 + 2

GnRH - Week 2 Days 3+4

These results show a significant increase in GnRH concentration in the blood using the Bentley Formulation with GnRH

B B B B B B BJ

JJ

J

J

JJ

H

H HH

H H H

FF

FF

F F F0 150 300 450 600 750 900 1050 1200 1350

0

50

100

150

200

250

300

350

GnR

H (P

g/0.

1ml)

Time (Minutes)

B 0% CPE 0% GnRHJ 2% CPE 1% GnRHH 0% CPE 1% GnRHF 0% CPE 2% GnRH

Week 2 Days 3 + 4

OutlineBackground

My Goal

Results

Conclusions

Summary

These studies showed that GnRH combined with the Bentley formulation

was successfully administered into the pig following transnasal application.

Implications

The results from this project can be eventually used by pharmaceutical industries

in development of drug applications.

• Dr. Stacia Sower• Scott Kavanaugh• Dr. Matt Silver and Geoff Bushold• Robert Gyurik, Zhengmao Li, and Carl

Reppucci• Dr. Roger Wells, Dean Elder, and Liz

Skovlund• Bentley Pharmaceutical Funds, UROP

Funds, and Funds From Dr. Sower’s Lab

Acknowledgements