ROLE OF GROWTH FACTORS IN DIABETIC FOOT ULCER

MANAGEMENT

DR ARUN BALPRESIDENT

DIABETIC FOOT SOCIETY OF INDIA

Moist Wound Dressing• It prevents tissue dehydration

(preserving the viability and proliferative potential).

• Increases breakdown of dead tissue and fibrin contributing to autolytic debridement.

• Potentiates interaction of growth factors with their target cells

• Reduces the incidence of infection.

• Associated with less pain.

HEMOSTASIS 1 hour

WOUNDING

PlateletsFibrin

INFLAMMATION days 1 through 7Proteoglycans

NeutrophilsMacrophages]

LymphocytesPROLIFERATION days 2 through 20

Normal wound healing

Fibroblasts[produce growth factors]

CollagenEpithelial Cells

Endothelial Cells

REMODELING 1 week to 6 months

Collagen Fibril Cross linking

Scar Maturation

Time from injury

Molecular environment of wounds

Healing woundsHigh Mitogenic activityLow inflammatory cytokinesLow proteasesMitotically competent cells

Chronic UlcersLow Mitogenic activityHigh inflammatory cytokinesHigh proteases

WOUND BED PREPARATION

• DYNAMIC & RAPIDLY EVOLVING CONCEPT• REDUCES THE WOUND HEALING TIME• REDUCES HE COST OF THE TREATMENT• TIME FRAMEWORK• T:TISSUE MANAGEMENT• I : INFECTION/INFLAMMATION CONTROL• M:MOISTURE CONTROL• E:EPITHELIAL(EDGE)ADVANCEMENT• DIFFERENCE BETWEEN SHARP & SURGICAL

DEBRIDEMENT

GROWTH FACTORS

VITAL FACTOR FOR WOUND HEALING

Growth Factors• Basically signal proteins released from

local tissues that activate target cells to replicate or migrate.

• Control wound healing by acting in a paracrine, endocrine or autocrine mode.

• Action of growth factors is complex and carefully controlled during the normal wound healing process.

• Growth factors at appropriate concentration are mitogenic to their target cells

Role of various growth factors

in wound healing

Growth factors

Growth factors of significance in wound healing are: Platelet Derived Growth Factor (PDGF) Vascular Endothelial Growth Factor (VEGF) Transforming Growth Factor- β (TGF- β) Keratinocyte Growth Factor (KGF) Epidermal Growth Factor (EGF)

Growth Factors• Basically signal proteins released from

local tissues that activate target cells to replicate or migrate.

• Control wound healing by acting in a paracrine, endocrine or autocrine mode.

• Action of growth factors is complex and carefully controlled during the normal wound healing process.

• Growth factors at appropriate concentration are mitogenic to their target cells

Growth Factor Characteristics Location

EGF Mitogenic for epithelial cellsAlmost all body (Epidermal Growth Fibroblasts, Endothelial cells

fluids \Plateletsfactor) Angiogenesis

Directs Epithelialisation Stimulates fibroblast collegenase secretionContributes to the scarless repair

KGF Potent mediator for kerotinocytesFibroblasts(Keratinocyte Monocyte maturationgrowth factor)

PDGF Mitogenic for fibroblasts Endothelial cell(Platelet derived Potentiates VEGF productionPlateletsgrowth factor) Monocyte maturationMacrophages

FGF Mitogenic for epithelial cells Fibroblasts(Fibroblast not fibroblasts or endothelialEndothelial cellgrowth factor) cells

TGF Inhibits mitogenesis of most Macrophages(Transforming cells Eosinophilsgrowth factor)

IGF Mitogenic for fibroblasts Fibroblasts (Insulin Endothelial cell Endothelial cellgrowth factor)

TGF-β

PDGF-BB

VEGF

TGF-β

VEGF

PDGF-BBTGF-β

IGF

TGF-βKGF

TGF-α

Multiple Growth Factors are Expressed Temporally in Human Wound Fluid

PDGF

bFGF

VEGF

TGF-

Cause of reduced expression of growth factors & receptors

in Diabetic Foot Ulcers

Diabetic foot lesion

•Repeated trauma

•Cell detritus/cell-fragments

• Increased & Prolonged inflammatory response

• Increased invasion of macrophages & neutrophiles

Increased activation of macrophages by cytokines and growth factors

TNF-α; IL-1β

Fibroblasts & inflammatory cells

Serin-proteases MMPs TIMPsDegradation of growth growth factors & receptorsfactors & receptors

Diabetologia. 2002 Jul;45(7):1011-6. Epub 2002 May 25

Chronification of Diabetic foot ulcers

The only growth factors available commercially, for the treatment of

diabetic foot ulcers is

1.recombinant human Platelet Derived Growth Factor (rhPDGF) 2.Epidermal growth Factor

Biology• Platelet Derived Growth Factor is structurally a 25 kDa

protein

• It is released during stages of wound healing by Platelets Macrophages Fibroblasts Endothelial cells Keratinocytes

PDGF and Receptors• PDGF represents a

family of growth factors consisting of 2 poly peptide chains (A & B) which forms the dimer (protein pairs).

PDGF-AA

PDGF-AB

PDGF-BB

• The PDGF receptor has a trans-membrane structure with extracellular ligand-binding domains and intracellular tyrosine kinase domains.

• Two PDGF receptors exist, R-α and R-β, with each possessing different specificities for their ligands.

• PDGF-BB can activate any PDGF receptor; therefore its therapeutic application can activate any configurations of it’s receptors.

• The proliferation rate of wound fibroblasts was higher by PDGF-BB than PDGF-AB and -AA.*

* Biochem Biophys Res Commun. 1995 Apr 17;209(2):393-9.

rhPDGF-BB

• Recombinant human Platelet Derived Growth Factor (rhPDGF-BB) is a homo-dimer produced by Recombinant DNA technology by inserting the human gene of PDGF-BB in E. coli.

• Can also be produced with Yeast (Saccharomyces cerevisiae).

• The biological activity of rhPDGF-BB is similar to that of naturally occurring PDGF.

Structure of rhPDGF-BB

Structure of PDGF-BB Monomers

(depicted in green and blue)

Role of PDGF-BB in wound healing process

Expert Opin.Biol Ther. (2002) 2(2):211-218

PDGF is released by Platelets & Macrophages

PDGF is released by Fibroblasts

* Adapted from 1.Supplement of Podiatry Today, October 2003. 2. Diabetes Care, vol, no.2, no.2, 17-23. 4.Diabetes Care August 1999, 22:8;1,354-60

PDGF is released by Endothelial Cells & Keratinocytes

PDGF Action at Cellular Level

Angiogenesis with PDGF-BB1) Binds to its receptor on

vascular endothelial cell.

4) Stimulates endothelial proliferation

2) Inducing production of other growth factors (VEGF)

3) Activates intracellular signal transduction pathways

5) Promotes endothelial migration

7) Recruits smooth muscle cells and pericytes to stabilize the newly formed vasculature

6) Facilitates vascular tube formation

1

23

4

5

6

7

Histological Evidence of Angiogenesis with PDGF

2nd week

4th week

Control PDGF

Summary of MOA of PDGF-BB

• Chemoattraction for neutrophils, monocytes & fibroblasts.

• Mitogenic for smooth muscle cells and fibroblasts.

• Stimulates endothelial cells proliferation & migration.

• Promotes vascular tube formation.

• Stimulates epithelialization.

• Induces the release of other growth factors.

Toxicology studies*

• Acute & chronic toxicity studies were performed on various animals showing no abnormal findings.

• Reproductive & teratogenic studies were not performed.

• Mutagenic studies were negative.

• In pharmacokinetic studies the level measured in blood samples were below the baseline even after repeated application.

*http://www.fda.gov/cder/biologics/products/becaomj121697.htm

Experimental evidence*• These results suggest that the levels of various growth

factors, particularly PDGF, may be limiting at wound sites and supplementation of wounds with these factors can accelerate the rate of new tissue formation.

Acceleration of new tissue in growth by PDGF (in rats). At day 10

*J Clin Invest. 1985 December; 76(6): 2323–2329

Control PDGF

30 & 100 μg/cm2

2.2 μg/cm2

1 & 3 μg

1,10

Dose (daily)

P=0.007(for 100 μg)

Diabetic382

Wieman et al.PDGF-BB

P=0.01Diabetic118

Steed et al.PDGF-BB

N.S.Pressure45

Mustoe et al.PDGF-BB

N.S.Pressure20

Robson et al.PDGF-BB

Results Target wound typeNo. of subjects

AuthorsGrowth Factor

Dosage Evaluation

N.S. = Not Significant

Double-blind, placebo controlled trials of PDGF

Pharmacokinetics• rhPDGF-BB 0.01% gel has biological activity similar to

endogenous PDGF and has a high affinity for PDGF-β receptor

• Daily application of rhPDGF-BB has only negligible systemic absorption.

• It is present in biological fluid for at least 12 hours in vivo and 24 hours in vitro

rhPDGF Experience

Complete healing after 11.5 weeks of GWC and rhPDGF1

After debridement, GWC and rhPDGF initiated1

1. www.regranex.com

IndicationIndicated for the treatment of lower extremity diabetic neuropathic ulcers* that extend into the subcutaneous tissue or beyond and have an adequate blood supply,

*(Stage III & Stage IV ulcers IAET Staging classification)

Good wound care & tight blood glucose control are very important in rhPDGF-BB therapy.

Contra-indications• rhPDGF-BB gel should not be used in patients with:

Known hypersensitivity to any component of the excipients used in the product.

Known neoplasm(s) at the site(s) of application.

Dose calculation The intended dose is around 7μg /cm² of ulcer per day for topical application. (For an average man of around 50kg weight)

Tube Size Formula7. 5g/15 g tube [Length (cm) X Width (cm) ÷ 4]*

*Greatest length of the ulcer multiplied with greatest width

Method of application• One tube for one patient

• Proper hand wash before application.

• Tip of the tube should not come into contact with the ulcer or any other surface.

• Before each application, the ulcer should be gently rinsed with saline or water to remove any residual gel and wound area cleaned.

• Applied once a day in a carefully measured quantity adjusted according to the size of ulcers.

• Calculated dose of gel should be squeezed out onto a clean, firm, non-absorbable surface (e.g. wax paper) in a linear fashion.

• Use clean application aid (Cotton swab, tongue depressor, etc )

• Measured quantity of gel should be spread evenly over the ulcerated area including the margins to yield a thin continuous layer of ~ 1.5 mm thickness.

• Gel should be covered with saline moistened gauze.

• Then gel can be gently rinsed off using saline or water and reapplied.

• Tube should be closed tightly after each use.

• After treatment is completed, any unused gel should be discarded.

(Contd…)

Algorithm for use of platelet derived growth factor (PDGF) in treatment of diabetic foot ulcer

Algorithm for use of platelet derived growth factor (PDGF) in treatment of diabetic foot ulcer (contd)

Epidermal Growth Factor

• EGF is a single chain polypeptide

• Comprising of 53 amino acids with Mol Wt of 6200 Daltons

Epidermal Growth Factor

• A cell proliferate growth factor

• Has wide role in cell stimulation – Cellular components activation – Has high interaction with plasma

proteins.

• Effective mediator that – Enhances cell locomotion – Contractility – Differentiation

All of which effect wound and wound healing process.

Mode of action

• EGF induces cellular proliferation through the EGF receptor, which has a tyrosine kinase cytoplasmic domain - a single transmembrane domain

• Binding of EGF results in EGF receptor activation, autophosphorylation of the receptor, and tyrosine phosphorylation of other proteins.

• EGF receptor activates various proteins, ultimately causing phosphorylation that contributes to proliferation.

EGF Signal Pathways

Role of EGF• Mitogenic for epithelial cells,

Fibroblasts, Endothelial cells

• Angiogenesis

• Directs Epithelialisation

• Stimulates fibroblast collegenase secretion

• Contributes to the scarless repair

Role of EGF• Potent mitogen for many types of cells1 • Shown a greater efficacy in ulcer healing2

• Attracts cells into the wound and stimulates their proliferation3

• Stimulates the rate of formation of granulation tissue4

• Enhances wound healing and increases collagen production5

1. Acta Endocrinol (Copenh). 1990 Sep; 123(3):326-30, 2. Irn J Endocrinol Metab 2003: Vol 5 No 2, 3. Journal of Trauma-Injury Infection & Critical Care 4(1):159-167, July 1996. 4. J Surg

Res. 1987 Oct: 43 (4): 322-8, 5. Biochem J. 1987 Oct 15; 24792:385-8

Multi centric, Phase III, Double Blind

• Product Developer: Bharat Biotech International Ltd

• Centers : 5

• Approved Indications» Diabetic foot ulcers» Donor site Skin Grafts» Ist & IInd Degree Burns

Regen -D (Epidermal Growth Factor) Diabetic Foot Ulcer Trial summary

• Total no. of patients - 57– Group 1 (EGF) - 29– Group 2 (Control) - 28

Group 1 Group 2 p value

No. of patients 29 28M / F 22 / 7 20 / 8

Mean age (in yrs) 58 1.9 59 2.0Ulcer size (in cm2) 13.3 ± 3.1 12.5± 2.2 No of Healed cases

at 10 weeks 20 6

No of Healed casesat 15 weeks 25 14 < 0.01

Mean healing time (days) 56.8 ± 4.7 81± 4.4 < 0.01

Week, w

Perc

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f pat

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cure

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k w

282420161284

100

80

60

40

20

0

Figure-5: Efficacy of Regen-D Gel

ControlTest

The healing takes place before the end of the 8th week in 50% of the patients under test; and it takes nearly 15 weeks to cure 50% of the patients under control.

Case no 8 : Healed in 8 weeksSource : Dr.Vijay Viswhanathan , MVJ center for Diabetes, Chennai

1st stage II nd stage

III rd stage Final stage

DFU Indication

GROWTH FACTORS• NEED TO USE SELECTIVELY• NOT USEFUL IN PVD• SHOULD BE USED ONLY WITH NORMAL SALINE• NEED TO BE USED DAILY• SHOULD BE USED ONLY IF WOUND HEALING

IS DELAYED BEYOND 3 WEEKS• STRICT OFF LOADING,GLYCEMIC

CONTROL,NUTRITIONAL CONTROL IS IMPORTANT

• NOT USEFUL WITH SLOUGH,DISCHARGE & INFECTION

PDGF IN DIABETIC FOOT PDGF IN DIABETIC FOOT WOUNDSWOUNDS

10DAYS AFTER PDGF USE

15 DAYS AFTER PDGF USE

25 DAYS AFTER PDGF

17 DAYS AFTER PDGF USE

BEFORE TREATMENT

8 DAYS AFTER PDGF

15 DAYS AFTER PDGF

FOOT ULCER FORMATION IS BEGINNING OF THE END

THANK THANK YOUYOU