Thermally targeted delivery of anticancer therapeutic peptides using elastin-like biopolymers

8th International Conference and Exhibition on March 07-09, 2016 Madrid, Spain Pharmaceutics & Novel Drug Delivery

Systems

Overview

• Localized tumors - current treatment

• Background of the ELP system

• Applications in cancer drug delivery

• Targeting c-Myc in breast and brain cancer

Localized Tumors - Current

Treatment

Surgical resection, followed by chemo- and/or radiotherapy

Limited by normal tissue tolerance and/or inherent tumor radio or chemo-resistance

Only a small fraction of the administered dose of drug reaches the tumor site, while the rest of the drug is distributed throughout the body

To make chemotherapy more effective and less toxic, site-specific drug delivery vehicles would increase the amount of drug that reaches the intended target and would simultaneously reduce nonselective cytotoxicity.

Targeted Drug Delivery Systems

Passive Targeting

uses the physicochemical properties (particle

diameter, hydrophilic properties, etc.) of a carrier

(transporter of the drug) to control behavior inside the

body

Active Targeting

adds special mechanisms to the passive type to

tightly control the directionality toward the target

tissue.

"missile drugs" that use carriers consisting of

combinations of ligands, e.g. antibodies, peptides,

sugar chains, etc., that have specific molecular

recognition features that can find target molecules of

certain cells that make up the target tissue

Elastin-like Polypeptide (ELP)

• Synthetic protein consisting of VPGxG repeats

• Thermally responsive

• Biologically inert

• Expressed and purified from E. coli

• Can be fused to therapeutic peptides or small molecule drugs

T < Tt T > Tt Urry, D.W. Prog. Biophys. Molec. Biol. 57:23-57 (1992).

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Temperature (ºC)

% o

f M

axim

um

OD

35

0 1 uM 5 uM

10 uM 20 uM 30 uM

37 42

ELP Expression and Purification

Cold Centrifuge

Discard pellet

T>Tt

Decant Supernatant

Warm Centrifuge

T<Tt

Start new cycle

Cel

l deb

ris

Lysa

te

PEI

pel

let

PEI

su

p.

1 c

ycle

2 c

ycle

s

3 c

ycle

s

4 c

ycle

s

Mar

ker

20

25

37

50

75

100 150 250

Heat

ELP as a Drug Delivery Vector

• Advantages of Polymeric Drug Carriers: • Passive targeting: Enhanced permeability

and retention effect. • Increased solubility and plasma half-life. • Systemic toxicity is reduced.

• Further advantages of ELP: • Active Targeting: Thermally responsive. • Genetically engineered, easy to

manipulate sequence, express in E. coli, and purify.

• Hypothesis: Systemically injected ELP will accumulate at locally heated regions, but will circulate and eventually be cleared in non-heated tissues.

• Hyperthermia can be applied in the clinical setting using high intensity focused ultrasound or radio-frequency radiation.

Heat Heat

Thermal Targeting

Tbody (37-38 ˚C) < Tt (~41 ˚C) < Thyperthermia (42-43 ˚C)

Complimentary with established advantages of:

Macromolecular Carriers

Increased solubility

Increased plasma half-life

High drug loading capacity

Hyperthermia

Increased chemo- and radiosensitivity

Increased macromolecular extravasation

Clinical Application of Hyperthermia • HIFU: High Intensity Focused Ultrasound

• HIFU technology uses a high-intensity convergent ultrasound beam generated by high power transducers to produce heat.

• As an acoustic wave propagates through the tissue, part of it is absorbed and converted to heat. With focused beams, a very small focus can be achieved deep in tissues.

MRI-guided HIFU

Therapeutic Peptides

• Advantages:

• Easy to design using “rational” strategies

• Can target nearly any protein or pathway

• Highly specific for their targets

• Disadvantages:

• Short plasma half-life

• Easily degraded in vivo

• Don’t penetrate biological membranes

• Can be immunogenic

Design of the ELP Drug Delivery Vector

Cell

Penetrating

Peptide

ELP Drug or

inhibitory

peptide

CPP Sequence

Penetratin RQIKIWFQNRRMKWKK

Tat YGRKKRRQRRR

Bac RRIRPRPPRLPRPRPRPLPFPRPG

SynB1 RGGRLSYSRRRFSTSTGR

Previous Applications of ELP for Drug Delivery in Cancer

Therapeutic Peptides Small Molecule Drugs Protein Target Peptide

Name Sequence

c-Myc H1-S6A, F8A

NELKRAFAALRDQI

p21 W10 GRKRRQTSMTDFYHSKRRLIFSKRKP

IKKβ NBD TALDWSWLQTE

p53 Peptide 46

GSRAHSSHLKSKKGQSTSRHKK

PRMT5 GRG GRGGRGGRGGRGGRGGRGGRG

Bad BH3 Bad NLWAAQRYGRELRRMSDEFVD

(mitochondrial membrane)

KLAK KLAKLAKKLAKLAK

O

O

O

OH

OO

O

HO

O

O

ONHO

O

OO

N

O

O

HN

O

N

C

Paclitaxel

Doxorubicin

C

Methotrexate

A c-Myc Inhibitory Peptide

Ras

MAP kinase Myc

Myc

Myc Myc Myc

Max

Max

Max Max Max

myc gene

myc transcript

Mitogen receptor + ligand

translation

Max Myc

odc

Cyclin D SCF

subunit E2F

ENTRY INTO S PHASE

plasma membrane

nuclear membrane

Myc Max ldh-a

In vivo Evaluation - E0771 Breast Cancer Model

• Medullary adenocarcinoma

• Isolated from a spontaneous tumor in C57BL/6 mice

• Very aggressive, and will invade the peritoneal cavity and metastasize to the lungs

Bac-ELP-H1 Tumor Uptake

Saline

No Tumor Unheated

Tumor Heated Tumor

Bac-ELP1-H1-Alexa 750

*

0.E+00

1.E+08

2.E+08

3.E+08

4.E+08

5.E+08

6.E+08

7.E+08

0 10 20 30 40 50 60 70 80

Rad

ian

t Ef

fici

ency

Time After Injection (h)

Bac-ELP1-H1

Bac-ELP1-H1 +Hyperthermia

Bidwell, G.L., Perkins, E., and Raucher, D. Cancer Letters. 2012.

Reduction of E0771 Breast Tumors

*

0

500

1000

1500

2000

2500

3000

0 5 10 15

Tum

or

Vo

lum

e (m

m3)

Days After First Injection

Saline

Saline + Hyperthermia

Bac-ELP1

Bac-ELP1-H1

Bac-ELP1-H1 +Hyperthermia

0

500

1000

1500

2000

2500

3000

3500

4000

0 5 10 15

Tum

or

Vo

lum

e (m

m3)

Days After First Injection

Saline

Bac-ELP1-H1

Bac-ELP2-H1

Bac-ELP2-H1 +Hyperthermia

Bidwell, G.L., Perkins, E., and Raucher, D. Cancer Letters. 2012.

The C6 Glioma Model

• C6 is a rat glioma model

• C6 cells were derived from methylnitrosourea-induced gliomas in Wistar rats

• C6 cells generate rapidly growing tumors when injected orthotopically in SD rats

• They mimic human glioblastoma in histology, rapid proliferation, and intracranial dissemination.

Delivery to Intracranial C6 Tumors

CPP-ELP-Rho FITC-dextran Merge EL

P1

Sy

nB

1-E

LP1

B

ac-E

LP1

Delivery to Intracranial C6 Tumors

Saline Bac-ELP1-H1 Bac-ELP2-H1 Unheated Heated Unheated Heated

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

Tumor Heart Liver Kidney Spleen Lung

Mea

n R

FU

Unheated Tumor

Heated Tumor

0.00E+00

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

Bac-ELP1-H1 Bac-ELP2-H1

Me

an R

FU

Unheated Tumor

Heated Tumor

*

A.

B.

C.

*

Inhibition of Intracranial C6 Tumor Growth

0

50

100

150

200

5 7 9 11 13 15 17 19 21 23 25

Tum

or

Vo

lum

e (

mm

3)

Days After Implantation

Saline

Saline+ Hyperthermia

Bac-ELP1-H1

Bac-ELP1-H1 + Hyperthermia

* *

Inhibition of Intracranial C6 Tumor Growth

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40

% S

urv

ival

Days After Implantation

Saline

Saline + Hyperthermia

Bac-ELP1

Bac-ELP1-H1

Bac-ELP1-H1 + Hyperthermia

*

Delivery of Doxorubicin (Dox) with ELP

• Doxorubicin is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas.

• Intercalates into DNA and induces double strand breaks by stabilizing topoisomerase II cleavage complexes.

• Acute adverse effects of doxorubicin can include nausea, vomiting, and heart arrhythmias.

• Doxorubicin's most serious adverse effect is life-threatening heart damage.

Delivery of Doxorubicin (Dox) with ELP

Dr. Kratz, CytRx Corporation,

Freiburg, Germany

Figure 1. Schematics of the ELP-based drug delivery vector. The delivery system consists of the

SynB1 cell penetrating peptide at the N-terminus, followed by the thermally responsive elastin-like

polypeptide with three terminal cysteine residues, and the thiol reactive prodrugs of doxorubicin,

paclitaxel or methotrexate,

SynB1

Cell

penetrating

peptide

Elastin-like

polypeptide

For thermal

targeting

Thiol reactive

maleimide

group

Acid-sensitive

hydrazone

linker

S

Drug

Thiol reactive

maleimide

group

Acid-sensitive

hydrazone

linker

S

Drug

Thiol reactive

maleimide

group

Acid-sensitive

hydrazone

linker

S

Drug

Prodrug

ELP CPP Dox

T<Tt T>Tt

T

T

0 2 4 6 8 100.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 50 100 150 200 250 300 350 400

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

No hyperthermia

With hyperthermia

Time post-injection (min)

Do

x p

lasm

a le

vel (

µg/

ml)

Do

x p

lasm

a le

vel (

µg/

ml)

Time post-injection (min)

0

1

2

3

4

5

Tum

or

dis

trib

uti

on

(%

ID/g

)

CPP-ELP1-Dox3 Free Dox

No hyperthermia

With hyperthermia

*

0

1

2

3

4

5

6

7

He

art

dis

trib

uti

on

(%

ID/g

)

Free Dox

No hypothermia

With hyperthermia

CPP-ELP1-Dox

ND

Free Dox Delivery of Dox with ELP

Plasma Clearance and Heart Toxicity

Tu

mo

r vo

lum

e (

mm

3)

Time post-treatment (days)

Saline no hyperthermia

Saline with hyperthermia

SynB1-ELP1-Dox3 no

hyperthermia

SynB1-ELP1-Dox3 with

hyperthermia

Dox no hyperthermia

Dox with hyperthermia

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14 16

*

* *

* *

* * *

*

Thermally targeted delivery of chemotherapeutics (Dr. Kratz)

Doxorubicin-EMC

Methotrexate-EMC

Paclitaxel-EMC

1.Moktan, S., Ryppa, C., Kratz, F., Raucher D.. A thermally responsive biopolymer conjugated to an acid sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis. Investigational New Drugs, in press.

Molecular Biology

Protein on Drug

Tissue Culture

Animal Treatment

Human Treatment

SUMMARY

Acknowledgements • Lab Members

– Dr. Gene L. Bidwell

– Shama Moktan

– Leslie Robinson

– Teuta Opacak Bernardi

– Rowshan Begum

– Rebecca Singlettery

– Maria Montano

– Ana-Matea Mikecin

• Collaborators

• Collaborators

• Dr. Eddie Perkins

• Dr. Majid Khan

• Dr. Judy James

• Dr. Jack Correia

• Dr. Michael Hebert

• Dr. Ana Levenson

• Dr. Felix Kratz

• Dr. Luis Martinez

• Dr. Lucio Miele

• Dr. Guri Tzivion

• Dr. Parminder Vig

• Collaborators

NIH, NSF, DOD, Wendy

Will Case Foundation

Future Studies and Collaborations

Delivery of chemotherapeutics

Targeting Ras Signaling Cascade

Thermally targeted therapy for prostate cancer

Targeting mutant p53 protein to increase selectivity for chemotherapeutics

Targeting Notch signaling pathway

Targeting splicing machinery components with therapeutic peptides

Biophysical characterization of an Elastin-Like- Polypeptide

Application of ELP in treatment of SCA-1

Tumor Heating with IR Light Tumor Heating with IR Light

25

27

29

31

33

35

37

39

41

43

45

0 20 40 60 80 100 120

Tem

pe

ratu

re (

⁰C)

Time (min)

Tumor Tempurature

Body Temperature

Skin Temperature

Bidwell, G.L., Perkins, E., and Raucher, D. Cancer Letters. 2012.

Bac-ELP-H1 Tumor Uptake

0

0.5

1

1.5

2

2.5

3

IV IP

% ID

/g

Unheated

Heated

0

0.5

1

1.5

2

2.5

Bac-ELP1-H1,Unheated

Bac-ELP1-H1,Heated

Bac-ELP2-H1,Heated

% ID

/g

FITC

- d

extr

an

Bac

-ELP

1-H

1-

Rh

o

Mer

ge

Unheated Heated

Bac-ELP1-H1 IP

Bac-ELP1-H1 IV

Unheated Heated

Bidwell, G.L., Perkins, E., and Raucher, D. Cancer Letters. 2012.

Bac-ELP-H1 Tumor Uptake FITC-

dextran Bac-ELP1-H1-

Rho Merge Hoescht

Un

he

ate

d

He

ate

d

Bac

-ELP

1-H

1

IP

Salin

e

Bidwell, G.L., Perkins, E., and Raucher, D. Cancer Letters. 2012.

Inhibition of GBM Cell Proliferation

0

20

40

60

80

100

120

0 10 20 30 40 50

% S

urv

ival

[Bac-ELP1-H1] (µM)

37 C

42 C

0

20

40

60

80

100

120

C6 D54 U-87 MG

% S

urv

ival

Saline Bac-ELP1-H1

37 C

42 C

* *

*

*

Heating Intracerebral GBM Tumors

35

35.5

36

36.5

37

37.5

38

38.5

39

0 20 40 60 80 100 120

Tem

per

atu

re (

⁰C)

Time (min)

Tumor Temperature

Body Temperature

Delivery to Intracranial C6 Tumors

0

20

40

60

80

100

120

140

160

180

200

ELP1 SynB1-ELP1 Tat-ELP1 Bac-ELP1

RFU

/Co

*

*

Autofl. ELP1 SynB1-ELP1 Tat-ELP1 Bac-ELP1

Delivery to Intracranial C6 Tumors

Syn

B1

-EL

P1-R

ho

B

ac-E

LP

1-R

ho

Hoescht FITC-dextran CPP-ELP-Rho Merge

Inhibition of Intracranial C6 Tumor Growth

10

1

5

18

2

2

Day

** **

Saline Bac-ELP1-H1

Bac-ELP1-H1 + Hyperthermia

Saline + Hyperthermia Bac-ELP1

Day 0 8 15

Polypeptide treatment

Imaging

9 10 18 22 11 12

0 2 4 6 8 100.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 50 100 150 200 250 300 350 400

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

No hyperthermia

With hyperthermia

Do

x p

las

ma

le

ve

l (µ

g/m

l)

Time post-injection (min) Time post-injection (min)

Do

x p

las

ma

le

ve

l (µ

g/m

l)

B

Plasma Clearance

Free Dox Delivery of Dox with ELP

Saline

Saline with hyperthermia

SynB1-ELP1-Dox

SynB1-ELP1-Dox with hyperthermia

Dox

Dox with hyperthermia

Tum

or

volu

me

(mm

3)

Day

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14 *

* *

* *

* * *

*

16

Saline

Saline with hyperthermia

SynB1-ELP1-Dox

SynB1-ELP1-Dox with hyperthermia

Dox

Dox with hyperthermia

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16

% o

f pre

trea

tmen

t w

t

Day

Tum

or

wei

ght

(g)

0.0

0.4

0.8

1.2

1.6

2.0

2.4

Saline CPP-ELP1-Dox Dox

No Hyperthermia

With Hyperthermia

*