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Hsp90 Inhibitor Drug Conjugates (HDCs): Payloads and Possibilities
#260
Dinesh Chimmanamada, David A. Proia, Patricia E. Rao, Dan Zhou, Junyi Zhang, Sami Osman, Teresa Przewloka, Jun Jiang, Genliang Lu, Dharma Vutukuri, Shoujun Chen, John Chu, Takayo Inoue,
Luisa Shin Ogawa, Ritu Singh, Noriaki Tatsuta, Jaime Aquaviva, Suqin He, Donald Smith, Jim Sang, Manual Sequeira, Yuan Liu, Josephine Ye, Andrew Sonderfan, Christian Cortis and Weiwen Ying
Synta Pharmaceuticals, Lexington, Massachusetts USA
Abstract
Retention with Minimal Hsp90 Inhibition:
Additional HDC Payloads
Multitargeted Kinase Inhibitors
Other potential HDCs--PARP Inhibitors
HDCs can be constructed in two ways: 1) keeping the Hsp90 inhibitor as a delivery vehicle or 2) using the
Multitargeted kinase inhibitors such as staurosporine or sunitinib are very potent anticancer compounds.
PARP1/2 enzymes play key roles in DNA repair pathways where they are recruited and bound at the site
Hsp90 inhibitor for synergistic or combinatorial PD effect. STA-12-8666 demonstrates the concept using
Due to their non-selectivity, they are also found to be toxic. Though the drug staurosporine is known for
of damage then further catalyze the synthesis of poly(ADP-ribose) to mediate DNA repair. Cancer cells
Hsp90 binding for delivery with minimal induction of Hsp90 inhibitor pharmacological effects.
decades, a clinical application was not possible due to the toxicity. Using the HDC mechanism, drugs of
may be dependent on these enzymes, and can therefore be targeted by their inhibitors. Hsp90 inhibitors
these kind can selectively be targeted to tumors which in turn can act on multiple pathways.
are also known to exhibit anti-PARP activity through mitochondrial membrane depolarization leading to
Hsp90 Binders vs Non-binders
Background: Despite the emergence of various therapies, chemotherapy still plays a significant role in the treatment of
BODIPY Probes: Active vs Inactive
cancer. Though toxic, the potency and broad applicability of chemotherapeutic drugs make compelling rationale for using
Inactive
conjugate
BODIPY-FL
them as payloads in drug delivery systems. Several recent technologies, especially antibody drug conjugates (ADC) have
Active
conjugate
been very successful, however for narrow sets of indications. We have recently disclosed the Hsp90 inhibitor Drug
Conjugate (HDC) platform, which utilizes the unique pharmacokinetic property of Hsp90 inhibitors, where the drug is
veh
Results: In this presentation, the proof-of-concept studies, the rationales for payload selection/applicability, preliminary
assessments and data for the most advanced candidate are discussed. Two types of HDCs can be conceived: 1) Hsp90
STA-12-8663
SN-38
STA-12-8663
+ SN-38
STA-12-8666
10 100 1000 10 100 1000
10 100 1000
apoptosis in SCLC cells. Due to the intrinsic properties of these PARP inhibitors and known synergistic
10 100 1000
veh
selectively retained in tumor in high concentrations while clearing from plasma and normal tissues relatively quickly.
effect of PARP and Hsp90 inhibition, a strong case can be made for the synthesis of a PARP inhibitor HDC,
where a selective and prolonged exposure of the drug in tumor can increase the therapeutic index and
inhibitor is used strictly as a delivery vehicle where the antitumor activity is achieved by payload only. 2) Antitumor effect
Hsp70
is obtained by the action of both Hsp90 inhibitor and the payload. Therefore, many highly potent small molecule cytotoxic
broaden the application.
HSP90 stabilizes mutant BRCA1: RR-1, RR-5, and RR-6 cells
were treated with vehicle (marked as “V”) or 50 nM 17DMAG (marked as “D”) in the presence of vehicle (marked as
“V”) or 100 nM rucaparib (marked as “R”), and colony
formation was assessed (n = 3,mean ± SEM of colonies
formed relative to vehicle + vehicle-treated cells). [RR =
MDA-MB-436+WT cells]
agents and those that synergize Hsp90 inhibitors qualify as payloads. Some examples are: taxanes, proteasome inhibitors,
camptothecins, antimetabolites, cell cycle inhibitors (CDKs) and pan-PI3K inhibitors etc.
CDC2
A lead SN-38 HDC for which detailed evaluations has been done showed that the payload is released in tumor for a
prolonged period of time (0.22uM and 0.38uM at 24h and72h respectively in tumor against 30nM at 24h and no drug
quantified at 72h for irinotecan, at their maximum tolerated doses (MTDs). In vivo efficacy in multiple tumor models also
GAPDH
exhibited remarkable activity for the HDC compared to irinotecan. Toxicological evaluations also revealed that the HDC is
safer than irinotecan at their efficacious doses.
(RT112 bladder cancer cells, 24 hr, conc. unit nM)
Conclusion: The HDC platform has the potential to utilize a number of commonly used small molecule anticancer agents
At 100 nM STA-12-8666 binds Hsp90
allowing HDC retention in tumor cells
as payloads; expanding applicability through improved efficacy and toxicity profiles. It also has the potential to resuscitate
the development of drugs that were abandoned due to toxicity and lack of efficacy.
BODIPY-FL
HDC Concept
BODIPY-FL
At doses / concentrations tested, enhanced antitumor effect of STA-12-8666 is attributable to payload
(SN-38) retention – no significant contribution from Hsp90 client degradation
Asp93
Asp93
Many small molecule inhibitors of Hsp90, including ganetespib, are retained in tumors longer than in
Inactive conjugate
blood and normal tissues. This property has been exploited for novel tumor imaging strategies in
Pemetrexed -fragment HDC: Active vs Inactive forms
patients. The same phenomenon can be exploited for the selective delivery of anticancer payloads to
STA-12-8666 is broadly efficacious
Active conjugate
In resistant pancreatic PDX model
This pair represents the molecular size and physical properties which minimizes chances for non-specific
retention. This pair represents a design that can be achieved for most small molecule drugs. Depending
tumors.
Hsp90i
PAYLOAD
LINKER
1000nM, the concentration needed to suppress
Hsp90 clients; 10x higher than the
concentration required for retention
Irinotecan (50mg/kg)
Vehicle
Active vs inactive
STA-12-8666 (150mg/kg)
Vehicle
Irinotecan
Inactive form: 100mg/kg
STA-12-8666: 100mg/kg
on the payload, the Hsp90 inhibitor fragment can be manipulated to generate an optimal HDC.
HDC
HER2 deg.
(IC50, nM)
Cytotoxicity
(BT474, nM)
HDC
Staurosporine
HDC1
HER2 deg.
(IC50, nM)
1,464
22
Sunitinib HDC1
1,386
Staurosporine
HDC2
59
27
Sunitinib HDC2
916
Additional HDC payloads--Proteosome Inhibitors
HDCs of CDK, Aurora Kinase, Eg5, PLK Inhibitors
The pharmacological inhibition of the ubiquitin–proteasome system has been established as an effective
Regulators of cell cycle such as CDKs, Aurora kinases, etc. have long been considered good targets for
anticancer strategy. However, the approved drugs in the class, namely, bortezomib and carfilzomib, are
anticancer therapy. However, due to their intrinsic on-target toxicity and lack of selectivity to tumor cells,
limited to hematological malignancies due to their poor pharmacokinetic properties. On-target cardio-
clinical attempts have been met with dismal results. Recent encouraging data from CDK4/6 inhibitor
toxicity also limits the doses of the drugs that can be given. An HDC of these drugs can not only stabilize
palbociclib in BRCA mutant breast cancer validates these regulators as good anticancer targets. HDCs of
these pharmacokinetically inferior drugs, but also help bring in a larger quantity of the drug to the solid
the inhibitors of these cell cycle inhibitors can not only improve the selectivity of the payload to tumor
tumors with selective prolonged exposure.
cells but also synergize with Hsp90 inhibitors.
Source: Fox Chase Cancer Center
(carfilzomib HDC1)
Tumor targeting arm
Cleavable linker
Anticancer agent
Preferential retention in
tumor cells
Optimized for controlled release
of payload in cancer cells;
multiple cleavage modes
Chemotherapies, targeted
agents; activity can be
masked ex-tumor
Source: Shapiro et. al., PNAS, 2013, 110, 17041-17046
HER2 (IC50)…..
77nM
Hsp90a
Binding (Kd)…..
0.6nM
Cytotoxicity
(BT474, 72h)…..
<10nM
enzymes are strong clients of Hsp90, which are
Proteosome inhibition
(WB, IC50)…..
0.47mM
Plasma stability
(% remaining in 1h)
Fig: Complex of Hsp90-CDC37-CDK4: CDK4/6
100%
activated by their complexation to the chaperone.
This proximity of the target to the payload can also
be utilized without the released drug being lost in
Arrows indicate conjugation sites on carfilzomib:
various linkers of varying strength can be used to
attach an appropriate Hsp90 inhibitor
the cellular components or pumped out.
Source: Vaughan et. al.; Molecular Cell, 2006,23, 697-707
Days of treatment
HDC
MW
500-1,500
Target
Intracellular Hsp90
Payloads
Most small molecule anticancer agents
Delivery of payload
Passive diffusion, active transporter
Tissue distribution data for pemetrexed-fragment HDC and its inactive form in a NSCLC xenograft model dosed at 50mg/kg
STA-12-8666, an SN-38 HDC
Tissue distribution of STA-12-8666 and its
components in H1975 tumor bearing mice.
STA-12-8666 was dosed at 150mg/kg
mM
Ganetespib vs its Inactive form
2.8nM
HER2 degradation
in BT-474 (EC50)
790nM
Molecular Weight
880
Vehicle
Irinotecan, 50mg/kg
S TSTA-12-8666,
A - 8 6 6 6 ( 1 0 0 m g /k g100mg/kg
)
1000
30
800
600
400
< 0.0001
Upon cleavage, STA-12-8666 produces the payload SN-38 and the Hsp90 binder STA-12-8663. In the same tissue
distribution experiment, irinotecan dosed at 60mg/kg retained 30nM at 24h and no quantifiable drug was found at 72h in
tumor.
20
0.5nM
>1000nM
5nM
>10,000nM
Tumor bearing mice (SCID female H1975 NSCLC).
Both ganetespib and inactive form were dosed at
50mg/kg once.
0.05
400
350
300
250
200
150
100
50
0
STA-12-8666
STA-12-8666 +
10uM CsA
0
10
20
hours
EORTC-NCI-AACR Symposium, Barcelona, November 18-21, 2014
0.06
30
Peak Area Ratio
>50,000nM
Entry of STA-12-8666 into H1975 cells
with or without cyclosporine (CsA)
nM in cell extract
Hsp90a
binding Kd:
Cytotoxicity:
(IC50, BT474):
30nM
exhibit anticancer activities. IDO1 which is responsible for the oxidation of tryptophan to kynurenine,
offer anchors (functional groups) for conjugation. The strength of Hsp90 inhibition and durability of the
when inhibited in tumor cells, restores the proliferation and activation of various immune cells, which are
linkage can be manipulated depending on the biology of the payload and intended payload release.
known to be suppressed in many cancers. An HDC of an IDO1 inhibitor may selectively decrease the
0.04
0.03
Uptake of STA-12-8666 by OATP1B1
SSTA-12-8666,
T A -8 6 6 6 (1 0 0 m g100mg/kg
/k g )
Irinotecan,
Irin
o te c a n (5 0 m g50mg/kg
/k g )
A few HDCs of known potent IDO inhibitors have been synthesized. Preliminary in vitro evaluations have
****
VVehicle
e h ic le
**
20
shown that such HDCs do inhibit cellular kynurenine production. Synthesis of optimized HDCs and further
ns
ns
****
evaluations are underway.
*
0
Other synthesized HDCs
Alkylating agents
bendamustine, temozolomide
Anthracyclines
doxorubicin
Antimetabolites
5-FU, pemetrexed, gemcitabine
Epigenetic modifiers
vorinostat , panobinostat
Hormonal therapy
fulvestrant, abiraterone
Microtubule stabilizers
docetaxel, paclitaxel
Platinums
carboplatin
Tyrosine kinase inhibitors
sunitinib, sorafenib
***
25
30
35
40
45
50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Synta has comprehensive intellectual property on various payloads including PARPi, proteosome inhibitors, pankinase inhibitors, cytotoxics, immunotherapeutics and many other molecularly targeted agents.
T im e (D a y s )
¼ Day
1 Day
3 Days
5 Days
7 Days
10 Days
15 Days
Summary
Vehicle
OATP1B1
Empty Vector
•
STA-12-8666
100 mg/kg
HDC is a unique idea utilizing a small molecule Hsp90 inhibitor as a targeting vehicle for the delivery of another small
molecule anticancer agent.
•
Irinotecan
50 mg/kg
Drugs that are rapidly pumped out of cells, losing potency, can be efficiently used as payloads in HDC, greatly
improving intracellular retention.
0.02
0.01
Category
10
D a y s a fte r M D A - M B - 2 3 1 c e ll im p la n ta tio n
Cell entry appears to be mainly via passive diffusion:
HER2 (IC50)
As mentioned in the abstract, an HDC can be created with any small molecule anticancer agent that can
0
0
INACTIVE
Immunomodulating agents such as indoleamine 2,3-dioxygenase (IDO1) inhibitors have been shown to
Ir in o te c a n ( 5 0 m g /k g )
200
ACTIVE
Synta HDC Platform
V e h ic le
3
Hsp90a binding
(Kd)
1200
(m m )
Drug Properties
A v e r a g e tu m o r v o lu m e
Hsp90 Binders are Retained in Tumors
Prolonged SN-38 activity seen following a single dose
Additional HDC payloads—IDO inhibitors
kynurenine production in tumor cells and dendritic cells (DC) that overexpress IDO1.
Concentration (mM)
Achieved payload concentration
STA-12-8666 has been tested in more than a dozen models where it has been shown to be
superior to irinotecan at the equivalent doses and at their respective MTDs
%  H 2 A X + / T o t a l N u c le a r A r e a
Features
-H2AX (20X)
Toxicological evaluations in rats and dogs have shown that STA-12-8666 is well tolerated. A canine Ph1 trial is underway
and the molecule is being considered for clinical development.
Figure: Inhibition of kynurenine production in HeLa cells by an IDO1i –HDC, and a
commercially available IDO1i. Ganetespib is a strong Hsp90 inhibitor.
•
Prolonged selective exposure of the payload by the HDC eliminates the need for frequent dosing, which may reduce
toxicity.
•
HDCs can improve the potency of a payload, making mildly potent payloads more effective.
For further information on HDC go to: www.syntapharma.com