The expanding role of liposome- based cancer nanomedicine : “Doxil” and beyond

Alberto A. Gabizon, M.D., Ph.D. Center of Nano-oncology / Shaare Zedek MC. Hebrew University-Faculty of Medicine, Jerusalem, ISRAEL

Séance: Les Nanomédicaments: d’où vient-on et où allons nous? Paris, 2019 PARIS 2010 Disclosures: - Founder & Director of Lipomedix Pharm. - Grant support form MERCK Co. - Sci.Adv.Board member of Cristal Therap. - 1964: Discovery of liposomes – Alec Bangham (Cambridge, UK)

- 1971: First in vivo applications of liposomes for drug delivery

- 1995: US-FDA approval of liposomal doxorubicin for cancer therapy

G. Gregoriadis, D. Papahadjopoulos, A. Bangham (2001) Liposomal Anti-Cancer Agents Clinically Tested Pegylated Non-Pegylated Regional therapy Non-cytotoxic DOXIL/Caelyx, Myocet (NPLD) DepoCyt Lip. MTP-PE Lipodox (PLD) (intrathecal) (Mepact) Nano- Irinotecan DaunoXome Lip. NDDP Lip. ATRA (Onivyde) (intrapleural) PROMITIL Lip. Annamycin Lip.Camptothecin BLP25 Lip. (MMC Prodrug) (aerosol) Vaccine Lip. Eribulin Marqibo (Lip. Lip. IL2 (aerosol) Lip.Nucleotides Vincristine) Lipoplatin Lurtotecan (OSI-211) Lip. E1A SPI-077 TS inhib. (OSI-7904L) (intra-tumoral)

S-CKD602 Lip. Paclitaxel FF- 10832 Dual Drug: Lip.AraC- (Gemcitabine) Dauno (Vyxeos) Targeted: αHer2-Doxil MCC-465 Consolidating Nanomedicine

Challenges and Key Considerations of the EPR Effect for Nanomedicine in Oncology Prabhakar U, et al., Cancer Research, 2013 Alliance for Nanotechnology Cancer nanomedicines - crossing the in Cancer / National Cancer translational gap Institute, USA Gabizon A, et al., Lancet, 2014 Integrating Nanotechnology into Cancer NCL: Nanotech Character. Lab Care Grodzinski P, et al., ACS Nano, 2019 “Conventional” “Leaky” “Stealth” Liposomes Liposomes Liposomes

-TUMORS-

There is a direct correlation between liposome circulation time and tumor uptake (Gabizon & Papahadjopoulos, PNAS 1988) Nanoparticle/Liposome classification system for characterization of liposome drug products

DOXIL-like liposomes

From: Hsu L and Huang J, Int J Clin Pharm Therap, 2014 Pegylated Liposomal Doxorubicin (DOXIL, Caelyx)

1. PEG Coating (Stealth Effect):  long circulation time 2. Ammonium sulfate drug loading gradient:  stability in circulation

PEG Doxorubicin Plasma Levels in Humans:

DOXIL vs. doxorubicin

90 nm 90

- 80

Hours After Infusion

Lipid Bilayer Phospholipid+Cholesterol DOXIL: doxorubicin remains in liposome- encapsulated form in circulation

25.0

10.0 Total Doxorubicin

Total Doxorubicin 2.5

1.0 Encapsulated Doxorubicin

Doxorubicin (µg/mL) Doxorubicin 0.2

0.1

0 1 2 3 4 5 6 7 Days After Infusion

Gabizon et al., Cancer Res. 1994 External medium

2x DOX-NH +Cl- Liposome 3 aqueous phase

2x NH + 3 2x DOX-NH2 2x DOX-NH2 + 2x H 2x NH Ammonia + - 3 + 2x H Cl escapes Neutral form of DOX + 2 -2 2x DOX-NH3 + (NH4) SO4 crosses bilayer + -2 2 (DOX–NH3 )2SO4 + (NH4) Cryo-TEM Rod-like precipitate (reversible) “coffee bean”

Remote Loading of Doxorubicin to form DOXIL Liposomes Cardiac Toxicity of Doxorubicin (Adriamycin)

• Limits the cumulative dose leading to early treatment discontinuation

• Irreversible, crippling and life-threatening

10/17/2019 Rationale for DOXIL: Heart vs Tumor: Liposome Tissue Access is the Key! Tumor Heart Muscle

Extracellular space

Cardiomyocytes and intercalated disks Enhanced Permeability and Retention (EPR) Extravasation and Release of Liposomal Drug in Tumor Interstitial Fluid

Tumor compartment: Interstitial fluid Tumor Cells

5

Vascular space

No Functional Lymphatics Blood Vessels: The Achyless Heel of Cancer

Extravasation of liposomes across tumor vessel: Skin-fold window in vivo model (R. Jain et al.) 111In-PLA

Mice bearing MDA-MB- 231 triple-negative Liposomes can be breast cancer tumors (10-90 mg) imaged with PET-imaged to select In111–labeled Doxil-like patients for therapy Liposomes with good targeting to in tumors.

Median tumor uptake: F. Man, A. Gabizon, ~25% ID/gram R.TM de Rosales et al. Marked Drug Deposition in Subcutaneous Tumor Implants After i.v. DOXIL Dox concentration: ~22% ID/gram tumor 30- fold increase in AUC M109 8 Human Tumor Model Skin = 0.5 µg/g

6

Tumor Halo 40 µg/g 4 86 µg/g DOXIL 2 µg Drug/gm Tumor Drug/gm µg Doxorubicin

0 50 100 150 200 Hours

Dose=20 mg/kg, i.v., 48h Gabizon et al., Vaage et al. Anti-Tumor Effect of DOXIL is >4-fold than doxorubicin (DXR)*

Values inside bars: • M109 mouse tumor % Tumor-Free Mice 800 (also observed in mouse 3LL and human N87 models) 700

600 P<0.05 Treatment given i.v. on 500 day 15 as indicated doses (mg/kg). 400 On day 36, mice 0% 17% sacrificed, tumors 300 11% dissected and weighed. 29% 200

100 Median Tumor Weight (% (% Weight Cures) Median Tumor 27% 0 Control DXR DXR DOXIL DOXIL 2.5 10 2.5 10 Gabizon et al, 2002 DOXIL Clinical Proofs of Added Value

• Cardiac function: Major reduction of cardiotoxicity as compared to free doxorubicin in all settings. (2000)

• AIDS-related Kaposi’s Sarcoma: Superior efficacy over former conventional therapy (1995)

• Recurrent Ovarian Cancer: Superior efficacy and improved safety profile over comparator drug (topotecan) (1998)

• Metastatic Breast Cancer: Equivalent efficacy and reduced cardiotoxicity compared to free doxorubicin (2003)

• Multiple Myeloma: Equivalent efficacy and improved safety profile compared to free doxorubicin combo. Superior efficacy in combination with bortezomib over single agent bortezomib. (2007) Reduced rate of cardiac events with DOXIL (vs doxorubicin)

PLD Doxorubicin, Hazard Ratio 50 mg/m2 q4w 60 mg/m2 q3w (95% CI) Cardiac 6.6% 25.7% 3.16 Events (1.58-6.31) CHF 0 5.3%

O’Brien M E R et al. Ann Oncol 2004;15:440-449 DOXIL (PLD) vs Doxorubicin: Change of toxicity profile

Side Effect Effect of liposome delivery Vesicant effect Absent (irritation only) Nausea/Vomiting Reduced Myelosuppression Reduced (no neutropenic fever) Stomatitis/Mucositis Increased Hand-Foot (PPE) Increased Cardiotoxicity Reduced or Absent Alopecia Reduced or Absent Slightly decreased Maximal Single Dose (50mg/m2 q4w) Maximal Cumulative Greatly increased Dose (>900mg/m2)

Doxil in Ovarian Ca: Major Improvement in Survival in “Pt-Sensitive” Patients*

100 DOXIL (n=109) 90 Topotecan (n=111)

80 P=.008 DOXIL 70 25.2 mths 60 50 40 30 20 Topotecan 10 15.6 mths 0 0 20 40 60 80 100 120 140

* Median survival for all patients: Weeks Since First Dose DOXIL=15mth; Topotecan=13mth (p=0.025) Gordon AN, et al. J Clin Oncol. 2001;19:3312-3322. 10/17/2019 3SP2# nozibaG tpp.37 23 Example of Response and Prolonged Remission on CAELYX™ Maintenance

1000 Relapse 6 months from ABMT

800 E.Q. 45 y.o. papillary serous Diagnosis 2/1/94 Cumulative dose=880 mg/m2

600

(U/mL) 125 125 400

CA CA New LLQ mass & liver mets 200 CAELYX™

0 Oct Dec Jun Dec Jun Dec Jun Oct 96 96 97 97 98 98 99 99 115 cycles of PLD (40mg/m2) during 9 years with stable disease: No Cardiac Toxicity! This is >10 times more than the maximal recommended dose of free Doxorubicin Caelyx in 1st Line Ovarian Cancer

No significant advantage of Doxil-based combo over Paclitaxel based combo! Doxil® (PLD) vs free doxorubicin (1st line) in Metastatic Breast Cancer No sig efficacy advantage of Doxil over free doxorubicin!

Progression-free Survival Overall Survival

O’Brien et al., Ann Oncol 2004 The DOXIL-Nanomedicine Efficacy Gap: Preclinical Results  Clinical results 

• EPR in human cancer limited or even insignificant. • Dose translation from animals to humans may differ for nanomeds. • Liposomes activate macrophages to enhance tumor growth and blunting the therapeutic advantage in humans. • Drug release rate from liposomes in the tumor bed is suboptimal in humans. • Poor clinical study design.

Despite its great safety, DOXIL has not been approved for 1st Line or Adjuvant/Curative Settings EPR in human cancer: present but variable iGamma Scintigraphy after Injection of [DTPA-In111] Stealth Liposomes

Kaposi Sarcoma

Lung Tumor

Liver, Spleen

Spleen

Bone Marrow

Harrington et al., Clin. Cancer Res. 2001 Posterior view 48h Controlling for the variability of EPR effect in Human Cancer: Real time imaging for Patient Selection

Grozinski et al., ACS Nano 2019 Imaging of EPR to predict Efficacy (X-ray enhancement = measure of EPR)

Control

Low tumor Treatment enhancement with DOXIL High tumor enhancement

Karathanasis et al., Radiology 2009 EPR imaging in pancreas tumor patients Tumor MRI signal and liposomal drug activity correlate!

Patient EPR effect stratification possible: Higher PET/CT signal corresponds with more favorable treatment outcome. Muco-cutaneous toxicity of DOXIL is the result of slower clearance upon retreatment with DOXIL

Clearance (Dose/AUC)

27.5 n.s. (p=0.56) p=0.0003 25.0 AUC Increase of 40% from 1st

SEM) 22.5 rd

 to 3 cycle 20.0 17.5 15.0 12.5 10.0 7.5 5.0

Clearance, mL/hourClearance, (Mean 2.5 0.0 30 mg/m2 60 mg/m2 1st Cycle 2nd Cycle 3rd Cycle

Gabizon et al., CCP 2007 Grenader and Gabizon, J Clin Oncol 2010 Effect of Dose: Increase in tumor accumulation of Doxil and saturation of liver uptake

Liver M109 Tumor

20 160

140

15 120 Free DXR 100 DOXIL

10 80

60

5 DOXIL 40

g doxorubicin-equiv. / g doxorubicin-equiv. g

g doxorubicin-equiv. / g doxorubicin-equiv. g

  20 Free DXR

0 0 0 5 10 15 20 0 5 10 15 20 Dose mg/kg Dose mg/kg

Hypothesis: Giving a high initial loading dose followed by a lower maintenance dose will increase efficacy without increase of toxicity Liposome-induced tumor growth enhancement is macrophage-dependent and can be abrogated by liposomal Alendronate (PLA) but not by free Alen.

1 2 0 0 L IP O )

3 1 0 0 0 L IP O + A le n m

m V e h ic le ( 8 0 0

e P L A

m u

l 6 0 0 * **

o

V

r 4 0 0

o m

u 2 0 0 T

0

3 6 9 2 5 7 9 3 6 8 1 1 1 1 2 2 2

T im e P o s t T u m o r Im p la n t (d a y s )

La-Beck NM, Rajan R, Wood LM, Gabizon AA, et al. (manuscript under revision) Dept. of Immunotherapeutics & Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center Nitrogen-containing Bisphosphonates

alendronate

Clezardin, P. et al. Cancer Res 2005;65:4971-4974 Rationale for combining bisphosphonates with doxorubicin in the same liposome

• Double attack on tumor cells and tumor-infiltrating macrophages • Different MoA’s, Non-overlapping toxicities • Immunological anti-tumor effects by gamma-delta T lymphocytes • Co-delivery ensures timely co-exposure to both agents • Bisphosphonates ammonium slats enable remote Dox Loading

Bisphosphonate Doxorubicin PLAD (Pegylated liposomal alendronate of doxorubicin) I. Passive Encapsulation: Ammonium ALD II. Active Loading: DOX Alendronic acid (ALD) Doxorubicin (DOX) HCl

+ - DOX-NH3Cl DOX-NH3 +Cl

+ Liposome aqueous DOX-NH2 +H phase Liposome aqueous

phase DOX-NH2

.. DOX-NH + + (NH )ALD NH4OH + ALD H ( NH4) ALD + H2O 3 4 pH 6.0 + DOX -NH3 –ALD NH3+ H HSPC: cholesterol: PEG2000 DSPE (Precipitate) 55:40:5 mole ratio External medium NH3

Shmeeda et al., J Drug Targ 2016 Alendronate Liposomal PLAD vs DOXIL

 More potent activator of the inflammasome pathway than PLD, leading to 40-fold greater secretion of IL-1b, and other cytokines

 Similar PK and comparable tumor PLAD drug levels  Greater therapeutic efficacy in immuno-competent mouse tumor model

PLAD appears to be a synergistic

DOXIL dual drug liposome formulation for chemo-immunotherapy of cancer

Shmeeda et al., J. Drug Targ. 2016 Promitil®, a “smart” nanomedicine for cancer chemotherapy and chemo-radiotherapy . Delivers Mitomycin-C Lipidic Prodrug (MLP)

Promitil® has a long circulation time and exploits the abnormal blood vessels of tumors for selective delivery of MLP to tumors by the EPR effect. Under clinical testing for gastro-intestinal cancer 39 Promitil Clinical Development

 Phase 1A/1B completd (88 patients) demonstrating Stealth PK, lower toxicity than MMC, and anti-tumor activity

 Randomized Phase 2B-3 study: Promitil vs. Regorafenib or other in metastatic Colo-rectal Ca to obtain proof of value

 Phase 2A study: Explore the clinical activity of Promitil in combination with radiotherapy, as radiosensitizer for multiple cancer types (ongoing). Challenges associated with translating the anticancer efficacy of carrier- mediated drugs from preclinical studies to clinical trials

Optimal Dose?

Drug Comparator? Acknowledgments: Collaborators: • Nano-oncology SZMC Lab: • Irene La-Beck, PhD (Abilene, TX) • Andrew Wang, MD (UNC)  Hilary Shmeeda PhD • Samuel Zalipsky, PhD (San Francisco)  Yasmine Amitay PhD • Franco Muggia, MD (NYU)  Yogita Patil, PhD • Nano Charact. Lab (NCI) • John Maher MD, Ana Parente, PhD,  Jenny Gorin, M.Sc. Rafael TM Rosales, PhD (KCL, London)  Lidia Mak • Thomas Andresen, PhD (DTU, DK)  Dina Tzemach • Yechezkel Barenholz PhD (HU, J-lem) • Lipomedix: Patricia Ohana, PhD

Liposome Community Ramon Crater (Israel)

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