‘COMMERCIAL FEASIBILITY OF THE MULTICOMPONENT NANOCHAIN
THERAPY’
By
CHAITANYA GOLLAKOTA
Submitted in partial fulfillment of the requirements
For the degree of Masters of Science
Thesis Adviser: Dr. Christopher Cullis
DEPARTMENT OF BIOLOGY
CASE WESTERN RESERVE UNIVERSITY
AUGUST 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Chaitanya Gollakota candidate for Master of Science
(Signed) Ed Caner
Dr. Christopher Cullis
Dr. Emmit Jolly
Dr. Roy Ritzmann
(Date) 07/06/2016
Acknowledgements
My thesis project and my graduate degree program would have been a lot less exciting and stressful without the following people:
My heartfelt thanks to Drs. Efstathios Karathanasis and Mark Griswold for letting me work on their area of research. Special thanks to Dr. Karathanasis for pushing me to the limit during the development of my thesis.
A huge thanks to Drs. Christopher Cullis and Emmit Jolly for instilling in me a lot of confidence during the course of my graduate degree program, and during the development of my thesis. Thank you Dr. Cullis, who was my thesis advisor, for being patient with me.
I cannot thank Ed Caner and Dr. Roy Ritzmann enough for joining my thesis defense committee with a very limited notice of time.
A special thanks to Bruce Terry and the rest of the STEP program faculty for all the fantastic mentorship I received.
The following people from the industry helped me shape my thesis by giving wonderful insights
• Suzette Dutch - Triathlon Medical Ventures • Neema Mayhugh - Wave Strategy LLC • Dr. Andrew Sloan - University Hospitals • Nicole Brey - Cleveland Clinic • Drs. Saunthararajah, M.D. Yogen and Velcheti, M.D. Vamsidhar - Cleveland Clinic • Raghuram Selvaraju - Rodman & Renshaw
And last but never the least, my parents - Jayashree Gollakota and Satyanarayana Gollakota. I don’t think any of this would have been possible without their support.
Special Thanks - James Brown. Your songs made my lonely days of writing this thesis much better.
Contents
Technology Highlights 1 Product Overview 6 Metastatic Breast Cancer 6 Matrix Metalloproteinase (MMPs) 12 Loss of Cell Adhesion is a Crucial Factor in Metastatic Cancers 13 Tumor Angiogenesis: 14 Epithelial-Mesenchymal Transition (EMT) 15 Metastatic Microenvironment 16 Organ Selection 17 Tiny Particles 18 Introduction to Nanotechnology 18 Evolution of Nanotechnology 19 First Generation Nanoparticles 20 Adagen 20 Zoladex 20 Interaction between the Complement System and the Nanoparticles 21 Second Generation Nanoparticles: 22 Doxil 23 Lipodex 24 Myocet 24 Third Generation Nanoparticles 25 Limitation with the EPR Effect 25 Factors Affecting the Uptake of NPs into Cancer Cells 26 Size and Charge 26 Encapsulating Liposome 27 Nanochains 27 Targeting Ligand 28 Role of Integrins in Metastasis: 29 TECHNOLOGY LANDSCAPE 30 Issues with Targeting Integrins/ Associated Ligands 30 Number of Receptors on the Cancer Cells vs. Normal Cells 30 Externally Modulated Release Mechanism 30 Magnetic Field Modulation: 32 ThermaDox 33 Results of Nanochain 34 Shape of the Nanoparticle: Extremely Crucial Factor 34 RGD Ligand Targeting 35 RGD Ligand Targeting causes Inflammation 36 Single Therapy vs. Combination Therapy 37 Advantages of a Combination Therapy 37 Designing Drug Combinations 38 Surface Modification of Nanoparticles 38 Circulation Time 39
Surface Charge 39 Clinical Trial Strategy 40 Market 42 Jobs To Be Done 43 Product Evolution 43 Cost 43 Hospital Costs 44 Function 45 Convenience 45 Reliability 45 Cancer Survival 46 Research Strategies 46 Co-Innovators 46 Incremental Innovation 47 Nanochain: Benefits to Costs 48 Competition 49 Glucose Inhibitors 49 p53 Pathway Activation 50 Monoclonal Antibodies (mAbs) 52 Market: 53 A Few Promising Products: 54 Keytruda: PD-1: Major Threat: Promising against TNMBC 55 Nanochain’s Opportunity 58 Pembrolizumab: Disease Indications 61 Avastin: 61 Liposomal Particles 64 BBB Therapeutics 64 Mebiopharm 65 Polymer-based Therapeutics 66 Engene IC 67 Immunoliposomes 69 Popular methods of making liposomes: 71 Anti-EGFR 75 Anti-RON Receptor Tyrosine Kinase: 76 Future Threats and Recommendations 79 A case of targeting multiple integrin-mediated pathways 79 Efficacy: 81 Competition in the Triple Negative Metastatic Breast Cancer Market 82 Problem with Metastatic Cancers: Multidrug resistance 86 Severity of the Disease: 87 Approved Therapies for TNBC: 89 Appendix 93 References 107
List of Tables:
Circulation times for various nanoparticles used in the nanochain therapy 39 Results of Glufosfamide 49 Results of Synergene’s Product 52 Results of an anti-EGFR-ILs-DOX developed by the University Hospital Basel 55 Results of Keytruda’s performance against Triple negative metastatic breast cancers 56 Results of 2B3-101 65 Results of MBP-426 66 Results of BIND’s trial 67 Results of Engene IC 68 Results of Intetuamab 79 Results from a Combination Therapy Trial 86
List of Figures
Missing receptors on triple negative breast cancer cells 2 Pipeline of Nanochain Therapy 3 Five-year Relative Survival Rates* (%) by Stage at Diagnosis, US, 2005-2011 4 Ras oncogene function in tumor metastasis 7 Son of Sevenless Pathway 9 Ras Protein Downstream Pathway 10 Rho Family of Proteins 11 Loss of Cell Adhesion a Major Cause for Metastasis 13 Increase in the number of nanoparticles developed during the past decade 19 Source: NIH Reporter 19 1 - Shows the nanoparticle engulfed by the phagocyte 2 - Shows a coated nanoparticle reaching its target cell 22 EPR Effect 23 Doxil 24 RGD amino acid sequence 28 Integrin pathway in tumor development 29 RGD amino acid binding sites 37 Structure of a positively charged nanoparticle, 40 Expired Patent of Docetaxel 47 A schematic representing the role of the p53 pathway in tumor invasion 51 Kaplan Meier Statistical Analysis of the Clinical Trial 60 Immunoliposome sketch 69 Receptor-mediated endocytosis 70 Various types of Immunoliposomal formulation 72
Commercial Feasibility of the Multicomponent Nanochain Therapy
Abstract by
CHAITANYA GOLLAKOTA
Abstract: One of the most dangerous forms of cancers are metastatic cancers for which there is no comprehensive treatment regimen. Drs. Karathanasis and Griswold at Case
Western Reserve University are developing a targeted cancer therapy called nanochain that accumulates exclusively near metastatic tumor sites delivering docetaxel and doxorubicin. In a recent animal model, the anti-cancer agent demonstrated close to 6% accumulation rates in an animal model. This thesis conducts a comprehensive analysis on the commercial feasibility of the nanochain product and comes to a conclusion that the product needs to prove that it can improve the overall survivability of metastatic patients compared to its competition like monoclonal antibodies and kinase pathway inhibitors.
This thesis suggests that it will be a good commercial strategy to conjugate the liposomal portion of the nanochain with a novel molecule like a monoclonal antibody (mAb).
Technology Highlights
• Market Area: According to the American Cancer Institute, close to 15 million
Americans were living with a history of cancer as of January 1, 2014. In the year 2016, about 1.7 million new cancer cases are expected to be diagnosed. What is even more dangerous is the fact that close to 600,000 Americans are projected to die of cancer in the year 2016. Cancer follows cardiac-related diseases in the number of fatalities each year.
There is an unmet need to treat tumor before it takes the lives of more people. A drug delivery platform developed by biomedical researchers at Case Western Reserve
University claiming to have a higher therapeutic index than therapeutic agents of this class Drs. Efstathios Karathanasis and Mark Griswold are fighting a compelling battle
against cancer using % of Cancer Deaths by Various Cancer Types in the Year 2016 a multicomponent Source: American Cancer Society 2% 3% 9% 6% nanoparticle called Breast Cancer 2% 0% 4% Nanochain. One of 1% Colorectal cancer 34% 15% 5% the therapeutic areas 5% Kidney Cancer 9% of focus is triple 6% 3% 11% negative metastatic breast cancer, a form of cancer without any comprehensive treatment. Approximately
40,000 deaths are expected to occur in the year 2016 due to triple negative breast cancer indicating that there is a need to develop an anti-cancer agent for this orphan disease class.
• The name ‘triple negative’ comes from the fact that these forms of tumor cells do not have receptors for estrogen, progesterone, and Human Epidermal Growth Factor
1 Receptor 2(HER2). Developing drugs against this type of cancer has been a challenge to
the scientific community due to the absence of these receptors. Herceptin is a triple
positive metastatic breast tumor-targeting monoclonal antibody. Trastuzumab, the drug’s
generic name, accumulated close to $7 billion in the year 2013, targeting all the three
receptors overexpressed on cancer cells - Her2, estrogen, and progesterone.
Figure: Missing receptors on triple negative breast cancer cells
Estrogen
Progesterone
HER2
2 Nanochain: A Pipeline in a Single Product: The product comprises of three iron oxide particles, a liposome, and a targeting entity on the surface. Nanochain targets a severe unmet need by accumulating precisely close to triple negative metastatic breast cancer sites inside the body. The product is also being tested for glioblastoma multiforme in a recent preclinical trial. The market for this therapeutic area is expected to grow by more than 10% over the next years and is predicted to reach $1 billion by the year 2022.
Metastatic tumors release several proteins during angiogenesis helping in the development of targeted therapeutics against different forms of cancer.
nanochain pipeline
Triple Negative Metastatic Breast Cancers
Potential Market : 30,000 patients per year
Glioblastoma Multiforme Potential Market: US $1 B by 2022
Cancers expressing integrins on the surface
Figure: Pipeline of Nanochain Therapy
•Controlled Delivery - Reducing the Amount of Dosage: Treatment of metastatic breast cancers is a major area of concern due to the lack of three main receptors, usually present in most forms of cancers - estrogen, progesterone, and HER2 oncogene.
Traditional chemotherapy is the standard of care for metastatic triple negative breast
3 cancer patients and has proved to be ineffective. It lacks the ability to accumulate specifically near cancer cells and is the cause for the lethal effects of cancer chemotherapy. Nanochain’s iron-oxide particles have a crucial function in drug delivery.
The research team is using a low power magnetic field (10kHz) that oscillates the iron oxide particles, in turn disrupting the liposome. In this way, a controlled mechanism is achieved enabling the team to deliver drugs at the best accumulation rate near metastatic tumor sites achieving accumulation rates close to 10-20 fold more than the standard care of treatment (chemotherapy). The survival rates of women with metastatic breast cancers are at a very low 26% when compared to 99% at local sites.
Figure: Five-year Relative Survival Rates* (%) by Stage at Diagnosis, US, 2005-2011 (Source: Cancer Facts and Figures 2016 - American Cancer Society)
Combination Therapy: One more advantage of this delivery system is that it is delivering two chemotherapeutics in one therapeutic cycle. Various research papers elucidated the efficiency of a combination therapy vs. the use of a single chemotherapeutic agent. The development of drug resistance by the tumor cells hampers the efficient delivery of a single therapeutic agent. Combination therapy has resulted in
4 better efficacy and overall survivability in patients with metastatic cancers. More details regarding this modality of chemotherapy will be explained further in this report.
OH O OH
O O Figure: Docetaxel H H Chemical O N Structure H O O O HO OH O O O
O OH O
OH
OH Figure: Doxorubicin Chemical Structure
O O OH O NH2
O OH
5 Product Overview
Metastatic Breast Cancer
Breast cancer is one of the most dangerous types of cancers in the world affecting women. Nine percent of all cancer-related deaths in the year 2016 are predicted due to breast cancer. Out of all the malignant tumors, metastatic cancers are considered to be the most dangerous ones. These tumor cells sustain proliferation, induce angiogenesis, and activate invasion and metastatic pathways in breast tumors1.
The spread of cancer cells from the primary to the secondary site results in mortality.
Cancer cells have a unique ability to escape immune response mechanism by spreading to nearby secondary sites and establish small tumor colonies in these tissues. Immune cells like natural killer cells, macrophages, and lymphocytes contribute to the eradication of antigens or any ‘foreign’ material entering the body. In the metastatic tumor condition, immune responses are severely mitigated due to the nature in which cancer cells escape to secondary tissues. This phenomenon is called ‘colonization,' an efficient mechanism displayed by the macroscopic metastatic tumor cells to evade antitumor mechanisms exhibited by the body.
Metastatic Related Genes: Normal genes that control the ability of the cells to divide are mutated during tumor conditions and are known as oncogenes. These genes render tumor cells capacity to evade the immune response and travel to the secondary tissues.
Not all the identified oncogenes can induce tumorigenic properties to the cancer cells.
Ras proteins are present on the inner wall of the plasma membrane and facilitate the
6 initiation of cancer. They indicate a larger group of proteins called the G proteins. All these proteins bind guanine nucleotides and hydrolyze GTP. The ras oncogene has an established role in cell growth and regulation, and its protein product affects functions like cell proliferation, apoptosis, migration, fate specification, and differentiation. K-ras
4B plays a major role in tumor micrometastatic progression by matrix metalloprotease-2 expression and cell migration. The K-ras 4A is known to possess a tumor suppressor effect.2
Figure: Ras oncogene function in tumor metastasis ( Source: Brian Ell and Yibin Kang, Transcriptional control of cancer metastasis)
7 Downstream and Upstream Regulation: There are several genetic/enzymatic pathways involved in the cascade of tumor-related pathways. This makes it difficult to target a particular molecular pathway while treating a tumor patient. Most of the therapeutics that
focus on
targeting MAPK Pathway molecular
pathways
fail to Binds growth factors to protein kinase receptors Cross phorylates tyrosine inhibit residues in cystolic domains tumor due
to this same
Son of Sevenless Grb2 adaptor proteins Phosphotyrosine reason. The
MAPK pathway involves the binding of growth factors to protein kinase receptors and cross phosphorylates tyrosine residues. This pathway, in turn, recruits Son of Sevenless (SOS) to the phosphotyrosine via Grb2 adaptor proteins. SOS also acts as a guanidine nucleotide exchange factor (gef) for Ras protein while binding to the plasma membrane.
8 Son of Sevenless
• SoS acts as a guanidine exchange factor for the Ras protein while binding to the plasma membrane Ras protein • Activated Ras reeruits Raf1 kinase
• Phosphorylates raf1 • Phosphorylated Raf1 in turn activates MEK Raf1 kinase
• Duel specific tyrosine/threonine kinase in turn phosphorylates and actiates Erk1 and Erk2 MEK
• Translocate into the nucleus and phosphorylate ELK 1 transcrption factor Erk1 and Erk2
c-Fos and c- Jun
Figure: Son of Sevenless Pathway Source: Brian Ell and Yibin Kang, Transcriptional control of cancer metastasis
Activated ras protein recruits Raf1 kinase phosphorylating raf1 leading to a downstream signaling cascade. The phosphorylated Raf1 ( an MAP3K) in turn phosphorylates and activates MEK, a duel specific tyrosine/ threonine kinase (MAP2K). This, in turn, phosphorylates and activates Erk1 and Erk2 that translocate into the nucleus where they phosphorylate ELK1 transcription factor. This process leads to the transcription of immediate early response genes. As an outcome of the Ras-MAPK signaling, c-Fos and c-Jun are, transcription factors are transcribed immediately in the vicinity. These pathways induce continuous growth of tumor cells by inducing downstream and upstream
9 pathways that express proteins responsible for the metastatic phenomenon. For example, mutations in the codon 12, 13 or 61, in the RAS gene encode a protein that continuously induces growth and progression of cells by linking tyrosine kinases to downstream serine and threonine kinases3.
Ras-GTPase signaling interacts with cell surface receptors directly getting upregulated.
Specifically, members of the Epidermal Growth Factor family of receptor tyrosine kinases (RTKs, including EGFR/ErbB/HER1 and ErbB2/Her2/Neu) or tyrosine kinases
(e.g. Bcr-Abl) are overexpressed in cancer cells. These receptors bind to the Ras proteins causing its activation in the absence of oncogenes.
• In the absence of oncogenes, Ras proteins Ras proteins bind to tyrosine kinases causing its activation EGF Family of • EGFR/ErbB/ Receptor HER1 Tyrosine • ErbB2/ Kinases Her2/Neu
Tyrosine Kinases • Bcr-Abl
Figure: Ras Protein Downstream Pathway Source: Brian Ell and Yibin Kang, Transcriptional control of cancer metastasis
Some of the most important factors in metastatic cancers are the irregularities in cell differentiation, migration, and cell adhesion. Rho GTPases regulate such activities in normal cells. While controlling cellular functions, Rho family of proteins either
10 cooperates or antagonize with each other. The family consists of Rho A, Rho B, Rho C,
Rho D, Rho E, Rho G, TC 10, Rac1A, Rac1B, Rac2, CDC42Hs, and G25K. Rho GTPase family members regulate loosening of epithelial cell-cell contacts, MMPs expression, and the plasticity of cell migration (EMT, MAT) points to a central role in cancer cell invasion and metastasis.
Figure: Rho Family of Proteins Source: Brian Ell and Yibin Kang, Transcriptional control of cancer metastasis
Loosening of epithelial cell-cell contents
MMPs expression
Cell Migration EMT and MAT Family of Proteins
Rho
The Caveolin protein regulated by the Cav1 gene regulates several cancer growth factors like cell migration, metastasis, cell death, multidrug resistance, and angiogenesis. It is a proven cancer suppressor. Decreased expression of this protein is also indicative of metastases head and neck squamous cell carcinoma while studies have shown that the restoration of this protein leads to the suppression of cancer metastasis. CAV1 gene
Phosphorylation on tyrosine-14 (Y14) favors the anchorage-independent growth of cancer due to Grb7 recruitment, integrin-dependent internalization of micro-membrane domains, activation of matrix metalloproteinases and cell invasion.
NM23 expression levels have also indicated the various levels of metastatic cancers.
Reduced levels of this gene correlated with high metastatic expression in tumor cells - generally indicated by the reduced disease or the increase in overall survival, presence of
11 lymph node metastases, poor differentiation grade in subsets of breast, gastric, ovarian, cervical, hepatocellular, carcinomas and melanoma cohorts4.
Matrix Metalloproteinase (MMPs)
Extracellular proteins like serine proteinases and metalloproteinases facilitate cancer cell invasion into the surrounding tissues by the breakdown of basement membrane and
ECM.
MMPs are a large family of proteins responsible for tissue remodeling and degradation of the extracellular matrix (ECM) that subsequently induces metastasis in various cancer conditions. They are responsible for tumor invasion, metastasis, and angiogenesis. MMP2 and MMP9 types are expressed more in cancer cells and are considered to have the highest activity against type IV collagen, which is the main component of the basement membrane. Extracellular proteins are responsible for activating MMPs secreted as inactive zymogens (Pro-MMPs). These enzymes are also directly involved in the regulation of growth factors like insulin-like growth factors facilitating the growth of tumor metastasis by releasing cell proliferation factors. There are kinds of MMPs known as Tissue Inhibitors of Metalloproteinases (TIMPs) that bind to the active sites of MMPs.
Several therapies are targeting TIMP complexes inhibiting tumor growth. Studies are demonstrating that TIMPs have pro-angiogenic features in them. These conflicting studies make it extremely hard to develop drugs focusing on targeting the action of
TIMPS. Plasminogen activators like urokinase-type (u-PA) and the tissue type (t-PA) identified as indicators of metastatic cancers.
12
Loss of Cell Adhesion is a Crucial Factor in Metastatic Cancers
E-cadherin is a crucial protein that enables proper adhesion between different cell types.
In a variety of cancers, loss of this protein leads to the spread of cancer cells to distant sites. Beta-catenins are another such cell adhesion proteins whose functioning is extremely crucial in the proper adhesion of cells inside a particular tissue.
Figure: Loss of Cell Adhesion a Major Cause for Metastasis
It also functions as a key intermediary in the Wnt signaling pathway. Loss of E-cadherin from the plasma membrane liberates -catenin molecules that will migrate to the nucleus
13 and associate with Tcf/Lef transcription factors, and induces expression of genes initiating the Epithelial-Mesenchymal Transition program.
Developmental down-regulation of E-cadherin is initiated by morphogen-mediated signals that cause a translocation of beta-catenin to the nucleus inducing an epithelial to mesenchymal conversion required for cell migration and tissue arrangements. The nuclear -catenin affects the TCF/LEF family transcription factors and consequently activates oncogenes such as cyclin D1, Myc, and many other downstream targets.
CD44 is a cell membrane protein associated with various cancer stem cells, also known as cancer causing cells. It is often overexpressed in several cancer cells and can cause cell migration and differentiation by interacting with several extracellular proteins. Being a
Cell-cell and cell-extracellular binding protein, it is a viable option to target in the patient.
Tumor Angiogenesis: Figure: Vascular Endothelial Growth Receptors are Responsible for Tumor Cancer cells spread to the circulation system of Angiogenesis a human body by initiating a process called angiogenesis. VEGFR2
The cancer cells are making use of proangiogenic VEGFR1 factors like vascular endothelial growth factors from several blood vessels with weak membranes.
VEGFR1, VEGFR2, and VEGFR3 are some of the growth factors that bind to the tumor cells inducing VEGFR3 tumor blood vessel formation.
14 VEGFs are potential targets for developing targeted therapeutics inhibiting the growth new blood vessels by the tumor tissue. Nanochain targets a type of protein that is specifically overexpressed by metastatic tumor vasculature. Thalidomide and its analogs act as influencers of tumor microenvironment by inhibiting the formation of tumor blood vessels. This anti-tumor effect of thalidomide is reported in several clinical trials inhibiting VEGF and Basic Fibro Blast Growth Factor (bFGF) induced angiogenesis.
Epithelial-Mesenchymal Transition (EMT)
EMT gives cancer the special ability to differentiate and invade normal tissue. This occurs mainly because, in tumor conditions, epithelial cells lose the property of cell-cell adhesion transforming into migratory mesenchymal-like cells. EMT is the hallmark of metastasis, and the loss of E-cadherin is identified as the main reason behind this effect.
There are several reasons behind this - somatic mutations, chromosomal deletions, proteolytic cleavage, and silencing of the CDH 1 promoter. The process of EMT is three- fold
1) Epithelial Cells lose the apical-basal polarity
2) E-cadherin and epithelial cytokeratin filaments are downregulated and express EMT based proteins like fibronectin, vimentin, and N-cadherin.
3) The phenotype of these cells changes from a stable form to a mobile form.
There are three types of EMTs
Type 1: This type of EMT is associated with implantation and embryonic gastrulation giving rise to the mesoderm and endoderm and mobile crest cells.
15 Type 2: This type of EMT is initiated by injury and results in the generation of fibroblasts to rebuild wounded tissues.
Type 3: This type of EMT enables epithelial cells to acquire invasive mesenchymal phenotype characteristics essential in metastatic spread.
When EMT takes place, invasion of local matrix takes place and intravasation into the vasculature. These EMT transitioned cells survive with the help of several pro-survival factors. Subsequent proliferation requires a mechanism called the mesenchymal-epithelial transition where the EMT transitioned cells transform into epithelial cells again. A clinical trial also suggested that TWIST protein is involved in the activation of EMT and reduction in cell-cell adhesion leading to cancer. A twist over -expression is observed in several tumors like breast, prostate, esophagus, lung, uterus, skin, liver, and melanomas.
Metastatic Microenvironment
Cytokines and enzymes secreted by the tumor cells are crucial in exhibiting favorable conditions in the tumor microenvironment. The matrix protein fibronectin, VEGFR1 expressing bone marrow-derived cells (VEGFR1 + BMDC), CD11b expressing myeloid cells, matrix metalloproteinases (MMP2 and MMP9), matrix crosslinking enzyme lysyl oxidase, S100 chemokines (S100A8/A9) and serum amyloid A3 (SAA3) are all important in the development of this micrometastatic niche. Essential extracellular proteins like
Tenascin-C (TNC), growth factors for autocrine signaling production, and the metastatic tumor cells themselves can also produce adhesion to growth factor rich platelets. Some of the factors present at the forefront and involved in the development of metastatic niches are:
16 • Proteolytic enzymes like MMPs and cathepsins
• Signaling pathways induced by TGFβ, hepatocyte growth factor, Wnt and the Notch signaling pathways play a major role in promoting Epithelial-Mesenchymal Transition
(EMT).
• Transcription Factor - Hypoxia Inducible Factor (HIF) is the key mediator of hypoxic response and induces expression of proteolytic MMPs, cross-linking LOX and various matricellular proteins.
Organ Selection
Chemokines are 8 to 12 kDa peptides that are chemoattractant cytokines that are active in cell activation, differentiation, and trafficking. These protein fragments select metastatic tumor secondary sites. The chemokine receptor CXCR4 has been found to be a prognostic marker in various types of cancer. CXCR4 and its ligand CXCL12 play a huge role in the metastasis of several cancer types including metastasis. Several cancers including breast cancers express these receptors on the surface providing an excellent targeting entity for the development of new cancer therapies5.
17 Tiny Particles
Nanotechnology is the science dealing with extremely tiny particles that are smaller than the size of an individual hair follicle. As this report indicates, several nanomedicines are available in the US market that has tried to alleviate the spread of tumors in the body. The difference between nanochain and the available nanomedicines is reported in the thesis.
Introduction to Nanotechnology
Dr. Richard P. Feynman, a renowned physicist professor at UC Berkeley, envisioned tiny therapeutic particles able to treat human diseases in the year 1959. Fifty-seven years after
his famous speech, there
are close to a dozen such
tiny particles out in the
pharmaceutical market
treating different forms
of cancer. It takes eight
hundred such particles;
each sized 100
nanometers, to cover the Figure: Richard Feynman width of a human hair.
These ‘nano’-sized particles are the exciting future of medicine. The classical definition of nanotechnology is the ability to manipulate materials at the atomic and molecular level.
18 Evolution of Nanotechnology
A nanometer is one billionth of a meter. Eric Drexler highly popularized the concept of small sized particles in his book ‘Engines of Creation’. He popularized the bottom-up approach in which particles are built from the atomic level. Nanoparticles, as the name suggests, are particles of size less than or equal to 100 nanometers. Ever since the first drug delivery system, a liposomal amphotericin, was approved in 1990, there has been a surge in nanotechnology research. There were 22 search results for “nanoparticles” in the year 2000 in NIH’s website (NIH reporter)6. The number rose to 568 when the same keyword was typed in for the year 2010, and almost 630 in the year 2015. The increase in the number clearly indicates the growing interest in developing nanoparticles. In fact, the
US drug delivery market has risen substantially from $75 million to over $50 billion by
2015.
Increase in nanoparticle research (Source: NIH reporter)
Nanoparticles
0 100 200 300 400 500 600 700 2015 2010 2000
Figure: Increase in the number of nanoparticles developed during the past decade Source: NIH Reporter
19 First Generation Nanoparticles
The first generation of nanoparticles comprised of novel materials with basic or no distinct surface chemistries. Lack of specific surface properties allowed the immune system to recognize them easily as foreign bodies and removed them within seconds.
Adagen
Adagen is the first polymer-based delivery systems approved for the treatment of severe immunodeficiency diseases. Early polymer-based delivery systems were proven to be toxic to the body, but a slew of new polymer nanoparticles have made claims to be clinically safe for market release. BIND Therapeutics is a prime example of such next generation nanoparticles that have the ability to target specific tumors.
Zoladex
Zoladex is used mainly for the treatment of breast and prostate cancer. The drug has poor protein binding capacity and a very low half-life period. It gets cleared from the system
20 very easily and does not stay long enough till the drug reaches the metastatic sites. The pharmacokinetic effects on the human body are insignificant and are not targeted to treat a specific disease.
Interaction between the Complement System and the Nanoparticles
The complement system plays a crucial role in the way nanoparticles function inside the body. Several factors are affecting the interaction of nanoparticles with the complement system. Pathogens are coated with opsonins (a type of proteins) by the immune system to render them useful in recognition by specific receptors. This phenomenon helps in the clearance by receptor-mediated endocytosis and phagocytosis. Immune cells are activated and recruited by releasing potent soluble mediators by the complement system. The complement system consists of over 30 plasma and membrane proteins, many of them being proteases that are activated by proteolytic cleavage. Nanochain effectively escapes from being captured by the immune system using a PEGylated surface. The PEG coating on the surface evades opsonization.
A large clinical trial suggested that there are cases of where individuals suffered from acute allergic reactions mainly due to the activation of the complement system in sensitive individuals. These responses include and are not limited to hemodynamic, respiratory, cutaneous and other subjective manifestations.
21
Figure: 1 - Shows the nanoparticle engulfed by the phagocyte 2 - Shows a coated nanoparticle reaching its target cell Second Generation Nanoparticles:
Modification of the surface with hydrophilic surface moieties helped in repulsion with proteins and fewer protein-particle interactions, leading to an increase in the residence/circulation time in the blood. The unique capability of these nanoparticles of avoiding Mononuclear Phagocyte System (MPS) sequestration makes them “Stealth nanoparticles” or second-generation nanoparticles. Hydrophilic polymers like poly
(ethylene oxide) or polyethylene glycol (PEG) are used to avoid recognition by the immune system. These nanoparticles use the Enhanced Permeability and Retention (EPR) effect to reach a particular tumor site. Most drug delivery systems like Doxil used the gaps in the tumor vasculature to slip their way into the tumor tissue. This phenomenon was a breakthrough in the field of nanotechnology. There were several 'toxicity' related deaths, and side effects are owing to the lack of specificity of this phenomenon. Leaky vessels are present at various parts of the body and do not provide a robust mechanism to reach the tumor tissue. There is also no guarantee that the particles will reach only the cancer cells. Various labs are developing the third generation of nanoparticles using highly optimized surface chemistries to target specific cancer cells. Nanochain, thanks to its targeting ligand belongs to the third and most effective generation of nanoparticles.
22 Figure: EPR Effect (Source: Martin Ragehly)
Doxil
Doxil is one of the first approved nano-sized drugs used to treat cancers like AIDS- related Kaposi’s sarcoma and metastatic ovarian cancer. It contains doxorubicin
(anthracycline group) enclosed in an 80-90 nm unilamer liposome. PEG coats the particle's surface allowing the drug to stay in the bloodstream longer than traditional chemotherapy. It is a control/standard in several studies employing nanoparticles (like nanochain). One of the biggest disadvantages with Doxil is the toxicity it causes in patients. If the concentration of Dxl is more than 550 mg/m2, the patient faces the risk of heart failure, comparable with therapies like epirubicin or cyclophosphamide.
23 Figure: Doxil Source: http://www.rxlist.com/doxil-drug.htm
Lipodex
It is the second generation of pegylated liposomal doxorubicin composed of distearyl phosphatidylcholine (DSPC) and cholesterol with a surface coating of PEG. It has a circulation half-life of 65 hours. Due to the high circulation time of the drug, inflammation of the mucous lining became the new dose-limiting toxicity. Doxil and lipodox accumulate at the tumor site by passive targeting mechanism. A corresponding increase in patient survival is not recorded with liposomal formulations. Toxicity issues such as skin reactions and hypersensitivity reactions are major areas of concern.
Myocet
Myocet is a non-pegylated liposomal doxorubicin citrate developed by Enzon
Pharmaceuticals for Cephalon in Europe and Sopherion Therapeutics in the US and
Canada. It is the first-line treatment for metastatic cancers along with cyclophosphamide.
Myocet, in combination with Herceptin and a taxol, is used for the treatment of HER2-
24 positive metastatic breast cancer. Studies showed that it is more efficient than other liposomal options in the market. This drug regimen shows an improved therapeutic index and less cardiotoxicity along with regular anticancer activity
A pH gradient is used to encapsulate the drug by maintaining an inner compartment pH of 4 using citric acid, and an outer compartment pH of 7 where doxorubicin hydrochloride is conjugated.
Doxil and Myocet have the same API in liposomes with different lipid composition, size, loading method, plasma and tissue distribution profiles as well as different dosing and safety profiles. They are not bioequivalent. Clearance of liposomal doxorubicin in breast cancer patients was found to be 5-9 fold lower than doxorubicin with a volume of distribution close to 10-25 fold lower. Half-life is reported to be between 16-50 hours and is significantly longer than conventional doxorubicin. These results are in agreement with the theoretical advantage of using a liposomal encapsulation for drug delivery.
Third Generation Nanoparticles
Surface functionalized nanoparticles allow for a ligand to target specific tumor cells.
Monoclonal antibodies, sugars, hormones, peptides (e.g. the tripeptide, arginine-glycine- aspartic acid (RGD)) are all suitable ligands on the “third generation of nanoparticles”.
The need for developing targeted nanoparticles arose because most of the passively targeted nanoparticles using the EPR phenomenon were not effective in treating cancer patients.
Limitation with the EPR Effect
The major limitations with the EPR effect are the fact that drugs are relying on the interstitial space between the tumor cells to accumulate near the target site. EPR effect
25 causes two major disadvantages leading to an urgent need for developing a targeted cancer drug. The first disadvantage is that this passive method of drug targeting leads to nonspecific accumulation of the drug resulting in the death of normal cells. Destruction of healthy cells is the primary reason why there are several side effects associated with cancers. In fact, it is estimated that the total cost of cancer to the United States government is close to $200B (till the year 2015) which includes the number of years lost due to years of disability. The next disadvantage is the concentration of drugs given to the patients is high due to non-specific interactions.
Factors Affecting the Uptake of NPs into Cancer Cells
Many factors are affecting the uptake of nanoparticles inside the tumor cells. Size, charge, and shape are vital factors influencing the total absorption of nanoparticles.
Size and Charge
The size of the nanoparticle plays an imperative role in determining the intake capacity of these platforms. Traditionally particles ranging from 30 to 100 nm have shown significant uptake capabilities. Due to the emergence of new targeting agents like monoclonal antibodies and specific molecular pathway inhibitors, the influence of size is slowly diminishing. Engene IC, an Australian nanotechnology company, has come up with a bi-specific nanoparticle that has the potential to target more than two binding sites on the tumor cell. The size of the nanoparticle is 400 nm and is still proving to be efficacious. Larger the size of the nanoparticle, the number of targeting ligands can be attached to the cell surface.
Long rod-shaped particles are shown to have more accumulation at cancer sites than other shapes. (Source:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405876/pdf/ijn-7-
26 3445.pdf) Short rod-shaped particles showed rapid clearance from the bloodstream via urine and feces compared with long-rod mesoporous silica nanoparticles. Also, it is proven that rod-shaped particles stimulate the phagocytic activity of macrophages than by spherical ones.
Fabrication of Nanochains
Co-precipitation is a common method to develop nanoparticles. Gradient-based nanoparticle formulation is also a popular method to fabricate nanomaterials. Nanochains are formed using the precipitation-based method as discussed below.
Encapsulating Liposome
Lipids and Docetaxel are dissolved at a ratio of 8:1 (w/w) in chloroform. Vacuum removes the solvent of the mixture at a temperature of 30° C.
After hydrating the thin films in ammonium sulfate, the formed liposomes are sized by sequential extrusion through a 50nm filter followed by sonication for 30 min at 30 C.
DOX will be loaded into the liposome using the difference gradient present between the inner phase and the extra-liposomal phase. Dialysis establishes an ammonium sulfate gradient. Liposomal DOX mixture is incubated at a ratio of 0.2:1 (w/w) drug/lipid for 30 min at 60° C. The final drug load will be 0.2/0.125 mg DOX/DTX per 1 mg of lipids.
Nanochains
Nanochains are fabricated using a solid two-step phase chemistry method. A crosslinker containing a disulfide bridge will attach amine-PEG functionalized IO nanospheres onto a CLEAR resin. Cleaving the nanosphere using a thermolytic cleavage will result in the formation of an uneven surface - one face of the surface having only thiols and the other has only amines. The uneven surface will serve as two unique fittings to link them into
27 chains using appropriate crosslinkers. The drug-loaded liposome will be attached chemically to the assembled chain. The thiol of the cysteine residue of the c(RGDfC) peptide will conjugate on the remaining PEG-NH2 portion of the nanoparticle helping in binding the integrin onto the nanoparticle. The β integrin-targeting nanochain particles will be cleaved off from the resin and recovered.
Targeting Ligand
The nanochain targets β integrins overexpressed in metastatic breast cancer vasculature.
Previously, nanoparticle ligands that target integrins were proteins like fibronectin, uPAR complex, and collagen I. One of the biggest disadvantages of these ligands is that some of them have the ability to activate the metastatic tumor pathways causing further proliferation of cancer to the secondary sites. Extracellular ligands like fibronectins are also responsible for the spread of cancer cells. In some trials, inactivation of these protein complexes resulted in the transition of the proliferating cells to a more dormant phase.
Animal tests also indicate that these ligands are responsible for the activation of integrin- based complexes responsible for tumor growth. Example: the fibronectin: integrin:uPAR complex in charge of the proliferation of cells in secondary and primary tumor sites.
Figure: RGD amino acid sequence Source: Wikipedia [https://en.wikipedia.org/wiki/Arginylglycylaspartic_acid]
28 Role of Integrins in Metastasis:
Two distinct chains were making up the subunits forming the large glycoprotein complex called integrins. In nanochain's case, the presence of integrins in the metastatic sites of a breast tumor is well established. α5β3 receptors are present on cells associated with invasive breast cancers and not normal mammary endothelium. Metastatic tumor samples have indicated a high percentage of such receptors on the cells (80%). In one trial, lung metastasis was impaired in a breast cancer mouse model using an inhibitory anti- β1 integrin antibody.
Figure: Integrin pathway in tumor development, Source: Trends in cell biology, cell.com
29 TECHNOLOGY LANDSCAPE
Issues with Targeting Integrins/ Associated Ligands
Targeting integrins and associated molecules is a seemingly good strategy. However, some reports suggest that targeting some of these molecules leads a dormant phenotype.
Some clinical trials have shown evidence that targeting these molecules leads to a multidrug resistant tumor site.
Number of Receptors on the Cancer Cells vs. Normal Cells
The number of receptors on the tumor cells varies for one receptor to the other. One of the biggest factors influencing the therapeutic index of a cancer therapy is the ratio of the targeted surface receptors on the tumor cells vs. on normal cells. The number of αβ
Integrins present on the surface of tumor cells should be higher than on normal cells.
Externally Modulated Release Mechanism
Once injected in a triple negative metastatic breast cancer mouse model, a magnetic field of strength 10 kHz is applied oscillating the iron oxide nanoparticles. The research team at CWRU optimized the most efficient delivery time to be 2 hours post injection of the nanoparticles in an animal model7.
Externally modulated nanoparticles have been in use for a long time. Several researchers used to heat as a means to disrupt nanoparticles for better drug delivery. Surface plasmon resonance observed in noble metallic nanoparticles helps in conferring unique tunable optical properties to the metallic nanoparticles. The plasmon is regarded as the coherent oscillation of the free electron density of the stationary ions in a metal and a dielectric
30 interacting strongly with light. When photons have impinged on the surface of such nanoparticles, a localized surface plasmon effect is caused. The photon can then decay radiatively by scattering or non-radiatively by electron-hole excitations (absorption).
This effect helps in inducing high temperatures inside the nanoparticles resulting in the destruction of cancer cells. Commonly, gold particles are used due to their suitable surface properties. In these particles, the plasmon resonance lies in the visible range of the spectrum. Gad et al. demonstrated that gold nanospheres of 150 nm in size are not toxic with good biodistribution studies. In vivo studies showed no toxicity or bio- incompatibilities. Several gold nanospheres are entering clinical trials using the same phenomenon. The table below indicates that there are externally modulated nanoparticles for treating cancers. Hirsch et al. in 2003 used a gold nanosphere in animal models and irradiated with an 820 nm CW NIR -laser (35 W/cm2) for 7 minutes. The results showed irreversible cell damage on tumor cells. The biggest disadvantage of using gold nonspherical is the fact that there is a patent filed in the US regulating its use.
Cholesterol is added to the liposomal particle to set the melting temperature at a specific value. There are also other ways to disrupt liposomes and subsequently releasing the chemotherapeutics lodged inside the particle. Smith used light sensitive antibody- conjugated nanoparticles effectively on HER2+ cells in 2011. Yavlovich et al., in the year
2010, worked on developing light sensitive liposomes using various lipid compositions.
Almost 80% of calcein was deposited by the liposomes when the team used different compositions of DPPC and the photo-triggerable DC PC. Exposure to a lamp with 254 nm for up to 40 minutes resulted in 80% accumulation when compared to none in the
31 control. In-vivo applications of these externally modulated liposomal particles will help reveal safety and efficacy8.
However, the use of such particles has two major limitations. The resonance peak falls in the wavelength range (520 and 580 nm) where human cells absorb light and converts them into heat causing damage to healthy cells. Tissue penetration using light might be limited thus not allowing tumor cells lodge deep in the tissue to remain alive.
Magnetic Field Modulation:
The famous Egyptian physician and philosopher Avicenna in the tenth century proposed the use of magnetite powder for internal application. If the size of the particle is below ten nm, it transforms into a superparamagnetic particle having multiple magnetic domains. In nanochain's case, the size is optimal for modulating via a magnetic field. The
Large particle has multiple magnetic domains, which means that there are several sub- domains in the particle consisting of different magnetization properties. All the magnetic sub-domains in the nanoparticle grow while others shrink under the influence of a magnetic field. This phenomenon is called 'domain wall displacements', and is an irreversible process where the materials are said to exhibit 'hysteresis behavior.' Iron oxide particles used in the nanochain are single domain particles. The advantage with these particles is that when an external magnetic field is applied, the magnetic moments start rotating and overcome the energy barrier. This energy is released when the particle relaxes to its equilibrium orientation. One of the biggest disadvantages of inducing hyperthermic effects using iron oxide is that huge quantities of the material are required to conduct effective heating. Also, several off-target effects have been reported with the
32 utilization of a magnetic field. There is competition in this space with the likes of
Hayashi et al. enhancing MRI contrast while reducing tumor growth simultaneously minimizing the intensity of the magnetic field used. The team used superparamagnetic iron oxide particles and clustered these particles post infusion. The clusters were modified using PEG and folic acid for better biocompatibility and to promote specific targeting. Mice were placed in a magnetic field of strength H = 8 kA/m, f = 230 kHz (Hf
=1.8 * 109 A/m*s) for 20 minutes. 35 days later, treated tumors had 1/10th of the tumor volume compared to the untreated tumors. One of the biggest drawbacks of using magnetic nanoparticles is the limited accumulation near tumor sites. Hepatotoxicity and nephrotoxicity were also comparatively less9.
Ultrasound-modulated drug delivery systems were also tested in animal models and have been found to have limited use as nanoparticles have not been fully developed that can absorb sonic energy. It has several advantages like high accumulation in cancer sites and low toxicity. Also, applying ultrasound also causes thermally induced heating near the tumor vasculature destroying the cancer cells. Most of these particles use the EPR effect to congregate at the tumor sites. This technology still needs further research and development to transfer it successfully to the clinic.
ThermaDox
This product is a temperature-sensitive doxorubicin pegylated liposome developed for liver cancer. The transition temperature of the lipids is between 40-45 C.
33
Results of Nanochain
The iron oxide component efficiently converts the magnetic energy to mechanical vibration leading to the disruption of the liposomal nanoparticles. Histological analysis assessed the accumulation of nanoparticles at the tumor site. Around 30 minutes after infusion, nanochain particles settled exclusively near the metastatic sites. Forty-eight hours after the injection of the nanoparticles and the subsequent application of the magnetic field resulted in cell death of the cancer cells. Cell death was analyzed using
TUNEL staining, an assay that quantifies the amount of programmed cell death occurring in the organism (apoptosis). Almost all of the control treatments showed minimal accumulation near the metastatic sites.
Shape of the Nanoparticle: Extremely Crucial Factor
Several factors are influencing the cellular uptake of nanoparticles. Size, shape, and surface charge are some of the most relevant parameters. The shape of the particle is paramount in determining the uptake of these particles into cancerous cells. It influences the circulation time inside the vessels. In a clinical trial, it was proved that rod-shaped particles demonstrated a tenfold higher uptake in tumor cells than spherical shaped nanoparticles.
In some tests conducted in animals, rods show the highest uptake, followed by spheres, and cube. In studies with sub 100 nm NPs, spheres show a major advantage over rods, which is where the shape of nanochain is advantageous.
The form of the nanoparticle is critical in determining the number of ligands presented to the target receptor.
34 Increasing the aspect ratio of the NPs decreased the overall cell uptake. Ligand coated rod-shaped NPs presented advantages, especially when presenting to cells consisting of two cell surface receptors. A localized decline in the Gibbs energy occurs when the receptors bind to the cell surface causing it to fold like an envelope.
The size of the NPs plays a significant role in attaching to the receptors on the cell surface. Above 50 nm, NPs bind such a large number that further binding of ligand- receptor is limited.
Receptor-specific peptides were shown to induce angiogenesis when they were bound to a nanoparticle surface rather than remain free.
RGD Ligand Targeting
Nanochain uses an Arginylglycylaspartic acid (RGD) peptide sequence to target metastatic breast cancer cells. Cheresh et al. used arginine-glycine-aspartic acid peptide sequence producing a modest reduction of the primary tumor growth (23%) and a significant decrease of metastasis (82%) in an orthotopic pancreatic tumor model that included metastatic lesions in the hepatic hilar lymph node. On the other hand, RAD-
Dox-NPs or free Dox did not provide apoptotic effects clearly indicating that metastatic cells have been effectively killed using the peptide sequence.
Increasing the aspect ratio of the NPs decreased the overall cell uptake. Ligand coated rod-shaped NPs presented advantages, especially when presenting to cells consisting of
35 two cell surface receptors. A localized decline in the Gibbs energy is observed when the receptors bind to the cell surface causing it to fold like an envelope.
The size of the NPs plays a significant role in attaching to the receptors on the cell surface. Above 50 nm, NPs bind such a large number that further binding of ligand- receptor is limited.
Receptor-specific peptides were shown to induce angiogenesis when they were bound to a nanoparticle surface rather than remain free. Denaturation of proteins occurs due to the binding of the NPs and the ligand.
RGD Ligand Targeting causes Inflammation
Nanochain uses an Arginylglycylaspartic acid (RGD) peptide sequence to target metastatic breast cancer cells. Cheresh et al. used arginine-glycine-aspartic acid peptide sequence producing a modest reduction of the primary tumor growth (23%) and a significant decrease of metastasis (82%) in an orthotopic pancreatic tumor model that included metastatic lesions in the hepatic hilar lymph node. On the other hand, RAD-
Dox-NPs or free Dox did not provide apoptotic effects indicating that metastatic cells have been effectively killed using the peptide sequence. There is a major disadvantage with the number of different pathways associated with these proteins (18 alpha and 6 beta subunit related pathways)
36 Figure: RGD amino acid binding sites, Source: AMD Future Perspectives: New promising drugs (http://amdbook.org/content/figure-3--and--subunits-integrins-are-transmembrane-proteins)
Single Therapy vs. Combination Therapy
A single line of treatment was used during the initial days of cancer treatment. It was unsuccessful in achieving long-term remissions, produced cell lines that were resistant to further drug therapy subsequently increasing severe or lethal toxicities. The most significant disadvantage is that the single therapeutic regimen found to improve tumor drug resistance. Improved therapeutic effects of the combination are due to the additive and synergistic effect of the drugs used.
Advantages of a Combination Therapy
It allows for maximal cell kill within the range of toxicity tolerated by the patient for each drug preventing the development of new cell lines that are resistant to chemotherapy.
Decrease in toxicity and better clinical results have successfully replaced combination therapy from a single agent based treatment. Combination therapy also reduces the burden of administering high amounts of a single agent. For example, doxorubicin alone
37 causes side effects related to the heart when the dose of the drug is more than 450-550 mg/m2.
P-Glycoprotein on the tumor cell surface acts as a pump so that cancer drugs enter the cell and are quickly pumped out from the cells leaving them undamaged.
Designing Drug Combinations
Important factors in designing combination drug therapy:
- Each drug should be personalized to individual patients since most of them have different tumor mechanisms.
- The mechanisms of the drugs should complement each other producing maximum cell kill
- Drugs producing toxicities in different organ systems should be combined so that maximal doses are administered without excessive morbidity
- Drug combinations having toxicities occurring at different times.
Surface Modification of Nanoparticles
Coating the surface of a nanoparticle with a surfactant or a polymer helps not only in stabilizing the particles using steric interactions but also prevents aggregation between the particles by inducing repulsion. PEG is one of the most common ways to avoid aggregation among a cluster of nanomaterials. It also immobilizes the protective coating layer on the nanoparticles. Thiol modified PEG is one of the most common ways to use
PEG as a surfactant. Hydrophilic and negatively charged particles proved increased circulation time in the circulation system and also helps in avoiding opsonisation by the immune system
38 Circulation Time
Studies in animal models demonstrate that nanomaterials within a diameter of 30 and 200 nm is the optimum circulation time inside a human body. In previous clinical trials, the average circulation time for liposomes was close to 24 - 48 hours.
Type of particle Circulation Time Example
(hours)
Liposomes 24-48 Doxil
Iron Oxides A study proved the Depends on each
time ranges from product
30 to 60 minutes
depending on the
surface coating10
Table: Circulation times for various nanoparticles used in the nanochain therapy Surface Charge
The charge of the nanoparticle determines the colloidal stability inside the body. In general, particles having high positive and negative zeta potential will not agglomerate easily in the solution. It also determines the distribution ability of the particles in the human body affecting the internalization of these particles inside the cells. Reports indicate that there were significant differences in the rate of internalization of uncoated and coated particles. In general, superparamagnetic iron oxide particles having a positive charge are known to be better internalized by human breast cancer cells than negatively charged cells. Particles having a hydrophobic surface are easily adsorbed at the protein surface (opsonization) making it easier for circulating macrophages to clear them from
39 the plasma. Surface engineering with hydrophilic particles like PEG will allow nanoparticles to evade the engulfment by the reticuloendothelial cells or circulating macrophages. PEG forms bonds with water molecules leading to a better therapeutic efficacy caused by the increase in circulation time.
Figure: Structure of a positively charged nanoparticle, Source: alaa essa, lecturer at zagazig university (http://www.slideshare.net/essaalaa/gene-therapyrr)
Clinical Trial Strategy
In the US, the primary mode of action of a drug-device combination is assessed before its approval. There are three centers of evaluation in the US that assess the regulation of these products
• Center for Drug Evaluation and Research (CDER)
• Center for Devices and Radiological health (CDRH)
• Center for Biologics Evaluation and Research (CBER)
The Office of Combination Products coordinates and oversees regulation of combination products.
Ideal clinical trials: FDA approval: Combination therapy
40 The FDA determines the regulatory pathway of drug/device components by evaluating the primary mode of action in these products. In the case of nanochain, the agency will determine whether the therapeutic effect is caused mainly due to the magnetic field inducing device, or the nanochain particle itself. The office of combination products is directly responsible for the regulation of combination products. The OPC also directs any queries related to the premarket review of such products. It also helps reviewers and applicants by guiding them through the regulatory pathway. As an example: the FDA evaluated The most important therapeutic action of drug-eluting stents before giving its approval. The agency determined that the device’s role in maintaining the patency in the blood lumen is crucial in the expression of therapeutic action of the product. The FDA uses an algorithm to determine whether the device/drug combination product is a first of its kind, Efficacy and toxicity data is key in determining the status.
Primary Outcome Measures:
• RECIST measurements: With new changes in the response evaluation criteria in solid tumors, nanochain needs to demonstrate its effectiveness in at least five tumor lesions.
Also, pathological lymph nodes incorporated into the study with nodes having a short axis of ≥15 mm are considered measurable and assessable as target lesions.
Confirmation of response in randomized studies is required if the trial needs a response as a primary endpoint.
Disease Progression:
Previous RECIST rules defined disease progression as an increase of 20% increase in volume (lesions). Now, it is required to have at least a 5mm increase that will help guard against over calling progression of disease when the total sum is too small.
41 Imaging Guidance:
New RECIST includes a new imaging appendix with updated recommendations on the optimal anatomical assessment of lesions.
RECIST measurements help in assessing the tumor size. These evaluations are carried out at baseline, week 6, week 12 and every 6 weeks after that. [Source: BIND’s clinical trial studies]
Secondary Outcome Measures:
Progression-free survival will analyze the change in tumor size using RECIST measurements. RECIST assessments will be carried out at baseline, week 6, week 12 and every 6 weeks after that (average of 18 weeks). Important factors in assessing the success of the clinical trial are
Overall Survivability
• Duration of response
• Time to response
• Safety and tolerability
• Objective response rate
Market The scientific team from Case Western Reserve University conducted studies using nanochain on Triple Negative Metastatic Breast Cancer (TNMBC) mouse models. Triple- negative breast cancers are those forms of cancers that lack three hormones associated with cancer cells; estrogen, progesterone, and HER-2. While there have been hormonal therapies developed in the past directed against these hormones, lack of these hormones on the cancer cells has made it hard for developing drugs against such tumor forms.
42 Approximately 30,000 - 40,000 patients are diagnosed with TNMBC every year, which is almost 15 - 20 % of all forms of breast cancers. Lack of a comprehensive treatment regimen has proved to be extremely costly to the entire U.S. health care system. The U.S. government on cancer treatment has spent approximately $190 billion so far including costs incurred due to lack of quality of life in cancer patients. There is a need for a treatment therapy that can increase the survival rate, cause fewer side effects, and also be inexpensive to the healthcare providers and patients.
Targeting cancer is extremely high risk. Treatment options for metastatic cancer have not been realized fully yet. Raising funds from not for profit entities like philanthropists and grant funding significantly reduces the risk of financial loss in the initial phase of drug development. Utilizing the funds in a country like India where there are several partners to support the development is a great strategy to bring the platform to the western market.
Jobs To Be Done
The need to develop targeted therapies rose due to the inability of existing therapies to specifically kill cancer cells. The job of nanochain will be to increase the therapeutic index of the drug once administered inside a human body
Product Evolution
Clayton Christensen mentions some of the key factors important in the development of a product. They are cost, function, convenience and reliability.
Cost
The U.S government has spent roughly $ 190B in cancer treatment so far that includes financial loss occurred due to the loss of human productivity. Recent blockbusters in
43 cancer treatment, including the likes of Herceptin; treatment for triple positive breast cancer, are priced at $70,000-$100,000 for a year's treatment cycle. Most of the recent treatments for cancer therapy have been monoclonal antibodies. The cost of these drugs is too expensive when we compare it to the national GDP and per capita income.
Traditional chemotherapeutics like doxorubicin and docetaxel are comparatively inexpensive, and cost the patient in the range of $700 to $6000 for one treatment cycle.
Clinical trial studies have indicated that mAbs are less toxic than the traditional chemotherapeutics nanochain is delivering. The cost of a monoclonal antibody is way too high as shown in the table below. Clinical trials are indicating that monoclonal antibodies are as effective as a combination of traditional chemotherapy. In a study comparing the performance of Keytruda and combination therapy, the mAb showed no greater efficacy than the chemotherapeutics. This indicates that the controlled delivery of nanochain has the potential to reduce toxicity by accumulating exclusively near the tumor sites.
Hospital Costs:
In a multicenter study comparing a group of patients with cancer and without cancer, it was found that the financial loss occurring to cancer patients was close to 88% greater than noncancer patients under noncancer stratified medical DRGs11. This study compared more than 5000 patients in 90 of the non-cancer designated DRGs. It proved that patients with cancer had greater resource allocation than non-cancer patients. They also had double the financial loss, more diagnoses and procedures than noncancer patients, all the while having a greater mortality rate than noncancer patients.
44
Function
As discussed earlier, the role of nanochain is extremely novel and helps in increasing the survival rates of patients. Patients with metastatic breast cancers have very low levels of survival rates ~26%, and the primary function of the nanochain would be to reduce toxicity and increase the overall survival rates of cancer patients.
Convenience
The convenience of administering a therapeutic must be experienced by three key stakeholders for it to get market approval - Hospitals/ Physicians, Insurance Companies, and the FDA.
Hospitals/ Physicians: Pills vs. I.V: some studies demonstrate that healthcare providers have the incentive to recommend infusion based medicines compared to pills.
Administering pills to patients will result in the loss of patient income garnered from the patient’s stay in the hospital.
Reliability
One of the biggest concerns with pharmaceutical products especially drugs is the toxic effects induced years after administration. Several drugs were recalled because of the severity of these long-term effects. On an average, 70mg/kg of traditional chemotherapeutics is the maximum tolerated dose observed in several clinical trials. The team is proposing to develop nanochain that can carry very less dosage of the drugs doxorubicin and docetaxel in human beings.
45 Cancer Survival
Over the past 40 years, cancer survival has increased significantly. However, there is still a huge need to increase these rates especially because more than 400,000 are predicted to die of the disease in the year 2016
• Survival Rate: These rates are measured by comparing the number of years the cancer patients survive vs. the survival rates of the general population.
• Quality of life: This is a measure of the quality of a patient’s life after all the treatment cycles.
Research Strategies
The first step of the process is to conduct trials in animal models which the team completed. The preclinical studies were carried out using a very aggressive form of tumor induced in a mouse. With close to 6% accumulation near metastatic cancer sites, the results are promising. The next step would be to conduct some more clinical trials before beginning clinical trials in human subjects.
Co-Innovators
As a platform technology, long-term partnerships with drug manufacturers will lead to success in the future. The choice of therapeutic agents used in Nanochain is Docetaxel
(DOC) and Doxorubicin (DOX). The hydrophobic core of the nanoparticles helps in the accumulation of chemotherapeutics like dox and dtxl inside the nanocarriers.
46
Figure:
Expired Patent of
doxorubicin
Figure: Expired Patent of Docetaxel
Incremental Innovation
As mentioned above nanochain belongs to the third generation of nanoparticle-based drug delivery systems. With a targeting moiety on its surface and a controlled delivery mechanism, nanochain has increased the efficiency of delivering already existing anti-
47 cancer drugs specifically to the tumor cells. It is a major step towards treating cancer as most of the nanoparticles belonging to the previous generations were not specific, which resulted in several non-specific toxic reactions in the body.
Nanochain: Benefits to Costs
A recent survey showed that the cost to improve a year's life of a cancer patient is roughly $300,000. Any reduction in this price will be a huge benefit to the cancer community. With traditional chemotherapeutics costing from $600 to $7000, it will be tough to come up with a reasonable price for the nanochain as most liposomal-based toxicity studies did not yield better results than naïve chemotherapy (w/o nchain or liposomes). There is no incentive for a pharmaceutical company to license the product when the chemotherapeutic agents have not proven efficacy. The cost of producing and marketing a product with a magnetic device is not feasible from a pharmaceutical company point of view.
48 Competition
Nanochain’s competition is ever increasing with more than 1,000 nanoparticle-based drug delivery systems tested in clinical trials. Competition to nanochain can be classified as the follows: - Nanocarriers that target specifically the integrins overexpressed on the tumor vasculature of metastatic breast cancers, and nanocarriers that have the ability and the surface charge to target any cancer. Promising technologies pose a major threat to nanochain as most of them have demonstrated high efficacy and low toxicity compared to traditional chemotherapeutics. The second aspect of the competitive analysis will focus on the nanocarriers developed with integrins as final targets. Concerning the development of targeted therapeutics for triple negative metastatic cancers, there is no clear evidence that traditional chemotherapeutics are more efficient than mAbs or checkpoint inhibitors.
Glucose Inhibitors Glucose is required to fulfill the energy requirements of several aggressive cancers.
Blocking certain glucose pathways is an alternative way of inhibiting tumor growth inside the body. Eleison Pharmaceuticals developed a drug called 'glufosfamide,' a glucose and alkylator conjugate, that can improve the overall survival rates by as much as
25% in a clinical trial. The drug is in phase III for the treatment of metastatic pancreatic cancers.
Table: Results of Glufosfamide Endpoints Phase II Side Effects Dosage
Primary Secondary
Objective Partial Total 5000
Response Response - 1 serious mg/m2 on
49 Rate Stable disease adverse day 1 of
- 7 effects - each three
Progressive 7/22 week cycle
disease - 11 (31.82%) for upto 6
cycles
Progression- N = 22
free survival No. of
months - 1.3
(1.3 to 3.9)
Overall N = 22
Survival No. of
months - 10.5
(6.3 to 13.6)
Note: This clinical trial was completed and no further trials were conducted. The company’s phase III clinical trials developing a drug for the treatment of metastatic pancreatic tumors did not yield efficacy levels superior to the standard of care due to which the company discontinued the remaining clinical trials. p53 Pathway Activation The p53 pathway is key in aggressive tumor development and progression to secondary sites. In almost all of the cancers, this pathway is inactivated leading to the development of metastatic cancers. A company called Synergene Therapeutics is developing a p53 targeting plasmid DNA with an antibody targeting the Tf receptors.
50 Figure: A schematic representing the role of the p53 pathway in tumor invasion bax Apaf-1
bel-2 Apoptosis bel-2
bax
LIGHT
ATM p p ATM p53 P21 Ubiquitin pathway GADD45 p53 P21 PCNA ub
p53 p Rb E2f CDK4 mdm2 Cyclin D1
CDK2 p ub Cyclin E mdm2 PCNA
Proteasome Rb p
ub p53
The size of the nanoparticle is at an ideal range of 90 nm. It also finished a clinical trial phase I study and has promising results with a mean survival period of 340 days. Seven of the eleven patients used in the trial showed stable disease conditions at week six. A patient showed high amounts of necrosis at metastatic sites. Biopsies were conducted to assess the presence of exogenous p53 near metastatic lesions. A patient administered with a dose of 0.6 mg/kg showed exogenous p53 expression in metastatic sites. A grade 3 adverse event was observed in a patient with chest pain and tachycardia; common side effects observed in patients. At present, the drug is in Phase 1b. SGT-53 was systemically
51 administered to eleven patients in four cohorts of 0.6, 1.2, 2.4, and 3.6 mg of DNA per infusion. The expression of DNA diminished after 7-10 days in the body.
Endpoints Phase I Side Effects Dosage
Primary Secondary
Overall 340 Grade 3 0.6 mg/kg
Survival days adverse demonstrated
event exogenous
occurred in expression of
1 patient p53 in a
patient
Stable 7/11 Chest pain Systemically
disease patients and administerd
conditions Tachycardia to eleven
patients in
four cohorts -
0.6, 1.2, 2.4,
and 3.6 mg of
DNA per
infusion
Table: Results of Synergene’s Product Monoclonal Antibodies (mAbs)
This decade is considered to be the era of immunotherapeutics that encompasses the development of targeted antibody-based drug delivery platforms. Several biotech
52 companies are developing monoclonal antibody-based drug conjugates treating solid tumors.
Market: The year 1986 saw the approval of the first therapeutic monoclonal antibody for commercial use. The year 2014 saw close to 47 monoclonal antibodies approved in the
US and European markets. Sales of monoclonal antibody products are estimated to be close to $125 billion. As later described in this report, conjugating a monoclonal antibody to the nanochain is proposed as an effective strategy to increase the efficacy while reducing side effects. Orthoclone OKT3 was the first approved antibody-based product used for a therapeutic indication. After the approval of the first humanized monoclonal antibody in the mid-1990s, the sales of antibody-based products increased astronomically. In the year 2013, the sales of monoclonal antibodies represented close to half of the sales of the total biopharmaceutical industry ~%75 billion. There are close to
300 monoclonal antibodies in the pipeline targeting multiple indications having the potential to increase the sales of this segment exponentially. In the year 2014, close to 44 products produced in mammalian cell culture were approved. Three of these antibody- based products are manufactured in E.Coli. Of the ones produced in mammalian cell culture, thirsty one are full-length naked monoclonal antibodies, one is a bispecific antibody, two are antibody conjugates, one is an antigen binding fragment, and eight of them are Fc-fusion protein. These products represent the highest approval rates for any biopharmaceutical product segments. A study demonstrated that from the clinical study phase II till the adoption of the product, it takes close to seven years for the product to get commercialized. The sales of these products have grown from ~39 billion in the year
2008 to almost $75 billion in the year 2013. These trends represent a growth of 90% in
53 sales which is the most in any therapeutic category. Humira achieved sales close to $11 billion which is the most by any biopharmaceutical product. In fact, the top six selling products in this segment - Humira, Remicade, Enbrel, Rituxan, Avastin, and Herceptin are compared to those of the two top selling recombinant protein products - the cytokines
Avonex and Rebif12. A comprehensive list is attached in the Exhibits
A Few Promising Products:
Making progress in the clinical trials is an anti-EGFR-ILs-DOX developed by the
University Hospital Basel. Like nanochain, the company is delivering traditional chemotherapeutic doxorubicin to the cancer sites. Nanoparticles were infused at escalating doses of 5, 10, 20, 30, 40,50, and 60 mg/m2 once in every four weeks for a maximum period of six weeks. The highest dose tolerated in patients was found to be 50 mg/m2 of dox. One complete response indicated 22 serious adverse events reported in 17 patients. There were also ten stable disease conditions that lasted for 2-13 months, with the median being close to 5.75 months. A biodistribution analysis of the clinical phase I data is missing. The study enrolled close to twenty-seven patients with the size of the particle being close to 85 nm.
Endpoints Phase I Side Dosage
Effects
Primary Secondary
Overall Complete Escalating
response response doses of 5,
rate :1 10, 20, 30,
Stable 40, 50,
54 disease and 60
condition mg/m2
- 10 once
lasting for every four
a median weeks
of 5.75
months
MTD - 50
mg/m2
Table: Results of an anti-EGFR-ILs-DOX developed by the University Hospital Basel
Keytruda: PD-1: Major Threat: Promising against TNMBC13
Keytruda is a PD-1 targeting human monoclonal antibody that has ben approved for the treatment of advanced melanoma. Recently there was a clinical trial that evaluated the efficacy of the monoclonal antibody.
Results: 37.5% of the patients in the trial experienced a decrease from the baseline tumor measurement. Of the 27 patients enrolled in the trial, the ORR was 18.5% within a population range of 93.7% to 61.9%. The disease control rate i.e. percentage of patients with the best response (CR), PR, or SD for >/ 24 weeks was 25.9% (95% CI, 11.1% to
46.3%).
Important to Note: Patients who experienced a CR had previously received eight lines of therapy for metastatic disease, including anthracyclines and taxane-based regimens
55 (Dox/Dtxl). The other drugs administered to this cohort were platinum-based regimen, capecitabine, and eribulin.
Table 1:Results of Keytruda’s performance against Triple negative metastatic breast cancers
Endpoints Phase I Side Dosage
Effects
Primary Secondary N = 32
Overall 18.5% Common 10mg/kg
response side for every
rate effects: 2 weeks
arthralgia
(n=6),
fatigue,
myalgia,
and
nausea
Five patients: Grade 3 toxicity
One treatment related death
56
(Source: American Society of Clinical Oncology, Pembrolizumab in Patients With
Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study)
57
(Source: American Society of Clinical Oncology, Pembrolizumab in Patients With
Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study)
Nanochain’s Opportunity
Of the four patients exhibiting PR, one of them received a first line therapy, one patient received three lines of treatment, and two patients received six lines of prior therapy in a metastatic setting. All the patients who exhibited a response to the therapy were administered with prior chemotherapy. Nanochain could be used as the first line of chemotherapy before the administration of next-generation molecules like monoclonal antibodies. On an average, the maximum tolerated dose of chemotherapy lies in the rang e of 55 mg/kg - 70 mg/kg. Nanochain will gain an easier market if it could provide better efficacy delivering lower than 55 mg/kg per dose.
58
Metronomic Therapy: This form of therapy regimen has been proved to be efficacious.
Contrary to the chemotherapeutic regimen, this form of treatment cycle is administered in low doses. Although not successful in all cancer forms, it demonstrated high efficacy in cancer types like gastric, liver, and lung. Nanochain could be used as a metronomic line of therapy in various cancers expressing any one of the biomarkers discussed in this thesis. The specificity of the nanochain will help in delivering lower amounts of the drugs to tumor sites.
Patients with high LDH at a disadvantage: The trial also proved that patients with mTNBC and high LDH would not benefit from the mAb therapy. This hypothesis was supported by the fact that patients receiving the mAb treatment were associated with a longer median time to response ( 18 weeks; range 7 to 32 weeks) than cytotoxic chemotherapy.
Median overall survival = 11.2 months
Median duration of SD = 17 weeks
SD>/ 24 weeks duration = Two patients
Median time to response = 17.9 weeks.
59
Kaplan Meier Statistical Analysis of the Clinical Trial
Keytruda is a PD-1 pathway inhibitor that is efficacious in the KEYNOTE-012 clinical trial. The PD-1 pathway or the programmed death receptor 1 pathway plays a crucial role in the spread of tumor cells in the human body. The PD-1 receptors are upregulated on T- cells, B-cells, natural killer cell, monocytes, dendritic cells, myeloid cells and a few thymocytes. It inhibits autoimmune activity of the immune system thereby helping cancer cells in metastasis. The PD-1 ligand is an immunosuppressant that is over expressed
60 during disease condition and inflammation. Previous research proved that primary breast cancers express PD-L1 with an increase during metastatic conditions. The PD-1 pathway uses mechanisms enabling T-cell exhaustion and immunosuppression, subsequently evading anti tumor pathways.
Pembrolizumab: Disease Indications
Pembrolizumab is approved in various countries as an effective treatment for melanoma and has also shown promise in other advanced malignancies. The Cancer Genome Atlas analysis demonstrated that higher PD-L1 mRNA expression was demonstrated in TNBC vs. non-TNBC samples (P<0.001). It was also reported that 20 to 30% of TNBCs expressed PD-L1 on the cell surfaces.
PD-1 Pathway: Mutation or Deletions in the PTEN/PI3K pathway is responsible for the upregulation of the PD-L1 and in the subsequent suppression of T-cell proliferation and survival.
Avastin: α3 β5 integrins are a popular target for many small molecules, peptide,
A monoclonal antibody developed by Genentech is directed towards inhibiting αβ integrins. The primary mechanism of action of this drug is to inhibit the action of bFGF and tumor necrosis factor - (TNF - ) induced angiogenesis. αvβ3 and αvβ5 have different mechanisms of action in promoting tumor growth and proliferation. αvβ3 activates tumor angiogenesis through the bFGF and TNF-a pathways.
61
The avb5 integrins use the VEGF and TGF-a pathways to activate the tumor angiogenic pathway. Avastin is a mouse anti-human integrin avb3 monoclonal antibody. The binding affinity of the antibodies performed after evaluating and optimizing the complementarity determining regions (CDRs) LCDR1, HCDR1, and HCDR3.
The human IgG1 antibody-based therapeutic Vitaxin I (MEDI-523) showed anti-tumor activity in a Kaposi’s sarcoma model and partially blocked the binding of the human immunodeficiency virus Tat protein to the avb3 integrins. Vitaxin I showed limited efficacy in patients with advanced malignancies in a phase I clinical trial indicating that monoclonal antibodies targeting these integrins have not been efficacious so far in clinical trials. This product was further developed using phage expression libraries, and the resulting antibody was named as Abegrin that was later licensed to MedImmune Inc.
In a clinical trial with 15 patients, the antibody proved safety but did not show efficacy.
In a cohort consisting of metastatic breast cancer patients, a dose escalation study did not prove efficacy in patients with metastatic cancers (n=3).
62 Limitations: Mab is targeting only one type of integrin-mediated angiogenic pathway.
Nanochain is also targeting the same integrin. Will it be effective? Will targeting various types of integrins be a viable option?
Abegrin (mg) n=3
250
200
150
Abegrin (mg) 100
50
0 0 50 100 150 200 250 Figure: Dosage of the Abegrin administered
CNTO 95 Centocor: It is a humanized monoclonal antibody binding to both avb3 and avb5 integrins mediating angiogenesis.
The mAb shows high binding affinity
(200pM) to the integrins. It showed good efficacy in a clinical trial consisting of patients expressing avb1 and not avb3, showing that it has the potential to be used as a multiple integrin targeting antibody. Nanochain’s capability to target just one integrin could prove ineffective compared to monoclonal antibodies targeting multiple integrin targeting pathways.
63
Liposomal Particles There are liposomal-based nanoparticle drug delivery systems at various stages of the clinical trial process. Some of the most promising ones are listed below:
BBB Therapeutics Nanochain's other indication is treating Glioblastoma multiforme, and the pre clinical studies showed that it exhibited a 2.6, 3.2, and 6-fold higher deposition in the tumor cells compared to nontargeted nanochains, targeted liposomes, and non-targeted liposomes.
The human study conducted by the company BBB therapeutics will be the closest to nanochain’s projected clinical trials because the control used in the pre-clinical trial is
DOXIL in both these cases. BBB’s product (2B3-101) showed a fivefold enhanced doxorubicin brain -delivery versus Doxil ( vs. 6-fold by nanochain). In a phase I trial conducted by the company, 28 patients received 2B3-101 at doses of 5-70 mg/m2, without DLTs. 15 patients had brain metastases from solid tumor, and 13 patients had recurrent malignant gliomas WHO grade III or IV. Twenty-three patients (82%) received
>/ 3 prior therapies before 2B3-101. No reports of cardiac or CNS toxicities. At doses of
>/ 40 mg/m2, adverse events were neutropenia (21%), thrombocytopenia (4%), mucositis
(4%), and PPE (18%). Grade I-II infusions were observed in 4/28 patients who were transient and manageable with standard treatment. At the same dose (40mg/m2), 12 of 16 patients, and more importantly, five glioblastoma patients ( a therapeutic area of interest of nanochain), demonstrated stable disease outcomes. This product is well-tolerated up to
70 mg/m2 in both brain metastases from solid tumors and recurrent malignant gliomas.
The drug well sustained a dose intensity of 15 mg/m2/week for at least four cycles. The company is basing the preclinical study to conduct a clinical trial (Phase II a) with
64 treatment regimens of 60 mg/m2 administered every four weeks for patients with recurrent glioma.
Table: Results of 2B3-101
Endpoints Phase I Side Effects Dosage
Primary Secondary
Overall Stable No DLTs were 5-70
Response Disease observed mg/m2
Rate Conditions
- 5
Doses greater
than 40mg/m2
expressed
neutropenia
(21%),
thrombocytopenia
(4%), mucositis
(4%), and PPE
(18%).
Mebiopharm This pharmaceutical company is developing lipid-based platforms with a targeting ligand on the surface. The ligand targets a receptor called the transferrin receptor, generally
65 overexpressed in breast cancer setting. The size of the particle is also optimal and lies in the range of 50 - 200 nm. A moderate tumor loss (TGI = 25%) and a reversible weight loss occurred when this drug was administered. The optimum dose of the drug is found to be 40 mg/kg of gemcitabine and 4mg/kg MBP-426 together. Therapeutic Indication:
Metastatic gastric, gastro esophageal junction, esophageal adenocarcinoma. High doses resulted in 6/9 deaths in one of the clinical trials indicating that the dosage needs to be optimized
Table: Results of MBP-426
Endpoints Phase Phase Phase Side Dosage
I II III Effects
Primary Secondary
TGI = High Optimum
25% doses dose: 40
resulted mg/kg of
in 6/9 gemcitabine
deaths and 4mg/kg
MBP-426
together
Polymer-based Therapeutics
BIND Therapeutics is a Boston based biotech startup that is conducting clinical trials to test a polymer-based nanoparticle with a small molecule targeting prostate specific membrane antigen. The study also tested a dose escalation is different cohorts of patients
66 (with doses ranging from 3.5 to 75 mg/m2 (n=28) and 15 to 45 mg/m2 (n=27). The product is in clinical trial phase II with recommended doses ranging from 60 mg/m2 for every three weeks, or a 40 mg/m2 weekly dose.
Endpoints Preclinical Phase Phase Phase Side Dosage
Trial I II III Effects
Primary Secondary
- - - 60mg/m2
every 4 weeks
or 40 mg/m2
weekly
Table: Results of BIND’s trial Engene IC The Australian-based firm is using a 400 nm particle having dual targeting ability i.e. the
NPs can bind to different targeting moieties. Some examples are multiple receptors binding capabilities and CD3 on T cells. There are past examples that showed the efficacy of such bispecific antibodies like revomab. The firm is recruiting patients to conduct a phase I clinical trial in the USA to receive an FDA approval targeting invasive brain tumors. The test also showed a reduction in the tumor volume close to 40 %.
Engene's nanocell technology has to be the biggest threat to nanochain because it targets multiple mechanisms inhibiting tumor growth. The size of the nanoparticle is 400 nm that
67 might prove to be a barrier. Some studies recorded efficient delivery of nanoparticles to the brain lying in the 30-100 nm range.
EDV nanocells target EGFR receptors and have a lipopolysaccharide coating and an anti-
EGFR antibody ABX-EGF (panitumumab) conjugated on the surface.
Endpoints Preclinical Phase Phase Phase Side Dosage
Trial I II III Effects
Primary Secondary
STILL CONDUCTING CLINICAL TRIALS AS OF JUNE 2016
Table: Results of Engene IC
68 Immunoliposomes
There is a need to eliminate the use of multiple lines of chemotherapy in treating metastatic cancer patients. Reducing the dose of the drug leads to a decrease in the number of side effects observed in the patient. Most biotech and pharmaceutical companies employ the drug rechallenge regimen during which traditional chemotherapeutics administered as the first line of therapy are again infused to the same patients after significant disease progression. For nanochain to be competitive in the future, the product needs to come up with an effective targeting strategy. One strategy would be to conjugate a monoclonal antibody on the surface of the liposome. There are close to 40 mAbs approved by the FDA and are the main forms of treatment for malignant tumors in the current cancer therapeutic landscape. Case studies are indicating that conjugating a monoclonal antibody onto the surface of a liposomal nanocarrier significantly increases the efficacy of the drug.
Figure: Immunoliposome sketch
Bangham et al. discovered and identified liposomes as efficient delivery agents more than
50 years ago. Liposomes are lipid vehicles with an aqueous phase inside and between the
69 layers. Immunoliposomes can encapsulate small molecules inside liposomal nanocarriers and use biological targeting techniques to induce cancer cell kill efficiently.
A hydrophilic head group and two hydrophobic chains make up the phospholipids used to prepare liposomes. It helps in the efficient encapsulation of both hydrophilic and hydrophobic drugs inside the liposomes. A few advantages of liposomes in delivering drugs are that they are water soluble, can be prepared of any size, and carry almost any substance in them14.
The first example of a therapeutic immuno-liposome was by Torchilin and used it to localize the particles in acute canine myocardial infarction. The surface chemistry of the liposome allows for the conjugation of several biological components like an antibody, antibody fragments, vitamins, glycoproteins, peptides, oligonucleotides, polysaccharides and others.
Figure: Receptor-mediated endocytosis
70 Popular methods of making liposomes: One of the simplest and popular methods of making liposomes is to hydrate lipid films in an organic solvent and later remove the film deposition via rotary evaporation.
Multilamellar vesicles are formed when the solid lipid mixture is hydrated with an aqueous buffer at a temperature above the gel-liquid crystalline transition temperature of the lipid causing them to swell and hydrate spontaneously leading to the formation of multilamellar liposomal vesicles. Ultrasound method prepares small unilamellar vesicles using ultrasonication of aqueous dispersions of phospholipids. A reverse phase evaporation method is also used to prepare large unilamellar and oligolamellar vesicles.
In this process, lipids are added to a round bottomed flask and the solvent is removed using distillation. They are re-dissolved in the organic phase resulting in the formation of vesicles after which evaporation of the solvent to a semisolid gel takes place, followed by the removal of non-encapsulated materials. Another popular method of protein encapsulation is the freeze-thaw extrusion, where the liposomes are formed in a thin lipid film with the solute to be entrapped present until the suspension of the film15.
With the competition of large pharmaceuticals lurking around, especially with several monoclonal antibody-based drug regimens on the horizon, nanochain's competitive advantage will be high only if it can efficiently reduce the use of monoclonal antibodies as a line of treatment. The liposomal-based antibody is prepared either after or before liposomes are synthesized. The ligand is bound to the surface of the liposomes by conjugating the hydrophobic anchor groups with functional groups on the liposomal surface.
71 Coupling of a ligand to the surface of liposomes is performed using thioether bonds.
Example; Reaction of thiols and maleimide groups. The sulfhydryl group is present in many proteins, however, often the number of –SH groups are low, or zero and they need to be generated from existing disulfide bonds or created by adding heterobifunctional crosslinking agents. SPDP and SATA are familiar crosslinking agents. For example, after modification with SPDP, a protein can be treated with dithiothreitol for disulfide to thiol reduction. A terminal –SH group is conjugated with a functionalized anchor containing sulfhydryl groups such as N-(4-(p-maleimidophenyl)butyryl) phosphatidylethanolamine
(MPB-PE)
Figure: Various types of Immunoliposomal formulation E. Paszko and M.O. Senge, Immunoliposmes
Antibodies are also attached to sterically stabilized long-circulating liposomal membranes in parallel with or at the distal end of the polyethylene glycol chain. An example is maleimide-PEG (where antibodies are thiolated and linked to liposomes containing MAL-PEG-DSPE through thioether bonds16. Formation of covalent bonding and a high coupling efficiency are two advantages with this type of conjugation.
Functionalization and thiolation of antibodies occur in most cases resulting in the random orientation of the antibody on the Immunolipsome (IL) surface causing faster circulation
72 and elimination in the body. Hydrazide PEG (Hz-PEG) method is based on oxidation of carbohydrates in the Fc region of the whole antibody to reactive aldehydes and then the formation of hydrazine bonds with hydrazide groups on the distal end of PEG chain.
Crosslinking of the carboxylic acid functional group on the surface of nanocarriers with primary amines of the ligands is also an efficient way in the development of ILs.
Example- Lipid with a carboxylic acid group at the distal end of the PEG chain, (DSPE-
PEG-COOH)
Antibodies can also be attached to the PEG-terminus of liposomes using an amphiphilic
PEG derivative such as pNP-PEG-PE, which is incorporated into the liposomes via phospholipid residues and binds primary amino groups containing ligands through its water exposed pNP groups17.
Type B immuno- liposomes are antibodies or antibody fragments conjugated to the surface of long-circulating liposomes with PEG on them. As a result, uptake by the reticuloendothelial system is less. The steric barrier of PEG decreases the efficiency of protein coupling and target recognition. Antibodies attached to the distal end of the PEG chain have improved circulation and better antibody accessibility. The pore size of tumors varies from 100 to 780 nm, and most of the ILs developed using this method make use of te EPR effect to accumulate specifically near cancer sites. The transport phase is the phase where ILs travel from the administration site to the desired tissue. The effector phase is the stage/ phase during which immuno- liposomes bind specifically to
73 an antigen or receptor, triggering receptor-mediated endocytosis where the whole particle is engulfed inside the cells.
Antibodies such as the anti-Her mAb or anti-ICAM-1 mAb attached to the surface of liposomes proved that the conjugation is an effective strategy to increase the efficacy of anti-cancer agents. These types of targeting candidates are used for drug delivery, imaging applications and also for the delivery of photodynamic therapy agents.
Monoclonal antibodies conjugated to the surface of liposomes, containing PEG and Gd coatings, improved the targeting efficacy of these agents. Surface bound nucleosomes released during apoptosis helped these systems to recognize various cancer cells accurately.
Mulder et al. designed pegylated Gd-DTPA-based paramagnetic ILs, modified with the monoclonal antibody H18/7 against human E-selectin. These particles stimulate human umbilical vein endothelial cells (HUVEC) along with cytokines such as IL-1 or TNF up- regulating the expression of the adhesion molecule E-selectin/CD62E resulting in a lesser metastatic spread of the primary tumors. MRI and fluorescence studies indicated higher specificity of such targeted particles compared to non-targeted nanocarrier systems
Cancer Therapy:
Most nanocarriers targeting the human epidermal growth factor receptors focus on the gene ERBB2. HER2 belongs to the same family and is overexpressed in 18-20% of all breast cancers. The HER2 protooncogene encodes a 1255 amino acid, 185 kDa receptor tyrosine kinase (RTK), which is a member of the class I-RTX family, along with the epidermal growth factor receptor (EGFR). Overexpression of HER2 results in an
74 aggressive form of cancer with significantly short overall survival periods. Nanochain is attempting to cure a form of metastatic breast cancer that does not express HER2.
Park et el. Developed anti-Her2 immune- liposomes using long-circulating liposomes coupled with monoclonal antibody anti-Her2 to provide targeted drug delivery. It showed close to 700 fold greater accumulation when compared to the non-targeted liposomes.
In a mouse model replicating TNMBC tumor conditions – MDA-MB-231, the efficacy of an IL conjugated with anti-HER2 antibody trastuzumab (Herceptin) and an encapsulated paclitaxel was tested. There was not a huge difference in the uptake efficiency between targeted and nontargeted liposomes.
Anti-EGFR
Since most of the examples presented targeted Her2 proteins, the following are examples of nanocarriers targeting other biomarkers expressed during triple negative metastatic cancer conditions.
Anti-EGFR based targeting ILs are promising candidates having the ability to treat
TNMBC. Several researchers are focusing their efforts on treating TNMBC tumors by targeting the human epidermal growth factor receptors.
Fab’ fragments of mAb C225 (cetuximab) were covalently attached to liposomes consisting of an anti-tumor drug targeting EGFRs.Irrespective of the mAb fragment present anti-EGFR immuno- liposomes showed longer circulation along with stable drug retention. The ILs were also internalized inside the tumor cells significantly - 92% of the cells compared to < 5% for non-targeted liposomes. Doxorubicin, epirubicin, and vinorelbine were used as controls in a test, where the targeted ILs proved more efficiency and less toxicity while targeting EGFRs.
75 Anti-RON Receptor Tyrosine Kinase:
The RON receptor tyrosine kinase receptor is a member of the proto-oncogene family and is expressed in various epithelial cells. Overexpression of this receptor is seen in colon and breast cancer cells. Immunoliposomes were generated with doxorubicin and were formulated by post insertion of mAb Zt/g4, Zt1/c1, or their Fab fragments, which have shown specificity towards the RON receptor tyrosine kinase. Flow cytometry analysis revealed that both Zt/g4 or Zt/c1 –IL bind to cancer cells and cause RON internalization resulting in significant uptake of the targeted liposomes. T-47 D breast cancer cells were used in a test determining the efficacy of ILs targeting RON receptors, with liposomes conjugated with IgG as the control. Zt/g4 IL encapsulated doxorubicin showed effectiveness in killing HCC1937 cancer cells, a cell line that is insensitive to pegylated liposomes. The efficiency of Fab fragments for targeted delivery was low compared to whole antibodies linked to liposomes containing doxorubicin inside them.
Anti- RoN antibody directed drug delivery is an effective means to deliver cytotoxic drugs.
A systemic administration of gemcitabine encapsulating immuno- liposomes with anti-
EGFR antibodies attached to the distal end of the PEG chain inhibited the growth of tumor in non-small lung cancer. RPCI-2E9 expressed integrins and were targeted with doxorubicin loaded liposomes conjugated with Fab’ fragment for mAb specific for human B1 integrins. Compared to doxorubicin alone, ILs with these targeting antibodies on the surface showed more internalization and cytotoxicity.
76 ILs also showed significant tumor suppression of tumor growth, prevention of the metastatic spread of the tumor, and longer survival times in tumor-bearing mice injected with 1F11 modified liposomes.
Hatakeyama et al. also proved that ILs conjugated with an anti-EGFR targeting monoclonal antibody showed superior internalization when compared with non-targeted liposomal-based carriers.
Doxorubicin loaded immuno-liposomes tagged with an antibody against the membrane type I matrix metalloproteinase (MT1-MMP) showed higher levels of specificity compared to non-targeted nanocarriers. MT1-MMPs play a major role in angiogenesis and are expressed on angiogenic endothelial cells as well as on tumor cells. It is a proven fact MT1-MMPs are expressed on triple negative metastatic cancer cells and are responsible for tumor invasion and growth. Immunoliposomes conjugated with the Fab’ fragment from humanized anti-EGFR mAb EMD72000 were presented on the liposomal surface. ILs conjugated with matuzumab or cetuximab exhibiting promising results when targeted to colorectal cancer.
ILs also expressed high affinity when directed against the vascular adhesion molecule 1
(VCAM-1). It is a glycoprotein that is expressed on activated endothelial cells during inflammation and cancer. In vitro and in vivo analysis showed specific binding of these
ILs to activated endothelial cells under various blood flow conditions (static and simulated). Endoglins are type I membrane glycoproteins located on the cell surfaces of cancer cells. They are part of the transforming growth factor receptor complex forming an attractive target found on the tumor vasculature. Endoglins are overexpressed during triple negative metastatic breast cancer conditions responsible for the proliferation and
77 spread of cancer. Single fragment chains (scFv A5) directed against the human endoglin
(IL 5A) showed rapid and strong binding to endoglin expressing endothelial cells. No binding was observed with various endoglin negative cell lines and also blood lymphocytes.
MCC-465, an immuno- liposome encapsulating doxorubicin, is coupled with the cancer reactive antibody GAh. When compared to non-targeted liposomes, it was observed to have more than 90% positive ratio in targeting specifically cancer cells. In preclinical studies, this IL presented more cytotoxic advantages than non-targeted liposomes. The maximum tolerated dose of MCC-465 was lower than that of Doxil indicating that these
ILs are an effective means of targeting tumors.
ILs conjugated with trastuzumab targeted against gastric cancer showed greater efficacy compared to docetaxel and non-modified liposomes.
Pastorino et al. demonstrated that liposomal drugs encapsulated with doxorubicin and conjugated with NGR peptides targeted the angiogenic endothelial cell marker: aminopeptidase N isoform effectively. The peptide is capable of delivering chemotherapeutics specifically to cancer cells. NGR targeted Ils demonstrated tumor regression, destruction of tumor vasculature and prolonged survival of orthotopic neuroblastoma xenografts using NGR targeted ILs were observed. Combined use of tumor and vascular targeted liposomal therapies enhanced the cytotoxic effects of the drugs in human neuroblastoma animal models and resulted in the dramatic inhibition of tumor endothelial cell density. Combined therapy, half dose of each single liposomal formulation, but equal total doses of DXR, were used. It showed better efficacy compared to a non-combination or single agent drug regimen.
78 Future Threats and Recommendations
A case of targeting multiple integrin-mediated pathways
Intetumumab, formerly known as CNTO 95, recognizes the different members of the family of αβ integrins. In this case study, a clinical study is conducted with patients with melanoma. It was well tolerated by patients suffering from invasive melanoma with doses ranging from 3 to 10 mg/kg. Patients with melanoma are administered interleukins-2 which are toxic in nature. This clinical trial compares the efficacy of intetumumab with and without the administration of dacarbazine, as compared to patients treated with dacarbazine monotherapy (stage IV melanoma). The primary endpoint was progression- free survival (PFS), which is defined as the time from randomization to the earliest date of documented PD, symptomatic deterioration, or death. Major secondary endpoints are complete response, overall response rate, PR, CR, and overall survival. Objective response rates were calculated using the RECIST criteria. Tumor biopsies were collected before and after the treatment with the monoclonal antibody. The protein expression levels of the av integrins were used to evaluate the amount of tumor inhibition. Treatment comparison was performed using the Kaplan - Meier method and the log-rank method.
Table: Results of Intetumamab Endpoints Preclinica Phase I Phas Phas Side Dosag
l Trial e II e III Effect e
s
Primary Secondar
79 y
Progressio No 3 to 10 n Free side mg/kg
Survival effects
(PFS) within
the
mAb
arm
Complete
response
Overall
response
rate
PR
CR
Overall Combined
Survival therapy: 11
months
mAb at
10mg/kg: 15
months
Only
chemotherapy
80 : 8 mg/kg
Efficacy: The overall survival of patients receiving only intetumumab showed an increase in the overall survivability compared to patients receiving only dacarbazine. Patients receiving the mab and the cytotoxic agent showed an OS of 11 months and those receiving 10 mg/kg alone had a median OS of 15 months compared with the control arm alone which showed a median of 8 months. Kaplan-Meier analysis proved that the control arms containing the antibody showed an increase in the overall survivability when compared to the arm with only dacarbazine alone. The estimated overall survival rate for 1-year also favored the use of the antibody therapeutic regimen compared to only the cytotoxic agent.
The rate was almost double for patients treated with only the monotherapy when compared to the arm with dacarbazine and was nearly 50% greater compared to patients receiving both. This study clearly shows that monoclonal antibodies are a much viable option when compared to the administration of a single cancer therapeutic.
Integrin Expression:
Expression of integrins in tissue biopsies collected from both the treatment arms was different. From the group of 36 selected for this test, stable disease condition was
81 observed in patients expressing higher levels of av integrins, and PD was witnessed in patients with lower levels of expression.
The treatment also showed a better safety profile compared to the administration of only cytotoxic. This study demonstrates the efficacy of monoclonal antibodies vs. that of single therapy administration of a cytotoxic agent.
C7E3 Fab (abciximab) and m7E3 (Fab) are the mouse-human chimeric and murine mAB fragments of the parent intact murine. It binds to aIIbb3 receptor on the platelets that are responsible for platelet aggregation. The advantage of this Mab is that it binds to the angiogenic tumor avb3 integrins preventing the spread of tumor cells to distant sites.
Animal studies representing human melanoma tumors suggest that the combined blockade of both these integrin receptors is efficacious than monospecific inhibition of avb3 antagonists alone especially after it is a proven fact that tumor angiogenesis and formation is caused by more than one integrin-mediated angiogenic pathway.
Competition in the Triple Negative Metastatic Breast Cancer Market
Women with a neoadjuvant treatment of platinum-based drug regimen have experienced a reduction in the tumor growth significantly.
After 3-5 years of treatment, patients are at a high risk of developing metastatic cancers accompanied by a sharp decrease in the survival rates. Patients with metastatic breast cancers are given adjuvant and neoadjuvant based regimens as first line or second line chemotherapy.
82 The clinical efficacy of the multidrug resistant phenotype was challenged during a clinical trial phase III of specific P-glycoprotein antagonists subsequently failing to show efficacy. The factors affecting acquired drug resistance to small molecule inhibitors of tyrosine kinase ( gefitinib, erlotinib, imatinib) is also unknown.
Pre-existing mutations in the genome have been identified to be primarily responsible for acquired drug resistance in cancer.
Defined mutations in molecular gene targets such as EGFR, BCR-ABL, or KIT, explain acquired resistance to anti-cancer agents.
Genomic sequencing data revealed that the heterogeneity of tumor genetic environment is also a reason causing the multi-drug phenomenon.
Patients develop sensitivity to the chemotherapeutics that are already administered as first-line therapy. Some of the common characteristics of the drug rechallenge are that patients respond to the first line of chemotherapy until disease progression. After some time into disease progression, the treatment is stopped. The same patients respond to the exact dosage of chemotherapy when administered at a later stage in the treatment cycle.
The introduction of a drug rechallenge regimen is crucial in treating invasive cancers like triple negative metastatic breast cancers.
Combination Therapy: Anthracyclines and taxanes are administered as drug rechallenge regimen in patients with 6-12 months of disease-free progression post- adjuvant chemotherapy and recurrence. A clinical trial testing the efficacy of a combination therapy regimen was conducted on 751 patients with advanced breast
83 cancer. The inclusion criterion also included patients previously treated with neoadjuvant
or adjuvant anthracycline therapy. These patients were assigned to receive either
docetaxel or Doxil followed by the cytotoxic docetaxel. A combination drug rechallenge
regimen yielded significant results in the clinical trial with a median survival of 10
months for the PLD-docetaxel therapy vs. the docetaxel monotherapy. Overall response
rates also varied significantly 35% for the combo vs. 26% for the docetaxel monotherapy.
Docetaxel was delivered at a dose of 75 mg/m2 (n=373) or PLD 30mg/m2 followed by
docetaxel 60mg/m2 every 21 days (n= 378) and continued until disease progression or
prohibitive toxicity. The primary endpoint of the investigation was time to
progression(TTP). The secondary end points were Overall Survival (OS), Objective
Response Rate (ORR), cardiac toxicity, and safety. Median TTP rose from 7 months to
9.8 months. ORR from 26% to 35% and the OS was similar between the two groups. The
occurrence of grade 3 or 4 adverse events was also similar in both the groups (78% v
72%).
Endpoints Phase III Side Effects Dosage
Primary Secondary
Overall (OS) HR=1.02, • Grade 3 or 4 Docetaxel
Survival 95% l, 0.86 to adverse events 78% was
(OS) 1.22 [DOX-DTXL] vs administered
72% [DTXL] at a dose of
84 • Higher incidence of 75 mg/m2
hand and foot (n=373) or
syndrome found in PLD
the PLD-DTXL arm 30mg/m2
(24% vs 0%) followed by
• Mucositis/stomatitis docetaxel
(12% vs 1%) were 60mg/m2
observed in the every 21
PLD-DTXL arm. days (n=
• Protocol-defined 378) and
left ventricular continued
ejection fraction until disease
decreases and progression
congestive heart or
failure were 5% and prohibitive
1% in both toxicity
treatment arms
respectively
Median
Time to increase from 7
Progression to 9.8 months
(TTP)
Overall ORR rose from
85 Response 26% (dtxl) to
Rate 35% (combo)
(ORR)
Cardiac PLD-DTXL
toxicity combination
showed
efficacy
without an
increase in
toxicity
Mucocutaneous
toxicity was
more common
Source: Sparano, Manikhas GM, et al.
Table: Results from a Combination Therapy Trial
Problem with Metastatic Cancers: Multidrug resistance
Previous clinical trials have shown that combination treatment with non-cross-resistant agents like capecitabine, gemcitabine, vinorelbine or albumin-bound paclitaxel are efficacious in anthracycline-pretreated patients having advanced breast cancer. Until the recent approval of ixabepilone, capecitabine was the only agent that was approved by the
FDA following the failure of anthracycline/taxane-based chemotherapy. In a randomized clinical trial, patients were pretreated with three gemcitabine-based regimens in
86 pretreated patients, with ORRs of 39% in gemcitabine/vinorelbine, 48% in gemcitabine/cisplatin and 35% in gemcitabine/capecitabine. Median survival rates were as follows: 17.5, 13.0, and 19.4 months respectively. The 2009 European School of
Oncology Metastatic Breast Cancer Task Force recommended sequential monotherapy for advanced breast cancer.
Patient and disease-related factors to used in determining which patients would benefit from combination treatment. There are research groups that have come to the conclusion that polytherapy should be administered with the risk of damage to inner organs.
Sparano, Manikhas GM, et al. arrived at the conclusion that because this type of tumor is aggressive and is hard to reduce in size, a multi-drug regimen should be administered for better efficacy. The administration of both docetaxel and doxorubicin is a good strategy to combat single therapeutic resistance.
Severity of the Disease:
In a recent multicenter trial, 111 patients were evaluated suffering from advanced metastatic breast cancers, and receiving various forms of treatment (67% - single-agent and 33% multi-agent palliative therapy). This test showed that the total duration of the first line therapy was 12 weeks. Eighty-seven patients received second-line therapy with a median duration of 9 weeks (range 0-121 weeks). Forty-nine percent of the patients
(55) received the third line of treatment for a median duration of four weeks, indicating that nanochain can generate value by reducing the number of cycles required for the chemotherapy during the treatment of metastatic cancers. The median overall survival of metastatic breast cancers was also lesser compared to the OS of the general breast cancer population ( 13 months vs. 2.0 - 35 years).
87 Platinum-based drug regimens have shown efficacy in TNMBC patients. With almost
90% BRCA1 mutations resulting in TNBC, platinum-based regimens have its advantages. The biggest benefit is that it is clinically proven that TNMBC tumors lose the ability to regain from the effects of DNA-damaging agents due to the mutation in germline BRCA1 mutation (~10% TNBC are due to BRCA1 mutations)
Platinum-taxane regimens indicated similar response rates when compared to on-TNBC but worse OS. In a recent randomized phase II study of iniparib, a novel anticancer agent for TNMBC, the gemcitabine/carboplatin combination arm had a progression-free survival of 3.6 months, exhibiting a median overall survival of only 7.7 months. 41% exhibited a response to this therapy in a randomized phase II study. Patients were divided per the addition of chemotherapy such as gemcitabine plus carboplatin either alone or with the addition of Iniparib. The phase III clinical data did not prove efficacious during the company’s presentation during 2011 ASCO Annual Meeting. There is also a risk that patients at risk from BRCA1 or BRCA2 mutation are more sensitive to this therapeutic regimen.
Metronomic, dose dense, or high-dose regimens with existing chemotherapies showed benefit in TNBC patients. In a clinical trial that administered metronomic doses of current chemotherapies to cancer patients, event-free survival benefit from high-dose chemotherapy ( HDC) at 1-year hazard ratio =1.8, P<0.00001) and at 5 years the hazard ratio =2.8, P=0.04) was reported in a total of 850 patients. Prognosis is very poor with already existing cytotoxic therapies at chemotherapeutic dosages.
88 Approved Therapies for TNBC:
The European Medicines Agency (EMA) approves Eribulin for patients that received prior anthracycline and taxane-based regimen. This drug was approved for patients that received at least two chemotherapeutic cycles for advanced malignancies. A global phase
III trial EMBRACE was conducted paving the way for approval in Europe. Close to 762 patients were studied and assessed for the two treatment arms (physician’s choice vs. eribulin). TPC was the physician’s choice, meaning - trastuzumab, paclitaxel, and carboplatin. The median overall survival was higher for the eribulin arm - with 13 months vs. 11 months in the TPC arm. 74% of the arm had patients with EGFR receptor negative, and 19% had TNBC. Hormone receptor- negative patients showed a 34% decrease in the risk of death compared to TPC arm. RNBC patients had a 29% risk reduction, and was less efficient in patients without a treatment cycle including capecitabine but was administered eribulin.
Ixabepilone is an epithilone antimicrotubule agent that was FDA approved in the year
2007. It is targeted for locally advanced or metastatic breast cancer in combination with capecitabine after the failure of anthracycline/taxane therapy. FDA granted approval following two pivotal studies
In the first study, close to 113 patients with metastatic breast cancers were evaluated who progressed after treatment with prior anthracycline, taxane, and/or capecitabine therapy.
This control arm received ixabepilone as monotherapy and showed an ORR of 18% with
14% patients showing stable disease conditions of >/ 6 months. The median duration of response was 5.7 months
89 PFS was 3.1 months
OS was 8.6 months.
This preliminary study was followed by a phase II trial (N=752) comparing ixabepilone and capecitabine with only capecitabine in patients pretreated with anthracyclines or resistant, and resistant to taxanes in locally advanced or metastatic breast cancer conditions. The arm treated with ixabepilone plus capecitabine showed a 25% reduction in the estimated risk of disease progression (HR=0.75, 95% CL 0.64-0.88) compared with patients who received capecitabine only. ORR was also greater for patients in the ixabepilone arm (35% vs. 14% for capecitabine). Grade ¾ related adverse events were reported in the ixabepilone group, with a greater rate of neuropathy (21% vs. 0%, fatigue
(9% vs. 3%) and neutropenia (68% vs. 11%)
Neuropathy associated risks vs. benefits outweighed and as a result, the EMA did not approve the drug for use in Europe.
The response to ixabepilone and capecitabine in clinical trials phase III subsets has been reported to be superior in TNBC patients. PFS improved (4.2 vs 1.7 months for capecitabine alone) HR = 0.63, 95% CI 0.52 – 0.77, P< 0.0001. Ni increase in OS was reported. Single partial response in a small study was reported in patients with taxane- resistant TNBC
Monoclonal Antibodies Conjugated with Chemotherapeutics is the Optimal Way mAbs like bevacizumab, and other antibodies are capable of targeting proteins
90 overexpressed during tumor like the vascular endothelial growth factor and have shown effectiveness in previously conducted clinical trials.
Anti-VGF antibody bevacizumab has shown efficacy in combination with cytotoxic agents and taxanes. Nanochain needs to prove that it is more efficacious than mAbs. The taxane and anthracycline combination treatment is quite a popular method of administering drugs in TNBC patients, but needs to show clinical efficacy. In a clinical trial called Ribbon 1, there was a significant increase in the PFR in patients administered with bevacizumab and an anthracycline/taxane regimen from 4.2 to 6.1 months in a patient population of 137. The overall survival did not see a significant difference with
6.5 vs. 6.2 months.
8.1 vs. 5.4 months was the median PFS in patients treated with bevacizumab plus chemotherapy than in those treated with chemotherapy proving that a monoclonal drug based drug regimen is efficacious.
In a trial called Ribbon-2, 159 patients were administered with and without the bevacizumab and chemotherapeutics based regimen. PFS with bevacizumab was 6.0 versus 2.7 months for chemotherapy alone. Bevacizumab was used in a large clinical trial, and the FDA concluded that the overall survival of these patients was not significant from the ones in the arm that did not have bevacizumab. Bevacizumab plus paclitaxel is administered as first-line therapy as recommended by the National Comprehensive
Cancer Network and also retains the label in a recent EMA decision as first-line treatment of metastatic breast cancer.
91 Anti-VEGFR Tyrosine Kinase Inhibitors Sunitinib and Sorafenib have shown anti- tumor properties in TNBC populations. A related clinical trial showed close to 15% response rate reported for sunitinib in a phase II trial. Neither of them is approved for metastatic breast cancer. EGFR directed monoclonal antibody cetuximab is FDA and
EMA-approved for the treatment of colorectal and head and neck cancer. EGFR is overexpressed in 60% of basal-like tumors and is a negative prognostic marker in TNBC.
Cetuximab in combination with cisplatin yielded an ORR of 20% vs. 10% cisplatin alone for the treatment of metastatic TNBC. PFS was 3.7 vs. 1.5 months. There were some adverse events reported like a rash, neutropenia, and fatigue. Activity was also reported in combination carboplatin and irinotecan. It is not yet approved for the treatment of
TNBC.
Apart from the competition mentioned above, there are several other monoclonal antibodies in various clinical trials. Another clinical trial with cetuximab added to carboplatin and irinotecan led to a response rate of 49% versus 30% of chemotherapy.
The study did not improve the PFS interval.
There are several promising novel molecules in various phases of clinical trials. Notably, numerous monoclonal antibodies in development have the potential to improve efficacy while reducing toxicity. As noted throughout this thesis, almost all the monoclonal antibodies are used as secondary or tertiary lines of therapy.
92 Appendix
Exhibit 1
Commonly used efficacy endpoints in oncology clinical trials: advantages and limitations
Endpoints Definition Advantages Limitations
Overall Time from Universally May require a larger survival (OS) randomization* until accepted measure trial population and
death from any cause of direct benefit longer follow-up to
Easily and show statistical
precisely measured difference between
groups
May be affected by
crossover or
subsequent therapies
Includes deaths
unrelated to cancer
Progression- Time from Requires small Validation as a free survival randomization* until sample size surrogate for
(PFS) disease progression or and shorter follow- survival can be
death up time compared difficult in some
Time to Time from with OS treatment settings progression randomization* until Includes Not precisely
(TTP) objective tumor measurement of measured (ie,
progression; does not stable disease (SD) measurement may be
93 include deaths Not affected by subject to bias)
crossover or Definition may vary
subsequent among trials
therapies Requires frequent
Generally based on radiologic or other
objective and assessments
quantitative Requires balanced
assessment timing of assessment
among treatment arms
Time to Time from Useful in settings Does not adequately treatment randomization* to in which toxicity is distinguish efficacy failure (TTF) discontinuation of potentially as from other variables,
treatment for any reason, serious as disease such as toxicity
including disease progression (eg,
progression, treatment allogeneic stem
toxicity, and death cell transplant)
Event-free Time from Similar to PFS; Initiation of next survival randomization* to disease may be useful in therapy is subjective.
(EFS) progression, death, or evaluation of Generally not
discontinuation of highly toxic encouraged by
treatment for any reason therapies regulatory agencies
(eg, toxicity, patient because it combines
preference, or initiation of efficacy, toxicity, and
94 a new treatment without patient withdrawal
documented progression)
Time to next Time from end of primary For incurable Not commonly used as treatment treatment to institution of diseases, may a primary endpoint
(TTNT) next therapy provide an Subject to variability
endpoint in practice patterns
meaningful to
patients
Objective Proportion of patients with Can be assessed in Not a comprehensive response rate reduction in tumor burden single-arm trials measure of drug
(ORR) of a predefined amount Requires a smaller activity
Duration of Time from documentation population and can response of tumor response to be assessed earlier,
(DoR) disease progression compared with
survival trials
Effect is
attributable
directly to the
drug, not the
natural history of
the disease
Source: [Oncology Endpoints in a Changing Landscape]
95 Exhibit 2: Sales of mAbs Source: Paszko, E. and M.O. Senge. "Immunoliposomes".
CMC 19.31 (2012): 5239-5277. Web
96 Appendix 3: Stages of Breast Cancer
(Source: http://www.breastcancer.org/symptoms/diagnosis/staging#stage0)
Stage 0 Stage I Stage II Stage III Stage IV
IA IB IIA IIB IIIA IIIB IIIC
Non Invasi No Invasive Invasive Invasive Inva Invasive Metast
Invasive ve tumor sive atic
Breast breast in tumors
Cancers cancer breast that
instead, spread
small to the
grops lungs,
of cells distant
larger lymph
than nodes,
0.2 skin,
milliete bones,
rs but liver,
not or
larger brain.
than 2
millime
ters are
97 found
in the
lymh
nodes
No Tumor Tumor No No No The There evidence measu in the tumor in tumor in tumor in tum may be
res up breast the breastbu the or no sign
to 2 and not breast, t tumor breast or may of
centim larger but > 2 the be cancer
eters than 2 cancer millimet tumor any in the
centime larger ers is can be size breast
ters. than 2 found in any size. and or, if
Presenc millimet 1 to 3 It is has there is
e of ers is axillary found in spre a tumor,
smaller found in lymph 4 to 9 ad it may
groups 1 to 3 nodes or axillary to be any
larger axillary the lymph the size and
than lymph lymph nodes or ches may
0.2 nodes or nodes in the t have
millime in the near the lymph wall spread
ters but lymph breast nodes and/ to the
not nodes near the or chest
larger near the breast skin wall
98 than 2 breast bone of and/or
millime bone the the skin
ters in brea of the
the st breast
lymph and
nodes. caus
ed
swel
ling
or
an
ulce
r
Ductul Not The the the carcino metast tumor tumor is cancer ma asized measures larger has
2 than 5 spread
centimet centimet to 10 or
ers or ers; more
smaller small axillary
and has groups lymph
spread to of breast nodes
the cancer
99 auxillary cells lymph (larger nodes than 0.2
millimet
er but
not
larger
than 2
millimet
ers) are
found in
the
lymph
nodes
Tumor the The larger tumor is cancer than 2 larger has centimet than 5 spread ers but centimet to not ers; lymph larger cancer nodes than 5 has above centimet spread or
100 ers and to 1 to 3 below has not axillary the spread to lymph collarbo the nodes or ne axillary to the lymph lymph nodes. nodes
near the
breastbo
ne
(found
during a
sentinel
lymph
node
biopsy)
The
cancer
has
spread
to
axillary
lymph
101 nodes or
to
lymph
nodes
near the
breastbo
ne
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106 References
1 Brian Ell and Yibin Kang, Transcriptional control of cancer metastasis
2 Ibid, page 3
3 Ibid, page 3
4 Ibid, page 5
5 Ibid
6 NIH Reporter
7 E. Paszko and M.O. Senge, Immunoliposmes, page 1
8 E. Paszko and M.O. Senge, Immunoliposmes, page 3
9 E. Paszko and M.O. Senge, Immunoliposmes, page 6
10 Scharlach C1, Warmuth C1, Schellenberger E Determination of blood circulation times of superparamagnetic iron oxide nanoparticles by T2* relaxometry using ultrashort echo time (UTE) MRI
11 Source: Hospital Cost, Cancer Patients, Medical Diagnosis Related Group, Oncology
45, Pages 401 - 405
12 The therapeutic monoclonal antibody market, Dawn M Ecker, Susan Dana Jones, and
Howard L Levine
13 American Society of Clinical Oncology, Pembrolizumab in Patients With Advanced
Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study
14 E. Paszko and M.O. Senge, Immunoliposmes, page 3
15 Ibid
16 Ibid
17 Ibid, page 4
107