DRUG DELIVERY: FUNDAMENTALS AND APPLICATIONS

This is the story of our low-temperature-sensitive liposome (LTSL) (Needham et al. 2000), subsequently called ThermoDox^® by the company Celsion Corporation which licensed it from Duke University in November 1999. It is an account of “lessons learned,” as told by me, as I saw it, experienced it, and lived it, and so much of this will be written in the first person. Everyone has their own story. I, myself, Celsion, and ThermoDox^® have ours. As Bernard Malamud says in his book, The Natural, “We have two lives, the one we learn with, and the life we live after that” (Malamud 1952). This certainly applies to me and Celsion. So I write this not for Celsion, or for Duke, but for the next generation of inventors, carers, and entrepreneurs, who have a life to learn with and perhaps have yet to make their own mistakes or achieve their own real and verifiable successes. This chapter contains some of what you might have in store. I encourage you to heed these “lessons learned.” See if you can benefit from the positive results and avoid the ones that, let’s say, made life more difficult. At least be aware of what could happen if you, as the inventor, trust anyone else with your precious invention, which you will have to do. I know this is a long and detailed story, but please try and make it all the way to, what will hopefully be, a happy ending.

23.1.1 IN THE LIPOSOME FIELD ITSELF

There are always “lessons to be learned,” and the liposome field is no exception, especially with its event-rich and inextricably linked journey from research to commercialization. Although slightly before my own time in liposomes,* but not in research (Eley and Needham 1984; Needham 1981), the liposome field rapidly progressed in the mid- to late 1970s. The challenge for the early pioneers was to identify where this seemingly high potential impact, biocompatible, emerging clinical technology could, in fact, find its therapeutic application. Immunology and especially anticancer drug encapsulation and targeting took the center stage. Thus, once liposomes had been “discovered” (Bangham and Horne 1964), much of this pioneering work was led by luminaries such as Dimitri Papahadjopoulos and Gregory Gregoriadis and included reports on making and characterizing liposomes in terms of structure and permeability (Papahadjopoulos and Miller 1967; Papahadjopoulos and Watkins 1967), the development of liposomes for encapsulation (Gregoriadis and Ryman 1971; Gregoriadis et al. 1974), as adjuvants (Allison and Gregoriadis 1974), and targeting (Gregoriadis and Neerunjun 1975), and even early patents (Allison and Gregoriadis 1977). Gregory was also an enthusiastic and prolific reporter of each new advance in liposomes, bringing together the relatively small community of researchers in the then nascent field, through his series of review papers (Gregoriadis 1976) and edited books (Gregoriadis 1979), including the extremely influential three-volume Liposome Technology (Gregoriadis 1984), a series that still continues in its third edition (Gregoriadis 2010).

In his 1975 paper on homing liposomes, the abstract reads as follows:

The possibility of homing liposomes to target cells was investigated. Liposomes containing an antitumor drug and associated with molecular probes, which exhibit a specific affinity for the surface of a variety of normal and malignant cells, were prepared. In vitro and in vivo experiments suggested that such probes were capable of mediating selective cellular uptake of the associated liposomes and the entrapped drug. It is anticipated that liposomes designed to home may become important tools in the control of cell behavior.

This is 1975! This kind of “homing” work is still going on today in liposomes and, indeed, with a whole host of “nanoparticles.” Drug encapsulation and targeting is still being researched, and attempts are still being made to test clinically and eventually commercialize these approaches. Forty years later, it’s still a challenge. While I was trying to give advice from our own experiences with ThermoDox^®, I suggest that these early papers still have something to teach us (especially the new generations entering the field of “nanomedicine”) about both research and the decision-making process during commercialization of all nanoparticles for drug delivery and imaging. So just because you and your advisor are choosing to work on nanoparticles of gold, iron oxide, chitosan, poly(lactic-co-glycolic acid), etc., and even antibodies, does not mean you should not look up, read, study, understand, and use this literature on liposomes to plan your studies and interpret your results.

23.1.2 EXAMPLE OF AN EARLY “LESSON LEARNED” IN LIPOSOMES: IS IT BEING HEEDED TODAY IN NANOMEDICINE?

For example, these early applications were hampered by the now relatively well-understood opsonization phenomenon that labels most nanoparticles in the bloodstream as foreign for removal by the reticuloendothelial system (RES) (Roerdink et al. 1981). In their early formulations (Gregoriadis et al. 1971, 1974), Gregoriadis and colleagues had used a composition of 40 μmol of phosphatidylcholine, 11.4 μmol of cholesterol, and 5.7 μmol of the negatively charged phosphatidic acid (molar ratio 7:2:1), giving liposome that was 10 mol% negatively charged. When Alec Bangham took me out to a Chinese dinner on the eve of my giving his 80th Birthday Lecture at Gregory’s 2001 5th International Conference on Liposome Advances, in London (Needham 2001a), he told me a story from those early days. Alec had explicitly advised that the initial strategies to make the liposomes “repulsive,” by including negative charge, which was thought to be electrostatically stabilizing, would conversely only result in liposomes going to the RES faster than ever. This has since been proven (Allen et al. 1988) and is fairly well understood (Liu et al. 1995) in serum and serum-free media (Rothkopf et al. 2005). As we now all appreciate, this opsonization to Kupffer cell removal of the liposome in the bloodstream was solved by applying earlier work on the PEGylation of proteins (Beauchamp et al. 1983), and the invention of the so-called “stealth” liposome (Allen 1989), with its sterically stabilizing polymer (Allen and Gabizon 1990; Klibanov et al. 1990), forming the basis for Doxil^® (see Barenholz’s review and references therein, Barenholz 2012). There is also a lesser-known mechanism of a mechanical effect of high cholesterol content in PC lipids or sphingomyelin (Kim and Needham 2001; Needham and Nunn 1990), which gives a similar long circulation half-life (Lasic and Needham 1995), used to good effect (slow leakage and long circulation) in the vincristine liposome (Boman et al. 1993, 1994; Kanter et al. 1994; Krishna et al. 2001; Webb et al. 1995, 1996).

I mention these early studies and “lessons learned” because newbies (and “oldbies”) to the field of nanomedicine would do well to read through some of these early reports and seek to inform their own current research strategies. “Nanomedicine” does seem to know all about PEG-derived steric stabilization, but is it as well appreciated in the nanomedicine literature that a tight lipid-surface is also not well opsonized and that an amount of negative charge of only 3 mol% or greater (Allen et al. 1988) can send your precious nanoparticle rapidly to its RES fate?

23.1.3 READ THE LITERATURE

As Frank Szoka (who entered the field in its “golden age”; Szoka and Papahadjopoulos 1978), famous for ranting in an editorial Commentary: Rantosomes and Ravosomes (Szoka 1998), reminded us, quoting the philosopher, essayist, and poet, George Santayana: “Those who cannot remember the past are condemned to repeat it.” The example Frank chose to illustrate this was liposomes carrying anti-HIV agents. He points out that

The difficulties with the approach could be anticipated based upon the early liposomal anti-cancer drug delivery literature. The failure of those working in the field is not because they repeated previously published studies. But rather because they did not translate the lessons from the early studies into a therapeutic strategy that complemented the pathophysiology of the disease, with the pharmacological aspects of the drug and the delivery properties of the liposome.

Thus, to reiterate in this editorial, Frank’s amendment to the Santayana quote was

Those who cannot translate the lessons of the past are condemned to repeat it.

Frank, correct me if I am wrong, but basically what I think you were saying is

Those of you who do not read the literature are condemned to repeat it! And guess what? We have already done it, and we are tired of hearing about your ‘new work’ at meetings, or seeing what you think is new and exciting in your grant proposals, that we have to take our precious time to read through, review, and reject.

Back then, the (liposome) world was certainly smaller, and as Szoka also points out, many of the potential uses of liposomes in drug delivery that have since come to be were discussed in those two 1976 review articles by Gregory Gregoriadis (1976). With only a few people involved, “discussions raged over materials, mechanisms, models, methods and structures” at the handful of liposome/drug carrier meetings that everyone attended.

Many lessons were, in fact, learned back then, face-to-face, between researchers during the meetings. But today, even with (and perhaps because of) easy access to the (voluminous) literature, this face-to-face contact is easily lost, and so the repetition of research and, indeed, the mistakes, still go on today. Frank, who rants on average about every 15 years, has another one. This time, it is directed at all nanomedicines and the people who research and develop them. Entitled “Cancer nanomedicines: So many papers and so few drugs!” (Venditto and Szoka 2013), folks would do well to heed it, as this most comprehensive review identifies a timeline to nanomedicine anticancer drug approval using the business model of inventors, innovators, and imitators. Unfortunately for today’s researchers who are still involved in “liposomes,” there is a lot of literature, some 48,772 publications currently listed (February 28, 2015) on a PubMed search for “liposome.” And incredibly, 339,246 patents that concern “liposome” at free patents online. “Lessons learned” from liposomes, like Chezy Barenholz’s landmark paper Doxil^®—the first FDA-approved nanodrug (Barenholz 2012), will undoubtedly apply to all nanomedicines being researched and developed today. With only 9422 papers found on PubMed for a search of “nanomedicine,” it is likely that “your precious nanomedicine” shares many of the problems that were already figured out by the liposomologists, maybe before you were born or, at least, while you were still in grade school. So where would a company turn to, in order to get the most expert and up-to-date knowledge about their licensed product? Would they sift through the 48,772 papers and pay a law firm $gazillions to search through the 339,246 patents? No. Maybe try asking the inventor, and first see what he or she has to say (and more about that relationship later).

23.1.4 EARLY COMMERCIALIZATION AND ACCESS TO A WEALTH OF EXPERTISE: JUST ASK US

Early commercialization efforts were considerable (as well as contentious; Free Library 2014; NeXstar Pharmaceuticals 1997). From my perspective, there were four principal liposome companies: three were established in the United States: Liposome Technology Inc. (LTI) in Menlo Park, California; The Liposome Company Inc. in Princeton, New Jersey; NeXstar, based in San Dimas, California; and Inex in Vancouver, Canada, first as a spin-off from TLC and then in its own right. These were the main entities that, for me, paved the way in mainly anticancer liposomal therapeutics, took relatively similar drug delivery ideas, and brought them through the development and translation to commercialization. Two companies focused on liposomal doxorubicin, LTI (Doxil^®) and The Liposome Company (Evacet^TM and Myocet^®), NeXstar went with daunorubicin (DaunoXome^®) and also successfully developed amphotericin B (AmBisome^®), and Inex developed liposomal vincristine (Marqibo^®).

With academic researchers involved at varying levels of invention, innovation, and continued support, there is a whole host of very experienced individuals (still) around the conference, industrial, and grant-review world, whom students, postdocs, and young faculty new to the field can call on for advice and informal consultation. So, if you see Chezy Barenholz (Barenholz 2012), Frank Martin, or Martin Woodle et al. (Barenholz and Haran 1994; Papahadjopoulos and Skoza 1980; Woodle et al. 1989) ex-LTI-Sequus; Andy Janoff et al. (Janoff et al. 1989, 1996; Mayer et al. 1997) ex-TLC; Pieter Cullis, Lawrence Meyer, Marcel Bally, or Murray Webb et al. (Bally et al. 1997; Webb et al. 1996; Wheeler et al. 1999), ex-Inex; or Gary Fujii, Bill Ernst, Jill Adler-Moore, or Su-Min Chang et al. (Adler-Moore and Chiang 1997; Adler-Moore and Ernst 1997; Proffitt et al. 1999; Schmidt and Fujii 1998) ex-NeXstar; or very knowledgeable and outspoken independents, like Frank Szoka (Papahadjopoulos and Skoza 1980), Theresa Allen (Allen 1989; Allen and Gabizon 1990), or Valdimir Torchilin (Torchilin et al. 2006), or me and Dewhirst, at a meeting or seminar, we are all inventors of some of the earliest or later patents and a host of peer-reviewed papers that have contributed to those 48,772 publications. You might ask us one or more of the questions I am hoping to address in this chapter: What was the original observation that motivated the product? How did you or your colleagues have it? What was the question you tried to answer? What was the scientific hypothesis that you and your colleagues addressed? What funds were available for initial development and how did you get them? How did you develop it, including preclinical animal studies, and what were the results? What collaborators did you work with? What was the initial licensing deal and how was it structured and with whom? How did you manage the scale-up of manufacturing? What particular disease did you decide to treat and why? What did the Food and Drug Administration (FDA) require you to write in order to obtain an Investigational New Drug (IND) application? What funds did you have to raise, in order to carry out the IND application and how did you get them? How did you manage the Phase 1 trials, and what was the result? How did you recruit the clinicians and the sites to carry out the trials? Were there any unexpected problems implementing the protocols? Was that next stage a Phase 2 or did you go straight to Phase 3 human clinical trials? What was the next documentation you had to provide to the FDA and how long did it take to move to this next stage? What was the protocol? How many patients and sites did this Phase 3 involve, and how was it all managed? How did it turn out? Are you still in business? If so, what are your future plans for your drug delivery technology? In your view, what’s next? What’s the next big thing in advanced drug delivery and why? The answers are all available if you would just ask.

23.1.5 THIS CHAPTER

Invented in 1996 (Needham 2004), the LTSL is now in Phase 3 human clinical trials for liver cancer. This chapter will describe the engineering design of the LTSL (Needham 2013) and the licensing and clinical testing that is moving the invention toward commercialization. LTSLs, in conjunction with local mild hyperthermia (HT), can release drug within seconds of entering the warmed tumor vasculature. The released, free drug diffuses into the tumor interstitium, reaching its nucleus target with greater penetration distance, and to much higher concentrations, than those achievable by either free drug or the more traditional long-circulating liposome formulations (Manzoor et al. 2012). Intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by drug diffusion. This establishes a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream, saturating neoplastic cells, as well as endothelia, pericytes, and stroma, with the anticancer drug.

This chapter will attempt to describe the process, the pitfalls, the good, the bad, and the ugly of translating what might be a good research idea through initial funding, development, and preclinical and clinical trials. Whether unique or not (and we suspect the issues highlighted in our particular case are probably quite ubiquitous), I will use, as the main example, the invention, development, clinical testing, and intended commercialization of the thermal-sensitive liposome, subsequently named ThermoDox^®. Additionally, an update of the recent progress in human clinical trials will be given, including Celsion’s Phase 3 for liver cancer, Phase 2 recurrent chest wall (RCW) cancer, and their new trials in metastatic liver cancer, ovarian cancer, pancreatic cancer, breast cancer, and glioblastoma, including the adaptation of high-frequency ultrasound (HiFu) as the source of targeted mild HT. I will say up front that the current Celsion administration should be commended for their pioneering work in taking on this invention, initiating this very impressive series of trials, with multiple sites, in different countries, and in particular for creating and carrying out what actually is the largest randomized double-blind trial ever with radio-frequency ablation (RFA). For example, following on from what was learned in the Phase 3 HEAT study on primary liver cancer, Celsion’s new OPTIMA trial is currently enrolling, with the first patient enrolled Q3-2014. It will include approximately 550 patients in up to 100 sites in North America, Europe, China, and the Asia-Pacific region.

Kudos to Celsion and in particular Mike Tardugno (CEO) and Nick Borys (CMO); it’s not an easy task visiting all these clinical sites and spending multiple days on airplanes.

Following the licensing of my (Oops, sorry, no, it’s not mine!) Duke’s invention by Celsion Corporation in November 1999, ThermoDox^® has now, just over 15 years later, completed a Phase 3 trial in 701 patients with primary liver cancer, where ThermoDox^® + RFA was trialed against RFA alone (Poon and Borys 2009; Wood et al. 2012). There were certainly lessons learned in the science (and all that is published in several reviews; Landon et al. 2011; Needham 2013; Needham and Dewhirst 2001, 2012) and the seminal paper on drug accumulation and penetration by Manzoor et al. (2012). But why we think there are real lessons to be learned in the development and commercialization from all this in particular, is the fact that when the 701-patient Phase 3 primary liver cancer trial results were unwrapped by Mike Tardugno and colleagues, on January 31, 2012 (Tardugno 2013), the headline read as follows:

ThermoDox^® failed to meet its primary endpoint of better progression free survival than the heating modality (RFA) alone.

As described later, data are still being analyzed and some encouraging results are being revealed, and further trials are being conducted including new heating modalities like HiFu. A lot of lessons have been learned (especially) by the inventor and also by his collaborators, the university licensing and ventures office, the faculty patent committee, and the company and the clinicians, which will still bring this particular treatment option to oncologists, their patients, and the clinic. Taken largely in chronological order, this chapter will detail the events and lessons learned and address many of the questions listed in Section 23.1.4 so that others, with potentially good ideas, compelling in vitro, and in vivo data, can perhaps have a smoother path to testing, and, if successful, commercialization of their own idea.

The dedicated Section 23.7 on “Lessons Learned” will present six main topics:

• Your university administration

• Your Office of Licensing and Ventures (OLV)

• Your license agreement

• Meeting the milestones

• Your (inventor’s) relationship with the company

• The distribution company

I hope this will form a basis for discussion, if not, identify pitfalls that others might avoid, or at least be aware of, and some of the reasons they could occur. There will also be a few lessons as we go, in dedicated boxes, just for good measure.

The chapter will end with a brief description of an approach we are currently pursuing to formulate especially hydrophobic anticancer drugs specifically to metastatic disease. This approach has again benefited from lessons learned in the liposome and other nanomedicine fields. It is an endogenous one, which is based on the way nature delivers her own hydrophobic molecules in the body, what we call “Put the Drug in the Cancer’s Food.” And it gives me one last chance to make a difference. With my group here in Odense, Denmark, I want to do it in such a way that it is not encumbered by the kinds of events that have hampered ThermoDox^® and other products. I therefore introduce what might be a surprising concept, translating drug delivery without profit and open-source pharmaceuticals, especially for cancer. Whether this approach might help to eliminate the negatives that sometimes cloud these translational issues of hidden agendas, incompetence, and greed and replace them with transparency and the right expertise for the right job, and an opportunity to provide healthcare to the people, which is not encumbered by the promise of huge profits, is still to be determined. This may not be possible in some countries, but the more socially minded ones, perhaps in Europe, indeed like Denmark, the “cancer capital of the world” (WCRF International 2015), could maybe lead the way in this new thinking about translating drug delivery and making our research advances actually available to the people who suffer, at cost.

23.2 WHAT WE KNEW IN 1995

The story proper starts with the initial motivation by Dr. Mark Dewhirst, the then director of the Duke Hyperthermia Program, who, in about 1995, quite explicitly said “Liposomes aren’t working, Hyperthermia isn’t working, I need something I can heat and it releases drug, damn it.” It was Mark then, who motivated the need to come up with a possible solution: a fast drug-releasing thermal-sensitive liposome, disclosed to Duke in 1996 (Box 23.1).

As shown in Figure 23.1, in 1995, three timelines intersected:

1. Dox was discovered and was one of the first choices of drug for encapsulation in some of the first liposomes to be made for testing against cancer.

2. HT as a treatment modality had been developed and was being used to heat tumors.

3. Lipid membrane mechanochemistry used micropipette manipulation techniques to measure the properties of single-walled lipid vesicles.

BOX 23.1 LESSON AS WE GO #1: LISTEN TO YOUR CUSTOMER

And here’s the first lesson. Listen to the people on the front lines, the clinicians, the veterinarians, and the directors of centers, and see what “your customer” might actually want. As I discuss at the end of this chapter, the new formulation we are now working on was requested, or at least motivated, by a need expressed by several actual medical oncologists, asking, “can you help us to reformulate lapatinib, niclosamide, fulvestrant, abiraterone, and orlistat?” So does your formulation fulfill an actual clinical need? If not, consider not doing it (at least not just for the papers) and challenge your PhD or postdoc advisor and ask them: Does what you want me to do pass the “so what test”? Is it useful knowledge? Does it even attempt to meet and unmet need? If you are not sure, you probably know the answer, but you still might want to canvas some end users over at your local cancer center.

FIGURE 23.1 In 1995, three timelines intersected. Doxorubicin and its encapsulation in liposomes, hyperthermia used to heat tumors, and lipid membrane mechanochemistry, using micropipette manipulation techniques to measure the properties of single-walled lipid vesicles.

23.2.1 DOXORUBICIN AND LIPOSOMES

When we decided to develop the LTSL technology, we simply inherited the “stealth” liposome concept and the cytotoxic drug, Dox, which had already been used in liposomal delivery. And so, as a prelude to discussing actual ThermoDox^®, it is worth a relatively brief (not exhaustive) review of what we knew about liposomes and Dox in 1995, highlighting a few comparisons, even a few lessons learned as we go, and exactly what it was about liposome technology and its performance at the time that motivated the LTSL invention and its development.

23.2.1.1 Doxorubicin, the Drug

Dox (also known as hydroxydaunorubicin and hydroxydaunomycin; trade name, Adriamycin^®) is an anthracycline antibiotic discovered in the early 1960s by the Farmitalia Research Laboratories of Milan. Clinical trials began in the 1960s, and the drug saw success in treating acute leukemia and lymphoma. It was approved for use in the United States in 1974. However, by 1967, it was already being recognized that Dox (and another anthracycline chemotherapeutic, daunorubicin), while active against cancer, could produce fatal cardiac toxicity.

FIGURE 23.2 Doxorubicin (Adriamycin). (a) Molecular composition and structure. (b) Diagram of two doxorubicin molecules intercalating DNA.

The structure and mechanism of action of Dox is shown in Figure 23.2. It is known to intercalate between DNA base pairs (Frederick et al. 1990), resulting in DNA and DNA-dependent RNA synthesis inhibition due to template disordering and steric obstruction (Abraham et al. 2005). By virtue of this ability to intercalate with DNA, it stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing DNA double helix from being resealed, stopping the process of replication (Frederick et al. 1990).

Dox has one of the widest spectrums of any neoplastic agent and is commonly used to treat Hodgkin’s lymphoma and some leukemias, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, and others. Toxicities exhibited include myelosuppression, alopecia, mucositis, nausea, vomiting, and cardiomyopathy. Due to the cardiotoxicity being additive, the cumulative lifetime dose is limited to ≈550 mg/m². The cardiotoxicity is most likely a consequence of free radical generation and binding of the drug to cardiolipin in the heart muscle.

Dox also interacts with the cell’s electron transport chain, to lead to the formation of superoxide anion radicals and hydrogen peroxide, which has a very damaging effect on the cellular components. Because of its anticancer activity and also because of its intense toxicity, its encapsulation in liposomes was one of the biggest early successes. Liposomal encapsulation reduced its toxicity. The flip side of reduced toxicity though could be reduced efficacy, and subsequent clinical data support this formulation conflict (O’Brien et al. 2004), where in first-line therapy for Metastatic Breast Cancer, PEGylated liposomal Dox (PLD) significantly reduced cardiotoxicity, myelosuppression, vomiting, and alopecia but only provided comparable efficacy to free Dox. Still, with the widespread prescribing of Dox, as O’Brien et al. suggest, for elderly patients and patients with specific cardiac risk factors, PLD was an important new therapeutic option.

As is well known, after intravenous (i.v.) dosing, Dox blood levels fall dramatically as the drug distributes into the tissues, followed by a slow elimination phase due to renal and biliary clearance and metabolism. Hence, worth reproducing, and as shown in Figure 23.3, Dox itself has a half-life of only ≈1 minute (Gustafson et al. 2002). But, as a chemical, Dox has some very interesting physicochemical properties. With a pKa of 8.3, it is a weak base cation, such that at pH 7.2, ≈10% of it is uncharged and 90% of it is positively charged. What this means is that the uncharged fraction of Dox can pass through the whole tissue, simply by concentration-dependent, Fickian diffusion. Permeation (and thus, loss) of the neutral form through the membranes drives the equilibrium disassociation reaction forward, releasing more uncharged fraction in the interstitial fluid, which is then available for further transit through each cell membrane. Uncharged Dox also partitions into the cytoplasmic and organelle membranes; it passes through and may have a direct, or indirect, toxicity effect there too. In fact, following Dox treatment, cardiotoxicity develops through the preferential accumulation of iron inside the mitochondria, due to Dox becoming concentrated in the membranerich mitochondria and increasing both mitochondrial iron and cellular reactive oxygen species levels (Ichikawa et al. 2014). Also, because of this dual solubility afforded to such weak base cations, once partitioned in, it takes a long time to get back out. It could be lethal in so many ways, if only we could get the drug just to the tumor vasculature, and in high quantities.

FIGURE 23.3 Plasma levels of doxorubicin in dogs, after a 20 minutes i.v. infusion at a dose of 30 mg/m². (From Gustafson, D.L., Rastatter, J.C., Colombo, T. et al.: Doxorubicin pharmacokinetics: Macromolecule binding, metabolism, and excretion in the context of a physiologic model. J. Pharm. Sci. 2002. 91(6). 1488–1501. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)

23.2.1.2 Doxorubicin Encapsulated in Liposomes

Dox was already approved and formulated in a non-PEGylated liposomal form (Myocet^®, formerly known as Evacet^™) (Abraham et al. 2005) but, as predicted by Bangham from the outset, “traditional” liposomal formulations showed too rapid a circulatory clearance and little efficacy. This was followed by Doxil^® (marketed as Caelyx^® in Europe), the PEGylated “stealth”* liposome (reviewed comprehensively in the edited book Stealth Liposome by Lasic and Martin, 1995; also Lasic and Needham, 1995 and also described in Chapter 5, Section 5.5.1.2). Although it was later than the traditional liposomes in its development, Doxil^® was the first liposomal pharmaceutical product to receive U.S. FDA approval in 1995, for the treatment of chemotherapy refractory, acquired immunodeficiency syndrome–related Kaposi’s sarcoma. Thus, as we were thinking about how to solve the problem that Mark Dewhirst identified, the field was buzzing with news of FDA approval for Doxil^®. Doxil^® certainly played a huge part in getting this liposomal technology into mainstream use, and as reviewed recently by Chang (Chang and Yeh 2012), as of 2012, there were 12 liposome-based drugs approved for clinical use and more are in various stages of clinical trials.

FIGURE 23.4 Intravital microscopy images of stealth liposomal injection, circulation, and extravasation into the tumor interstitium. (a) Bright-field image of tumor vasculature through window, (b) 1 minute after injection, (c) 90 minutes after injection (reduced camera gain). Larger blood vessels are about 30 μm in diameter. (Reproduced from Wu, N.Z. et al., Cancer Res., 53, 3765–3770. Copyright 1993, American Association for Cancer Research. With permission.)

The success of Doxil^® and its stealth, PEGylated polymer design was to escape RES uptake and create a long-circulating liposome that could stand a chance of extravasating into the tumor interstitium via the purported enhanced permeability and retention (EPR) effect. We had even already shown this ourselves in 1993 (Wu et al. 1993). Figure 23.4 shows the intravital microscopy images after fluorescently labeled stealth liposomes were injected i.v. into rats bearing Dewhirst’s dorsal skin-flap window chambers, containing a vascularized mammary adenocarcinoma.

The bright-field image in Figure 23.4a shows the complex and resurgent flow of the tumor vasculature, where, for scale, the larger blood vessels are about 30 μm in diameter. Figure 23.4b is taken just 1 minute after i.v. injection of the 82 ± 24 nm diameter stealth liposomes into the rat’s tail vein (Wu et al. 1993). Switching the microscope to epifluorescence illumination, the liposomes are clearly flowing in the blood vessels and, even at this early time point, have started to extravasate into the tumor interstitium. Ninety minutes later, not yet having set our optimal low-light-level camera settings, we had to turn the gain on the fluorescent camera down to get the last image in Figure 23.4c, where there was a significant, but heterogeneous, accumulation in the tumor perivascular space.

As shown in Figure 23.5, when the data were quantitatively analyzed, the longer circulation halflife correlated with there being more stealth liposome accumulation in the tumor interstitium than for the more conventional non-PEGylated (91 ± 41 nm diameter) liposomes.

And with all this actual in vivo data, we were part of helping Doxil^® to get approved, by showing that the extravasation actually happened in real time within 90 minutes or less. In fact, normal vasculature showed no extravasation at all. The importance of these data then was that it seemingly supported the Doxil^® data from Vaage and Mayhew (Vaage et al. 1992) that had prompted our window chamber study in the first place. The EPR effect was alive and well (in this subcutaneous animal model). The passive accumulation of long-circulating stealth liposomes was an unqualified success (again, it must be emphasized “in this subcutaneous animal model”).

So, why would anyone need to improve on this?

It didn’t take long for data to start accumulating in the literature that even though there was now a long enough time to allow for passive extravasation, there were other challenges:

• Stealth liposomes might not be able extravasate to the extent that we had been seeing in subcutaneous animal tumor models.

• If they did, “this is as far as your liposomes can go,” i.e., only as far as the perivascular space.

• As Yuan et al. in 1994 had shown, using a similar window chamber technique (Yuan et al. 1994), the intramural accumulation of liposomal fluorescent spots (observed within 5 minutes after liposome injection) could be continuously observed for up to 2 weeks.

• No one knew exactly how leaky a human tumor vasculature really was to these ≈100 nm liposomes.

• No one was really sure to what extent, and at what rate, the liposomes, which had been well optimized for the best encapsulation of Dox, could even release their drug in the tumor interstitium.

• In very recent studies that have sought to provide a mechanism whereby Doxil^® could potentially leak its drug, in an environment of ammonia (Silveram amd Barenholz 2015), it was found that PLD without ammonia had a “very poor cytotoxicity,” demonstrating again that Doxil^® does a very good job of retaining its drug, but is not designed to release it.

FIGURE 23.5 Time courses of normalized vascular and interstitial liposome amounts for stealth and conventional liposomes in tumor tissues. (a) Averaged vascular decay and interstitial accumulation of stealth liposomes from nine tumor preparations. (b) Averaged vascular decay and interstitial accumulation of conventional liposomes from seven tumor preparations. (Reproduced from Wu, N.Z. et al., Cancer Res., 53, 3765–3770. Copyright 1993, American Association for Cancer Research. With permission.)

So, quietly, between ourselves, Mark Dewhirst and I had questions in 1993–1994, born out of our own preclinical animal studies: “could Doxil^® really deliver enough drug to human tumors to achieve significant efficacy?”

Attempting to improve Doxil’s efficacy by using HT to increase the vascular permeability, the Hyperthermia Center at Duke also carried out two “Doxil^® + HT” clinical trials in ovarian and breast cancers (Secord et al. 2005; Vujaskovic et al. 2010). Unfortunately, the clinical trial evaluating the combination of Doxil^® + HT in patients with persistent and recurrent ovarian cancer did not demonstrate increased efficacy, compared with prior reports using Doxil^® alone. And in a Phase 1/2 study of paclitaxel in patients with locally advanced breast cancer in the preoperative setting, the pathological complete response (pCR) rate was only 9% for liposomal Dox (Evacet^™) + local HT, although the combined (i.e., paclitaxel + liposomal Dox [Evacet^™] + local HT) pCR rate was 61%.

Although we did not know it at the time, therapeutically all these concerns were, in fact, borne out in subsequent human trials, including a trial that was instrumental in gaining Doxil’s approval for ovarian cancer. A trial for metastatic breast cancer showed that Doxil^® really was no better than free drug alone (O’Brien et al. 2004). That is, even though Dox has a half-life of only 2 minutes and Doxil^® has a half-life of 73.9 hours:

• The overall survival was comparable with both treatments.

• Median: PEGylated liposomal Dox 21 months, versus doxorubicin, 22 months; hazard ratio (HR) = 0.94 (95% CI 0.74–1.19).

• At the time of the analysis, approximately 56% of patients in each group had died. When adjusted for potential imbalances in prognostic variables using the Cox regression analysis, the HR was 0.94 (95% CI 0.75–1.19), similar to the unadjusted treatment HR.

Doxil^® was approved for ovarian cancer (Gordon et al. 2001) in 2005, but again, with little therapeutic benefit, when compared to topotecan:

• Time to progression (TTP): Doxil^®, 4.1 months, and topotecan, 4.2 months

• Overall median survival: Doxil^®, 14.4 months, and topotecan, 13.7 months

• 18% reduction in risk of death (hazard ratio [HR] = 1.216)

• Overall tumor response rates: 19.7% (47 patients) in the Doxil^® arm and 17% (40 patients) in the topotecan arm

Compared to topotecan then, improvement in TTP for Doxil^® was 0.1 month, which is only a 2.4% progression-free survival (PFS), and it was approved. TTP was also a measure for ThermoDox^®, and we will see later how it faired (when taken in total, it didn’t fare well), in relation to the FDA-required %PFS stipulated for its approval.

23.2.1.3 New Toxicities for Doxil^®

As mentioned earlier, the initial success of non-PLD (Myocet^®) was to reduce the drug’s cardiotoxicity, by simply encapsulating and retaining it; Doxil^® certainly achieved the same retention and reduced cardiotoxicity profile. However, the long circulation half-life also introduced new toxicities: hand–foot syndrome or palmar–plantar erythrodysesthesia (PPE). PPE is a common dermatologic toxic reaction associated with certain chemotherapeutic agents, including continuous infusion Dox, cytarabine, floxuridine, high-dose interleukin-2, docetaxel, capecitabine, vinorelbine, and gemcitabine. It presents as a redness, swelling, and pain on the palms of the hands and/or the soles of the feet; sometimes blisters appear. In a study in 2007 by Lorusso et al. (2007), the incidence of PPE is increased in patients receiving PLD compared with conventional Dox. In studies that utilized the currently approved dose of PLD (50 mg/m² every 4 weeks), ≈50% of all patients receiving PLD experienced PPE and ≈20% experienced grade 3 PPE. It has been hypothesized that following local trauma associated with routine activities, PLD may extravasate from the deeper microcapillaries in the hands and feet. Interestingly then, that passive accumulation in tumors—which could help in its therapeutic effect—may be compromised because of limited extravasation, whereas a similar extravasation of PLD in active hands and feet introduces a new toxicity (Box 23.2).

Given these successes of the Doxil^® formulation:

• Good encapsulation and retention of a toxic chemotherapeutic during the blood-borne delivery phase

• A reduction in the cardiotoxicity for the drug

• Long circulation half-life that at least gives the chance of EPR

but coupled with its discovered limitations of:

• Low EPR in human tumors

• Introduction of known drug-associated toxicities for the liposome formulation

BOX 23.2 LESSON AS WE GO #2: DESIGN, DESIGN, DESIGN

Do as much forward engineering design for your formulation as possible before and during the research and development phase. A function that has been designed for therapy can often lead to a second negative performance characteristic. In fact, I would encourage you to follow the design methodology I laid out in a review chapter for a different Kinam Park–edited book: “Reverse Engineer the Low Temperature Sensitive Liposome (LTSL)” (Needham 2013). In it, I take the LTSL as an example and outline a design process that not only describes all aspects of the liposome and how it is used, but also provides a general scheme that, when followed for any device or product or “problem that has already been solved,” allows a person to formally reverse engineer that product. In doing so, the process itself becomes an invention generator for material improvement or inspiration for new designs and, when used in a forward engineering sense, is the template for your new research and development effort. As discussed further in Section 23.8, we did that for the LDL project we are pursuing now in Denmark, and this process ensures a more knowledgeable design.

• Limited penetration of the 80 nm diameter liposome into the tumor interstitium

• Lack of effective delivery to all tumor cells (due to the same good retention of the drug)

I felt that in trying to respond to Mark’s need of “something I can heat and it releases drug,” the main challenge was to try to keep the good parts, i.e., the retention of Dox in the liposome while in the bloodstream (although this was somewhat compromised, as we will see later), but to also get the drug out, fast, and only at the tumor. Since there was a well-established maximum tolerated dose (MTD), the challenge was actually less about reducing toxicity and more about increased bioavailability in delivery, thereby gaining actual efficacy.

23.2.2 HYPERTHERMIA

While all this liposomal development was going on, we also knew that tumors could be heated using HT, or at least Mark Dewhirst did. Mark and his colleagues had been working in HT since the early 1980s. In a monograph from 1983 (Oleson and Dewhirst 1983), Oleson and Dewhirst focused on progress at the time regarding “major aspects of the biologic effects of elevated temperatures both in vitro and in vivo, on the physical methods clinically used to produce HT, and on the results of treatment in large animals and humans.” It is well recognized that HT can have at least three different effects on cells:

• Direct cytotoxicity at elevated temperatures, where the degree of cell killing is both time and temperature dependent

• Heat sensitization of radiation, where HT reduces cancer cells ability to repair sublethal and potentially lethal radiation damage

• Synergistic effects with certain drugs, including alkylating agents, nitrosoureas, cisplatinum, bleomycin, and adriamycin, which showed marked synergism above 43°C

There were also distinctions between the temperatures attained and the desired effect. Mild HT is a therapeutic technique in which cancerous tissue is heated above the body temperature to induce a physiological or biological effect but often not intended to directly produce substantial cell death. The goal is to obtain temperatures of 40°C–45°C for time periods up to 1 hour (Issels et al. 2010; Viglianti et al. 2010). In contrast, ablative HT is commonly greater than 55°C, but for shorter durations of 20 seconds to 15 minutes (Wood et al. 2002). An example would be the relatively recent, HiFu (Kennedy 2005), and the older and more traditional, RFA, in treating lesions in primary liver cancer (Tateishi et al. 2005). RFA is used in conjunction with imaging techniques (e.g., ultrasound, computed tomography, or magnetic resonance imaging) to help guide a needle electrode into a cancerous tumor. High-frequency electrical currents are then passed through the electrode, creating heat that destroys the abnormal cells. An area of ≈30 cm² (≈3 cm diameter tumors) can be ablated with a single application of RFA (Goldberg et al. 1996). While HiFu is a technique that Celsion is now planning to use, RFA was the heating modality Celsion chose to use in the human clinical trials, in order to raise the temperature of the one or more liver lesions that would be exposed to ThermoDox^® (more on this later in Section 23.6).

Given that there are at least 25 different sites for cancers, the ability to heat and provide HT is only limited by the engineer’s ability to design and build an applicator, catheter, or focused radio-frequency (RF) array, to provide the heat. Thus, most parts of the body can be heated by HT using especially designed applicators for breast and head and neck cancer, a focused RF array for abdominal tumors, a catheter for prostate cancer, a microwave cap for brain cancers, and an RFA probe for liver cancer.

As will be discussed later, while RFA is a widely used technique and certainly has the capacity to burn away the center of the tumor, Celsion’s plan was to target the micrometastases at its edge, thought to be responsible for disease recurrence, with ThermoDox^®. However, the control over the temperatures attained at the tumor margins was not well characterized, and so this became a “heat-and-hope” strategy. Also, the continual heating that we had applied using a simple “leg-in-the-water-bath” technique in the mouse studies was not possible here. RFA is applied full-on for 6 minutes, and additional heating can only be given in a cycled fashion.

In contrast, Celsion had already fully characterized their Prolieve^® Thermodilatation System (Prolieve^®) technology for benign prostatic hyperplasia (BPH). This catheter-based system was approved for use in BPH in February 2004 as being capable of heating the whole prostate. Prolieve^® has undertaken a Phase 4 postmarketing study to evaluate the long-term safety and effectiveness in the treatment of BPH (Weiner 2015). So with an FDA-approved treatment, Celsion started a Phase 1 study with William Gannon as the CMO, which could easily have provided good solid data on temperature and drug release and even resulted in a chance at approval for ThermoDox^®. So, what went wrong here? Despite encouraging efficacy data in Phase 1, Celsion decided against finishing it for “business reasons,” which are described further in Section 23.6.1.

The bottom line for HT though, in the context of the LTSL, is that now we had a drug–device combination, with several devices that could effectively heat many different parts of the body, where tumors might grow. If the engineers could heat it, LTSL would release its drug, which was the underlying tumor-targeting strategy.

23.2.2.1 Effects of Hyperthermia

As shown in Figure 23.6, HT has some effects alone (Dewhirst and Sim 1984; Oleson and Dewhirst 1983), is synergistic with drug (Storm 1989), increases blood flow and vascular permeability to macromolecules (Wu et al. 1993), and enhances liposome extravasation (Kong and Dewhirst 1999; Kong et al. 2000, 2001; Li et al. 2013; Matteucci et al. 2000). But the new concept (Needham 1999, 2001b, 2004) was HT-induced rapid triggered release of drug from the LTSL (Anyarambhatla and Needham 1999; Mills and Needham 2005; Needham 2001b, 2013; Needham and Dewhirst 2012; Needham et al. 2012; Wright 2006) that was only initiated in the bloodstream of the tumor (Needham and Ponce 2006), resulting in deeper penetration of Dox to all cell nuclei throughout the tumor interstitium (Landon et al. 2011; Manzoor et al. 2012), leading to an increased therapeutic effect in in vivo preclinical models (Kong et al. 2000; Needham 2001b; Yarmolenko et al. 2010) and in canine (Hauck et al. 2006) and human clinical trials (Poon and Borys 2009; Vujaskovic 2007; Zagar et al. 2014).

So what was the underlying science? What were the observations and questions that generated the invention? It was 15 years of lipid membrane mechanochemistry.

FIGURE 23.6 Hyperthermia interacts with liposomes, and at other levels, to generate a therapeutic effect, but the new concept was rapid triggered release in the bloodstream of the tumor, leading to an increased therapeutic effect. (Reproduced from Kong, G. and Dewhirst, M.W., Int. J. Hyperthermia, 15(5), 370. Copyright 1999, Taylor & Francis Group.)

23.2.3 LIPID MEMBRANE MECHANOCHEMISTRY

Following the pioneering characterization of the red blood cell (RBC) membrane in the 1970s (Evans and Hochmuth 1976), “lipid membrane mechanochemistry” as a field of study for pure lipid bilayers was really initiated in the early 1980s by Evan Evans, when he moved in 1980 from Duke Biomedical Engineering to the University of British Columbia, Vancouver, Canada. But he started endogenously, by checking out nature’s own designs for lipid-based capsules.

23.2.3.1 Starting with Red Blood Cells

While at Duke (1973–1981), Evans had developed and used the micropipette manipulation technique to characterize the mechanical properties of RBCs. Interestingly, the biochemists (Steck 1974) were only just starting to identify the composition and organization of the spectrin membrane cytoskeleton (Bennett 1982) at the same time that Evan was measuring with the micropipette technique (Evans 1973a,b; Evans et al. 1976). Thus, sophisticated mechanical models of the RBC membrane were introduced in the early 1970s by both Skalak (Skalak et al. 1973) and Evans (Evans 1973a). Evans unified a new material concept for the RBC membrane, which provided the capability of large deformations exhibited by normal discocytes as a two-dimensional, incompressible material, and a general stress–strain law was developed for finite deformations (Evans 1973). What followed was a period of intense activity (Evans and Hochmuth 1977, 1978), culminating in the seminal book by Evans and Skalak Mechanics and Thermodynamics of Biomembranes (Evans and Skalak 1980). Thus, for over 50 years, these micropipette techniques have provided the unique ability to apply well-defined stresses for dilation, shear, and bending modes of membrane and cellular deformation. They laid the foundation for similar micromechanical experiments applying well-defined stresses to single-giant unilamellar vesicles (GUVs) that, in turn, eventually generated the “idea” for the LTSL. This is how that happened.

23.2.3.2 Giant Unilamellar Vesicle Experiments

The classic materials engineering approach to either understanding an existing design (like the red or white blood cell) or creating a new design for a nanoparticle drug delivery system like a liposome is the same. It involves as complete an understanding as possible for the composition–structure–property (CSP) relationships of the materials involved in the component design. To be fair, most liposomologists did not have access to the more mechanochemical techniques we were using, and so in contrast to the development of most liposome and nanomedicine formulations, we didn’t just come up with an all-encompassing series of lipid–drug compositions and evaluate them for performance; we came up with one. Of course, this was based on liposomes that had already been developed and tested, especially the stealth version (Allen 1989). But with over 20,000 papers in the literature on liposomes at the time, new innovation can often require a deeper understanding of CSP relationships. And so, by the time Mark Dewhirst asked for a formulation he could “heat and it released drug,” we (and Evans before me) had been studying membrane materials science for over two decades and had already evaluated many of their CSP relationships using the micropipette technique. These micromechanical methods and the resulting data on single-bilayer vesicles have allowed us to characterize the lipid bilayer membrane in both a liquid and a solid state, including the influence of cholesterol on membrane elasticity and tensile strength. It was these kinds of direct measurements of the “two-molecule-thin” material that were, literally, instrumental in helping to characterize liposomal systems and explain much of the processing and performance of the liposome.

Starting in the 1980s, the micropipette technique was adapted and developed by Evans and Kwok (Evans and Kwok 1982; Kowk and Evans 1981) and then established by (Evans and Needham 1987), to study individual GUVs of various lipid compositions. A particularly historical and newly insightful perspective on the mechanics and thermodynamics of lipid biomembranes has been given recently (Evans et al. 2013), which emphasizes “the inherent softness of fluid–lipid biomembranes and the important entropic restrictions that play major roles in the elastic properties of vesicle bilayers.” In particular, the properties of importance for the LTSL were

• The membrane elastic modulus and other mechanical properties that determine drug loading and retention

• The nature of its main acyl-melting phase transition, which was the key trigger for drug release

• The behavior of the gel-phase membrane in shear and in particular its degree of yield shear and shear viscosity, as a result of in-plane shear deformations of membrane-grain structure

• Molecular exchange with water-soluble and membrane-soluble molecules like lysolipids that modified the membrane to allow it to have such a rapid release of encapsulated drug

• The adhesive and repulsive interactions that help maintain stability in blood circulation

Tables 23.1 through 23.5 show each of the most important and influential micropipette experiments that directly led to the LTSL invention. These key experiments will now be briefly described, along with the video images of the micropipette and GUV on which the experiments were carried out and their typical results. Students, please take note of the depth of inquiry, understanding, and especially mechanism, which such measurements and analyses have revealed. When I say you have to understand as much as you can about structure–property relationships of your nanomedicine material, in order to put mechanism between composition and performance, this is what I am talking about.

The first experiment and analysis that was carried out on single-walled GUVs by micropipette was to measure the elastic expansion and failure of egg lecithin bilayers (Kwok and Evans 1981). Summarized in Table 23.1, this experiment was similar to what had been used to measure the elastic modulus of RBC membranes (Evans et al. 1976)—a favorite inspiration for liposomes. It was used again by Needham and Nunn to characterize SOPC* and SOPC/cholesterol systems (Needham and Nunn 1990) and was further refined to include the contribution of membrane undulations by Evans (Evans et al. 2013), again, as a function of lipid composition including cholesterol content. For comparison, typical monounsaturated dichain lipids such as SOPC form lipid bilayers that are relatively soft (K_A = 200 mN/m), weak (T_lys = 6 mN/m), and somewhat permeable to water and other materials (Bloom et al. 1991). The inclusion of 50 mol% cholesterol in bilayers composed of saturated chain lipids like DSPC, or hydrogenated soy lecithin (again a favorite compositional strategy for making liposomes), have K_A ≈ 2000 mN/m, being 10× as stiff and 10× as strong, and relatively impermeable to water (Bloom et al. 1991). In their review of the Stealth Liposome, Lasic and Needham correlated such micromechanical lipid membrane data with liposome circulation half-life, showing that it is not just PEG and steric stability that can underlie extended half-life of a liposome in the bloodstream (Lasic and Needham 1995). The tighter the interface, the longer the liposomes could circulate. This explains the successful “stealth” effect of Cullis et al.’s (non-PEGylated) sphingomyelin–cholesterol–vincristine liposomes, with a measured elastic modulus of ≈2000 mN/m (Needham and Nunn 1990). These data helped us understand the role of mechanics in liposome-behavior: how it retains the drug and aspects of membrane permeability to water (at least), and how it evades the body’s defenses, with both steric, and mechanical, mechanisms.

TABLE 23.1
Elastic Expansion and Failure of Membranes

By osmotically shrinking a single vesicle by just a few percent, thereby creating sufficient excess membrane area (compared to a sphere of the same volume) and supporting this membrane under low tension in the micropipette, I was able to take a single-vesicle membrane through its complete liquid-to-solid main acyl phase transition, T_m. For the SOPC/POPC-mixed system shown in Table 23.2, by measuring the area changes of the single vesicle, we could provide a phase diagram for this lipid mixture as shown in the plot. In an earlier study (actually my first ever study using the micropipette system and GUVs with Evans) reported in a very comprehensive paper on the thermomechanics of DMPC vesicle membranes (Needham et al. 1988), we showed how the liquid membrane underwent a tension-free transition into the L phase of over 30% change in lipid membrane area, consistent with the corresponding single lipid molecular area change from DMPC from 64 to 42 Å². We also showed how the membrane entered the known Pβ′-rippled phase with tilted acyl chains, measured the mechanics of this rippled deformation, and, from the slopes of the area vs temperate plots, obtained the thermal area expansion coefficients in each liquid (Lα), semisolid (Pβ′), and solid (Lβ) phases. It was interesting to note that the changes in relative area per molecule at T_m and the pretransition T_p of 22% and 4% were in a similar ratio to the respective excess-specific heats reported from differential scanning calorimetry (DSC) measurements (Lentz et al. 1978; Mabrey and Sturtevant 1976), i.e., in the ratio 5:l, thus showing how our single-vesicle mechanocalorimetry agreed with traditional DSC. We also studied the influence of cholesterol in broadening and eventually obliterating any main acyl chain–melting transition, using again DMPC GUVs (Needham et al. 1988).

TABLE 23.2
Main Acyl Solid–Liquid Phase Transition Video Micrographs

During this DMPC phase transition work (Needham et al. 1988), unpublished observations showed that a single, tension-free vesicle below the phase transition was faceted in appearance. Additional evidence for the presence of grains was actually seen in this transition experiment. If you look closely at the magnified insert of the solid lipid–melting image (Table 23.2b circled for your attention), there is a slight “kink,” which represents a grain boundary. Holding the vesicle with just a slight supporting suction pressure, as the temperature was raised and the lipid vesicle melted, its area per molecule expanded, the total area of the vesicle expanded, and it reversibly returned to its original melted liquid area. The (even more) interesting event was that, as it expanded, it clearly had to go through the mixed phase region of solid plus liquid domains, and we actually saw a lumpy-bumpy motion of the outer vesicle portion as “icebergs” of still frozen lipid in the melting bilayer moved past the glass pipette tip. Thus, for this SOPC/POPC lipid mixture, we showed that a vesicle became solid below its main acyl transition. But the next question was, “How solid?” In fact, the more mechanically oriented question was, “Is it a solid-like material (with elasticity), or does it have liquid-like character (and flows)?” It turns out it has both, behaving like a classic Bingham plastic with an initial elastic region, a plastic yield, and a viscous flow when deformed in in-plan shear. Next then is this experiment and the result that tested and measured this property of gel-phase bilayers, a state our LTSL would eventually be in, when injected into the bloodstream and then warmed through this transition by the local HT; whether in the blood stream or in a test tube, a property is a property.

By considering the gel-phase vesicle again with excess membrane area (over a sphere of the same volume), deformed into a micropipette, Evans came up with a membrane-mechanical analysis of the shear deformation of this single gel-phase vesicle that would measure the yield shear and shear viscosity of such DMPC membranes (Evans and Needham 1987). Then, as usual, it was my job to do the experiment and make it represent exactly the boundary conditions and assumptions in the theory. Table 23.3 shows this experiment for a smoothed out DMPC vesicle below its main acyl chain phase transition at a temperature of 13°C* (Needham et al. 1988).

In the experiment, a gel-phase vesicle is aspirated into the micropipette and formed into an outer spherical portion with a long projection into the pipette itself (as shown in Table 23.3d). I then release the pressure and gently blow out the vesicle, turn it around, and reaspirate it, axially with the projection. In applying a small suction pressure, the idea is to then find the point at which the elastic shear deformation of the membrane is exceeded, i.e., its elastic limit (Table 23.3a) at its plastic yield. This occurs when the projection in the pipette just reaches one pipette radius (1 R_p). Below this point, the material would return elastically to its original shape; beyond this plastic yield, the membrane would flow in shear. And so, a suction pressure of six times this yield pressure is applied and the vesicle duly flows into the pipette (Table 23.3b and c), with plug flow at the projections and positive and negative shear near the mouth and at the back end, respectively, until it is completely reaspirated (Table 23.3d). This behavior, of an elastic yield and viscous flow, is similar to more traditional materials that are termed “Bingham plastics” (Bingham 1922). In our highly cited 1987 review paper^† (Evans and Needham 1987), we suggested that the shear rigidity and shear viscosity primarily reflected the density and mobility of crystal defects in the membrane bilayer surface. Thus, even a two-molecule-thick bilayer in its solid Lβ gel phase can deform with in-plane shear by grain sliding at intergrain boundaries (Kim et al. 2003). Ole Mouritsen and Martin Zuckermann (1987) had theorized that grains existed for single-phase membranes, in their analysis of the phase transitions for another similar PC lipid, DPPC, and we will see where this fits in later, when I discuss mechanisms of drug release at the phase transition of the LTSL. In fact, grain structure is clearly evident in the LTSL itself, as in the lower panel showing a transmission electron micrograph (TEM) of a Dox-loaded LTSL of only 100 nm in diameter (Ickenstein et al. 2003). The faceted nanostructures are as evident as similar grain structures in monolayers identified by fluorescent lipids that phase separate to the grain boundaries, as studied by Dennis Kim (Kim 1999; Kim and Needham 2001; Kim et al. 2003). Importantly then, the microparticle diameter is ≈5 μm with 0.5 μm grains; in the lower panel, the liposome is 100 nm, with 20 nm grains. Thus, grain structure is commensurate with total domain material size.

TABLE 23.3
Yield Shear and Shear Viscosity of Solid Membranes

Now comes the experiment that generated the concept for lysolipid modification of the phase transition, to generate such rapid drug release from the thermosensitive liposomes. At the time, we were not studying heat-triggered drug release, but we were studying molecular exchange with lysolipids (Needham et al. 1997). As shown in Table 23.4, by using three micropipettes in the microscope chamber, our very talented postdoc, Natalia Stoicheva, could hold a single-test vesicle with one pipette and then introduce one of the two flow pipettes in order to either deliver a lysolipid solution (the lysolipid was MOPC) or, after such delivery, wash the vesicle with MOPC-free solution at controlled flow rates. As shown in Table 23.4a, the initial vesicle projection length (Lp) inside the pipette is established when the lower pipette is used to flow MOPC-free bathing solution over the vesicle. Then, the lower pipette is replaced by the upper pipette, and MOPC solution is made to flow over the vesicle, causing an increase of the vesicle projection length, ∆Lp (Table 23.4b). This ∆Lp then gives the membrane area change due to individual molecules of lysolipid entering the outer monolayer of the vesicle, and the plot shows uptake of MOPC monomer reaching 6 mol% before the MOPC-free bathing solution rapidly washes back out the MOPC.

TABLE 23.4
Molecular Exchange with Lysolipids

Source: Reproduced from Biophys. J., 73(5), Needham, D., Stoicheva, N., and Zhelev, D.V., Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol) lipid, 2615–2629. Copyright 1997, with permission from Elsevier.

So, what was the idea? “If lysolipid can be included in a liquid bilayer and then trapped in the bilayer in its gel state, would it desorb or form defects that would enhance the release of encapsulated membrane-impermeable contents?” The answer is yes, as described in Section 23.3.2.

Finally, the whole story about stabilizing vesicle–vesicle adhesive contact and aggregation, as well as protein binding due to a stealth effect (as opposed to mechanical stiffness), starts with fundamental measurements and theory of what it is to be adherent and what it is to be stabilized against adhesion, i.e., the accumulation of all the adhesive and repulsive potentials involving colloidal long-range forces (Evans and Needham 1987, 1988). This is what we were studying in the mid-1980s: by bringing two lipid vesicles together in salt solutions. Much of this work is described in a series of papers with Evans, where we completely stripped back the nature of the attractive and repulsive potentials at lipid bilayer interfaces and introduced them one at a time: measure one thing (adhesion energy) and change one thing (interactive potentials by changing the lipid, salt solution concentration, or polymer composition) in the system. In 1980, Evans had already modeled the vesicle adhesion experiment and trialed some preliminary systems, but again, it was my job to make an experiment that matched exactly his required boundary conditions of enough excess membrane so that—as shown in the Figures in Table 23.5—the left-hand vesicle tension could be gently lowered, allowing the vesicle membrane to spread on the tensioned, right-hand vesicle of the same material (SOPC).

TABLE 23.5
Adhesive and Repulsive Interactions Involving Colloidal Long-Range Forces Video Micrographs Typical Results

In the experiment, we first measured a fundamental force of nature, van der Waals attraction, limited at PC membrane surfaces by a very short-range hydration repulsion. We then started adding in all the other potentials, as described in Evans and Needham (1987). By adding in charged lipids, this attraction could eventually be overcome by ≈5 mol% phosphatidylserine, which fitted according to the well-known DLVO theory for colloid stability. For neutral vesicles, adding extraneous nonadsorbing polymers, like PEG or dextrans, generated large attractive stresses and highly measured adhesion energies, which again were well modeled by depletion–flocculation theory (Evans and Needham 1988). These attractive potentials were so large they could overcome even mutual negative repulsions at the interfaces of up to 30 mol% charged lipids. Finally, on this baseline, the inclusion of PEG lipids caused separations of the membranes and overcame all long-range colloidal forces and generated the “stealth” effect, as described in more detail with Dan Lasic (Lasic and Needham, 1995).

Although not part of the LTSL story, we (with Dorris Noppl, a student exchange from Germany) used the same two-vesicle experiment to observe and measure receptor-mediated adhesion (Needham and Kim 2000; Noppl-Simson and Needham 1996), an effect that could have some bearing on ligand targeting.

To summarize then, and as described more fully in another recent review (Bagatolli and Needham 2014), these experiments have characterized a two-molecule-thick membrane:

• It is a soft elastic material with a compressibility somewhere between that of a bulk liquid and a gas.

• It is stiffened considerably by the inclusion of cholesterol to levels equivalent to polyethylene.

• It is permeable to water in relation to its compliance.

• It displays a 25% change in area when taken through its main acyl chain freezing transition.

• As a gel-phase material, it shows the yield shear and shear viscosity of a Bingham plastic.

• It can exchange small amounts of other lipids and surfactants with its surrounding milieu.

When manipulated in pairs, lipid vesicles have also been found to be subject to the same range of attractive and repulsive colloidal interactions as many other colloidal particles, including

• Van der Waals attraction, limited by a very-short-range hydration repulsion and the variable-range power law of electrostatic repulsion

• Steric repulsive barriers due to the presence of bound aqueous polymers like PEG, incorporated as, for example, PEG lipid

• Depletion and flocculation by water-soluble polymers like PEG and dextrans when free in surrounding solution

• Intimate mixing (fusion) when manipulated into contacts that reduce the hydration barrier and allow membrane–membrane fusion

DMPC is not much smaller (2× (CH₂)) than DPPC, the lipid used in ThermoDox^®. And so, even in 1983–1987 (over 10 years before I invented the LTSL), these thermomechanic studies of GUVs were solidifying in my mind exactly what happens when a liposome is taken through a phase transition by changing the temperature, in terms of its physical behavior, the membrane area change, the broadening by a second lipid or cholesterol component, and the shear and viscosity as a result of sliding grain boundaries in the membrane surface. All that was needed now to provide the “light bulb” of the LTSL idea was a fundamental understanding of what happens when a lipid vesicle is exposed to a soluble lipid that can partition into the membrane and can be washed out again, upon changing the bathing medium.

This section may seem arduous, in the context of a chapter on commercialization, but it is absolutely necessary, in order to create survivable patents and to understand the mechanism that the technology is based on (as opposed, again, to patenting a whole series of compositions and not having much clue at all about CSP relationships that influence performance and indeed are part of processing). This then is the level of understanding that is needed, in order to fully characterize your nanomedicine and develop one that actually works in the way it was designed to. I would, therefore, gently suggest that you might need at least some of the same kinds of insights, if you are going to invent anything of use in nanomedicine. There is another lesson to be learned here (see Box 23.3)

BOX 23.3 LESSON AS WE GO #3: FULL CHARACTERIZATION OF ANY NANOMEDICINE INVOLVES A COMPLETE UNDERSTANDING OF COMPOSITION–STRUCTURE–PROPERTY RELATIONSHIPS

Attending many of the drug delivery meetings as I do, it is often the case that a “new” nanomedicine is presented in terms of its material composition, and this is compared directly to its performance in vitro or in vivo experiments. “This is what it’s made of and my graduate student carried out these studies showing that this is the IC₅₀ for these particular cancer cells,” or “this is the tumor growth delay compared to other systems that also do not work.” While these kinds of feasibility studies are certainly advancing the field, it is left to others to gain the deeper understanding of if, and why, the design works or, most often, does not work, especially in humans, if it even gets that far. IMHO simply is not good enough for researchers to proceed like this. I appreciate that not everybody has all the expertise or techniques required for a complete understanding of structure–property relationships (or is lucky enough to be working with Evan Evans), but that does not mean you cannot collaborate with people who do, or that you “read the damn literature.” At least recognize that not only it is necessary to more fully understand your system, but also the design process itself is your invention generator. If you and your advisor do not know the mechanisms, then simply carving out as big a compositional space as possible to protect your idea, and trying to persuade the patent examiner that it’s okay to patent every composition that you (or your lawyer) can think of, is cheating. And do not let me catch you at a meeting, or worse still, in your PhD defense if I am an examiner, presenting something you don’t understand, or have not at least tried to understand, mechanistically.

23.3 ENGINEERING DESIGN OF THE LTSL

Invention generation, to my mind, relies on an ability to make connections of fundamental characteristics of materials involved in the new design. It is then a design methodology process to generate the whole concept from functions to evaluating performance in service. Placing this kind of knowledge in the context of a design scheme is the invention generator I keep referring to. I have shown how to go through the whole process of “reverse engineering” a design that already works, using the LTSL as the prime example (Needham 2013). If you do this yourselves and adhere to the rigor of the process, who knows? You might generate an invention yourself; furthermore, this process of reverse engineering a design guarantees that you at least have a mechanism, and not just a series of compositions.

23.3.1 LYSOLIPID EXCHANGE WITH MEMBRANES GENERATES THE “IDEA”

The LTSL formulation comprises gel-phase DPPC membranes, containing nonbilayer-forming lipids like the lysolipids MPPC (Anyarambhatla and Needham 1999; Needham 2001b) or later, MSPC (Mills and Needham 2005; Wright 2006), as well as DSPE-PEG. Our optimized formulation consists of DPPC/MSPC/DSPE-PEG²⁰⁰⁰ in the molar ratio of 86.5:9.7:3.8 mol% (Mills and Needham 2005; Wright 2006). As described earlier, and in reference to the Figures in Tables 23.1 through 23.5, the idea for the lysolipid incorporation to potentially generate a more rapid and complete release of an encapsulated drug came from a series of micropipette experiments, theory, and interpretation, spanning at least 15 years. Taken together, in the context of lipid membrane CSP relationships, they explain much of the mechanisms (further described next) that provide for Inex’s mantra of “load, retain, avoid, target, fuse” and our innovation of “release.”

23.3.2 MECHANISM OF RELEASE

The mechanism of release relies on three main features of gel-phase DPPC membranes containing nonbilayer-forming lipids like MSPC and DSPE-PEG:

1. The membrane composition can freeze with the formation of grains and grain boundaries.

2. Lipid-chain-compatible lysolipids, like MSPC, are readily incorporated and trapped in the gel-phase DPPC membranes and form porous defects as the membrane is warmed into its acyl-melting phase transition.

3. Coincorporated PEG lipids, while not generating enhanced porosity themselves, do seem to stabilize the equilibrium pores.

As first shown by Anyarambhatla (Anyarambhatla and Needham 1999; Needham 2001b), confirmed in more detail mainly by my graduate students Jeff Mills (Mills 2002; Mills and Needham 2004, 2005) and Alex Wright (2006) and supported by data from other labs (Banno et al. 2010; Li et al. 2010; Negussie et al. 2011; Tagami et al. 2011), the release of encapsulated Dox and other drugs like cisplatin (Woo et al. 2008) can occur within only a few seconds of reaching the transition temperature of the lipid bilayer (mixture) around 41°C.

The mechanism has been described in detail in a paper we wrote for the Faraday discussions (Needham et al. 2012) and, in the context of its design, in the reverse engineering chapter (Needham 2013). To summarize briefly here, using the example illustrated in Figure 23.7, which shows an LTSL formulation consisting of MSPC and DSPE-PEG²⁰⁰⁰ in the DPPC host bilayer, the enhanced permeability in the phase transition region is through MSPC pores.

That is, as the transition temperature is approached and the grain boundaries begin to melt, the lysolipid forms lysolipid-lined nanopores at these now liquid boundaries. These MSPC pores also seem to be stabilized by the presence of PEG lipid, since in the absence of PEG lipid, the release is slightly slower. As a consequence, the hydrogen ion gradient rapidly equalizes and DoxH⁺ comes out in seconds (large red arrow), as does any remaining embedded Dox in the bilayer.

The presence of only a few mol% of lysolipid contained in the gel-phase DPPC bilayer of the LTSLs significantly increases both the rate and amount of drug released, allowing for a “burst” release in vivo, in only a matter of seconds, upon the application of HT directly to the tumor. Furthermore, compared with DPPC alone, the slight lowering of the bilayer transition temperature by the presence of the ≈10 mol% MSPC is offset by the inclusion of the disaturated acyl chains of the ≈4 mol% DSPE-PEG, which slightly raises T_m compared to DPPC alone. The result is that the transition temperature of the optimized LTSL bilayer maintains the transition around 41.3°C, thereby maximizing the release parameters for mild HT clinical use.

FIGURE 23.7 Proposed mechanisms for thermally triggered release at 41°C: grain boundaries melt, lysolipid forms, PEG lipid stabilizes the nanopores, and doxorubicin is released. (From Needham, D. et al., Faraday Discuss. (Lipids Membr. Biophys.), 161, 515, Copyright 2012. Reproduced by permission of Faraday Division, Royal Society of Chemistry.)

To get an idea of the pore size, we tested for permeability through the LTSL membrane at its phase transition for a range of ions and molecules including dithionite, cisplatin, carboplatin, carboxyfluorescein, Dox, manganese porphyrin, and both 10,000 and 40,000 Da dextran. The results showed that only the 40,000 Da dextran could not pass through the membrane. Thus, the conclusion is that the pore is at least 5 nm in diameter and less than 10 nm (Wright 2006).

23.4 PERFORMANCE IN PRECLINICAL STUDIES

23.4.1 ALL 11/11 MICE ARE “CURED”

As presented in two papers in Cancer Research in 2000 (Kong et al. 2000; Needham 2001b), the first preclinical assessment of the Dox-containing LTSL (Dox-LTSL) formulation could not have gone any better. The first growth delay study we did showed that the Dox-LTSL could actually “cure” mice from an implanted tumor (FaDu, a squamous cell carcinoma) out to 60 days, after just a 1 hour heating to mild HT temperature of 42°C with LTSL in the bloodstream. We tested an unheated saline control against several treatments including a nonthermal-sensitive, Doxil-like liposome (NTSL), both with and without heating to 42°C for 1 hour. In the experiment, FaDu tumors implanted on the leg of the test mice grew to five times the original tumor volume (of 5–7 mm diameter) in 10 days in the unheated control. Depending on the test “treatment,” the growth of the tumor was delayed by

• 3.5 days for a single dose of free Dox at normothermic (34°C) skin temperature (13.5 days)

• 10 days for a heated tumor with no drug (20 days)

• 14 days for a heated tumor with drug (24 days)

• 11 days for a normothermic tumor with NTSL (21 days)

• 1 day for a normothermic tumor with LTSL (1 day)

• 22 days for a heated tumor with NTSL (32 days)

• 40 days for a heated tumor with LTSL (40 days)

Thus, while free drug could only delay the growth by 3.5 days, HT alone had a significant effect, causing a delay in growth of 10 days. When the two approved treatments were combined, heating to 42°C with injected drug, the delay was 14 days. NTSL had some effects (11-day growth delay) at normothermic temperatures, showing how the EPR effect must have been operating for this long-circulating liposome, in this particular implanted tumor. But LTSL at normothermic was less than useless. Producing only a 1-day growth delay, its lack of effect was comparable to saline and worse than free drug. This showed that LTSL must be leaking drug and not even performing like a stealth liposome. This was later confirmed by others who measured the drug-to-lipid ratio (i.e., a measure of the encapsulated drug compared with the circulating lipid of the liposomes) showing a half-life of Dox retention in vivo of only 1 hour (Banno et al. 2010), which was confirmed also in humans (Wood et al. 2012). When the tumor was heated to 42°C for 1 hour, NTSL had an even greater effect, causing a growth delay of 22 days. Since it is not thermally sensitive, this was probably a direct result of the increased extravasation enabled by mild HT, which is known to enhance vascular permeability (Gaber et al. 1996; Kong et al. 2000) but could also have included some increase in membrane permeability to H⁺ ions and the deprotonated Dox that would follow through the membrane.

The main event though came with the LTSL cohort, where the tumor was heated to 42°C, the Dox-LTSL was injected, and the tumor heating was continued for 1 hour (Dox-LTSL + HT). In the second paper in this series, all 11/11 tumors remained regressed out to the full endpoint of the study, which was 60 days, with a minimum 50-day growth delay. It was this result, published in 2000 (Needham 2001b), but obviously obtained earlier, that sealed the license agreement with Celsion in November 1999. It is time for another quick lesson (Box 23.4).

BOX 23.4 LESSON AS WE GO #4: IN VIVO, IN VIVO, IN VIVO

As an inventor, you will not see your precious idea be even remotely appreciated by anyone with investment capital unless you have very positive in vivo data, and quite rightly so.

23.4.2 NOT ALL CANCERS RESPOND THE SAME

So, did we get lucky with FaDu? Kind of. Expanding the preclinical testing to a series of other tumors, the efficacy of the commercial formulation (ThermoDox^®) was reexamined in FaDu and compared with HCT116, PC3, SKOV-3, and 4T07 cancer cell lines (Yarmolenko et al. 2010). It turned out that variations in antitumor effect of Dox-LTSL + HT are primarily related to in vitro doubling time. In all five tumor types, Dox-LTSL + HT increased median tumor growth time, compared with untreated controls and HT alone (Yarmolenko et al. 2010). Compared with the Dox-LTSL without heating, Dox-LTSL + HT yielded significantly higher drug concentrations in the tumor. Thus, heating is critical for drug release; the EPR effect alone for this formulation is simply not good enough.

The study also evaluated the cell lines for sensitivity to the drug. FaDu was the most sensitive to Dox (IC₅₀ = 90 nM) in vitro, compared to the other cell lines (IC₅₀ = 129–168 nM). Again, of the parameters tested for correlation with efficacy, only the correlation of in vitro doubling time and in vivo median growth time was significant. Slower-growing SKOV-3 and PC-3 had the greatest numbers of complete regressions and longest tumor growth delays, which are clinically important parameters. Thus, while in this second series of preclinical model tests, we did not get the same 11/11 tumors “cured” and the results were still incredibly positive. Dox-LTSL + HT again resulted in the best antitumor effect in each of the five tumor types. Interestingly, these variations in efficacy were most correlated to in vitro cell doubling time. It was suggested in this 2010 paper that in the clinic (and perhaps Celsion should consider this), the rate of tumor progression must be considered in the design of treatment regimens involving Dox-LTSL + HT. Another study from Brad Wood’s lab at National Institutes of Health (NIH) largely confirmed the ability of LTSL to release Dox in vitro and in vivo, by combining LTSL with noninvasive- and nondestructive-pulsed HiFu exposure. This study also showed enhanced delivery of Dox and, consequently, its antitumor effects (Dromi et al. 2007). More on these clinical studies will be discussed later.

23.4.3 NEW PARADIGM FOR LOCAL DRUG DELIVERY: DRUG RELEASE IN THE BLOODSTREAM PROVIDES FOR GREATER AMOUNTS OF FREE DRUG AND DEEPER PENETRATION INTO TUMOR TISSUE AND SHUTS DOWN THE TUMOR VASCULATURE

Thus, from the in vitro studies, the release time of Dox from the LTSL at only mild HT temperatures was faster than the transit time of liposomes through the warmed tumor vasculature. Drug release could therefore occur in the bloodstream of the tumor! The problematic EPR effect (for such large 80–100 nm liposomes) was not needed at all. We had previously shown the importance of dosimetry (Kong et al. 2000), achieving approximately 50 μM Dox in the tumor tissue. When compared to the IC₅₀ for FaDu of 90 nM, drug concentrations by release in the bloodstream were 500 times the IC₅₀. Tumor drug levels for Dox-LTSL + HT were up to 30 times higher than those achievable with free drug administration and 3–5 times elevated compared to NTSL, which must rely on extravasation to deliver any drug at all to the tumor interstitium (Needham and Dewhirst 2001; Yarmolenko et al. 2010), and their leakage of drug would be slow at best. Moreover, bioavailability was also key; heating the tumor to 42°C and continuing to heat for 1 hour while administering Dox-LTSL resulted in half of the Dox being bound to DNA and RNA of tumor cells after only that one 1 hour of treatment (Kong et al. 2000). In stark contrast, the amount of Dox bound to DNA and RNA of tumor cells after free Dox + HT or NTSL + HT was not even detectable (Kong et al. 2000) (Box 23.5).

BOX 23.5 LESSON AS WE GO #5: STAND AND DELIVER AND RELEASE

From a drug delivery perspective, it is not enough to deliver drug to the perivascular space of the tumor interstitium: that drug must be bioavailable. It is also not enough to deliver what seems to be sufficient drug to cause tumor growth delay and abolish tumors over the first 10 days, if not enough drug is delivered to prevent them from growing back. It will be important to remember these data when discussing the inherent limitations that may have been responsible for the underperformance of the subsequent Celsion-run, Phase 3 liver cancer trial using ThermoDox^® with RFA.

So, if drug was being released into the blood vessels of the tumor, what effects could it be having, perhaps on the vasculature itself? Fan Yuan, a close collaborator of ours and ex-Rakesh Jain’s group, measured the RBC velocity in tumor vasculature before and after the 1 hour LTSL “treatment.” Using fluorescent red cells as “tracers bullets” (Chen et al. 2004), he found that the average RBC velocity was reduced by almost 150 times, from 0.428 to 0.003 mm/s and the microvascular density was reduced from 3.93 to 0.86 mm/mm². In addition, blood flow stasis and severe hemorrhage occurred immediately after treatment and there was no blood flow in microvessels in five out of six tumors at 6 and 24 hours after the treatment. Thus, at 24 hour, after just a 1 hour treatment, tumor blood flow can actually be shut down by Dox-LTSL + HT in FaDu tumors.

FIGURE 23.8 Tumor uptake of doxorubicin vs time. Time sequence images of blood vessels (green) and doxorubicin (red) for preinjection and at 1, 5, 10, and 20 minutes after injection. Shown are injections to a warmed tumor (42°C) of free doxorubicin (free Dox + HT) and the doxorubicin-loaded LTSL (Dox-LTSL + HT). Scale bar = 100 μm. (Reprinted from Manzoor, A.A. et al., Cancer Res., 72(21), 5575. Copyright 2012, American Association for Cancer Research. With permission.)

Finally, the most compelling and dramatic evidence for not only release in the bloodstream, but deeper penetration of Dox into a tumor than has ever before been achieved and measured in vivo, was presented by Manzoor et al. (2012). As shown in Figure 23.8, adapted from that paper, real-time confocal imaging of Dox delivery to the FaDu xenograft in window chambers illustrates that compared to administering free drug alone, intravascular drug release from Dox-LTSL in the prewarmed tumor massively increased the amount of free drug in the interstitial space, after only 20 minutes of heating. Clearly, this increased both the time that tumor cells were exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional PEGylated liposomes. Maximum measureable drug penetration from tumor vasculature vs. treatment group shows that drug delivered with Dox-LTSL penetrates twice as far as Doxil^® liposomes (78 vs 34 μm). The released, now free, drug diffuses into the tumor interstitium, reaching its nucleus target with greater penetration distance, and to much higher concentrations, than those achievable by either free drug administered alone or the more traditional long-circulating liposome formulations (Eley and Needham 1984). Intravascular drug release provides a mechanism (see properties of Dox above, Section 23.2.1.1) to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by drug diffusion. This establishes a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream, saturating neoplastic cells, as well as endothelia, pericytes, and stroma, with the anticancer drug.

23.4.4 THERMODOX^® PHASE 1 IN CANINE PATIENTS

A Phase 1 canine trial of the Dox-LTSL was also carried out in spontaneous tumors. Of the 20 dogs that received 2 doses of Dox-LTSL, 12 had stable disease (SD) and 6 had a partial response (PR) to treatment (Hauck et al. 2006). The conclusion from this work was that “doxorubicin-LTSL offers a novel approach to improving drug delivery to solid tumors. It was well tolerated and resulted in favorable response profiles in these patients. Additional evaluation in human patients is warranted.”

23.4.5 NEW DATA SHOWS A SYSTEMIC EFFECT: HEAT ONE LEG TUMOR AND THE OTHER TUMOR ALSO SHOWS GROWTH DELAY

As we continued to explore the potential for the LTSL to advance local tumor drug therapy, anecdotal evidence from the chest wall recurrence trial seemed to indicate there was even some systemic effects. The MD carrying out the clinical studies, Dr. Kim Blackwell, told Mark Dewhirst about this and Mark immediately established a new study to determine whether treatment of a tumor site with systemically administered LTSL, with HT for triggered release, would have a dual antitumor effect on the primary heated tumor and an unheated secondary tumor in a distant site (Viglianti et al. 2014). They also wanted to determine the ability of noninvasive optical spectroscopy to predict treatment outcome. As discussed in their paper, mice were inoculated with SKOV3 human ovarian carcinoma in both hind legs. Only one tumor was selected for local HT and subsequent systemic treatment, and the size and characteristics of both would be measured.

Data for the four treatment groups (control, Doxil^®, and two different LTSL formulations containing Dox) showed that similar to previous studies (Kong and Dewhirst 1999; Kong et al. 2000; Needham 2001b; Ponce et al. 2007), tumor growth delay was seen with both Doxil^® and the thermally sensitive liposomes, in the tumors that were heated. As before, there was significant growth delay with the Doxil^® and two LTSL treatment groups on the primary tumor side, since HT enhances both EPR and Doxil^® uptake into the interstitium (Gaber et al. 1996), as well as releasing drug from the LTSL in the tumor vasculature. The data are also consistent with Mark Dewhirst’s previous work showing that growth time correlates with intratumoral drug levels (Kong et al. 2000; Palmer et al. 2010). However, the startling result was that tumor growth delay was also seen in the opposing tumor in the thermally sensitive liposome–treated groups, but not with Doxil^®. This mechanism of the so-called abscopal effect * of Dox-LTSL is most likely due to recirculation of intravascularly released drug. Thus, these thermally sensitive liposomes affect the primary heated tumor and also bring systemic efficacy. Learning from the past, as you will read about next, we have sent this paper to Celsion and informed them to look out for these effects in their continued clinical trials. We hope they do (listen that is) (Box 23.6).

23.5 LICENSING

LTSL was licensed by Duke University to Celsion Corporation in November 1999. Here is a brief historical synopsis about Celsion. Augustine Y. Cheung, a well-known microwave expert with a PhD in electrical engineering from the University of Maryland, established Cheung Laboratories, Inc. in the State of Maryland in 1982. It was a device company that focused on HT and developed various types of HT equipment, including a balloon catheter technology for enhanced thermotherapy of BPH, called Prolieve^®. We started talking with Dr. Cheung (an old friend of Mark Dewhirst’s) in the mid-1990s, as we were starting to develop our LTSL. Boston Scientific exclusively distributed Prolieve^® from 2004 and eventually purchased the technology from Celsion, in 2007. They then licensed it back to Dr. Cheung in 2013. Dr. Cheung was the president and CEO of Celsion, but as Celsion transitioned from “just” a device company to a biotech company, he “stepped down” in 2005, to be replaced by Lawrence Olanoff (ex-Forest Laboratories); Dr Cheung eventually became president and CEO of Medifocus. Olanoff himself (whom I did not get a good feeling about, right from the first time we met) only lasted 14 months and quit Celsion to go back to Forest Labs in October 2006. Celsion Executive Vice President, Chief Operating Officer, and Chief Finance Officer Anthony P. Deasey, replaced Olanoff on an interim basis, until a replacement was found. Luckily, for the project, this turned out to be Mr. Michael Tardugno, who joined Celsion on January 3, 2007, as president and chief executive officer. Mike is really the person (with his team including Nick Borys, CMO), who eventually moved the technology along to where it is today. Celsion is finally under a stable and strong leadership in Tardugno and Borys. But the aforementioned is typical of the merry-go-round of small company wheelings and dealings, ins and outs, and ups and downs, which your invention and potential product might be exposed to. If it is, little will get done until senior management is capable and stable, competent, and free from hidden agendas and greed.

BOX 23.6 LESSON AS WE GO #6: HEAT TUMOR, ADMINISTER LTSL, KILL TUMOR (SIMPLE, RIGHT?)

From a drug delivery perspective, it is not enough to inject liposomes and hope to deliver drug to the perivascular space using the LTSLs, imagining that they would release Dox inside the tumor vasculature. To reiterate, the tumor has to be first heated to 42°C (Manzoor et al. 2012) and be at the release temperature before the liposomes are administered. Otherwise, the loss of drug from the liposomes and loss of liposomes themselves from the circulation can deplete the reservoir of Dox-LTSL in the blood stream, and so reduce the amount of doxorubicin that can be available and be released in the tumor vasculature. So, from all the preclinical studies that we did, both in vitro (Anyarambhatla and Needham 1999; Ickenstein et al. 2003; Matteucci et al. 2000; Mills 2002; Mills and Needham 2005; Needham 2001b; Wright 2006; Wu et al. 1993a,b) and in vivo (Chen et al. 2004, 2008; Gaber et al. 1996; Kong et al. 2000a,b, 2001; Manzoor et al. 2012; Matteucci et al. 2000; Needham 2001b; Ponce et al. 2007; Wright 2006; Wu et al. 1993a,b; Yarmolenko et al. 2010)—and these are original peer-reviewed manuscripts and PhD theses—not just reviews, which we also published (Kong and Dewhirst 1999; Landon et al. 2011; Mills and Needham 2004; Needham 2013; Needham and Dewhirst 2001, 2012; Needham and Ponce 2006) (for Celsion and others to read), here is our recommended, research-derived protocol:

• Heat tumor to 42°C.

• Administer LTSL while maintaining this mild HT level for preferably 1 hour.

• Kill tumor.

• Release drug in the bloodstream, enough to kill a horse.

• Reduce tumor blood flow within the first few hours.

• Shut down the tumor vasculature in the first 24 hours.

• Penetrate the drug deep into endothelial, pericyte, stroma, and neoplastic cells of the tumor in the first 20 minutes.

• Half the Dox in the tumor is already bound to the DNA of every cell in the tumor within the first hour.

Simple, right? You would think so, but as we will see later, this was not what Celsion did.

The license agreement is actually available on line. As an example of a license agreement, just search Celsion license agreement Duke and this will pop up: Home > Sample Business Contracts > Celsion Corp. The contracts are at the Onecle web page (Onecle 1999): “Business Contracts from SEC Filings.” Thus, the license agreement—Duke University and Celsion Corporation—is there for all to see (why this is public or who put it there; I have no idea).

Celsion got this “field”:

“FIELD” shall mean the use of the HEAT TRIGGERED DELIVERY TECHNOLOGY and PATENT RIGHTS in thermally sensitive formulations designed to release, activate, or express pharmaceutically active agents locally, such release, activation, or expression being initiated by local application of heat and being made for the purpose of treating any disease or altering a physiological process in animals, including, without restriction, in humans.

All drugs, all diseases, for the lifetime of all the patents: not a bad deal. So what did they do with it?

23.6 CLINICAL TESTING

Having licensed this “technology,” Celsion, in their various guises, first as a device company and then as a full-blown biotech company, to their credit, started a series of human clinical trials. These included a Phase 1 study in prostate cancer, a Phase 2 RCW, and a Phase 1 for liver cancer that rapidly progressed to a Phase 3 trial, but without the usual intermediate stage of a Phase 2. Some of this is newly reported in this chapter (see prostate cancer trial in the following section), while some—the RCW and liver trial data—has already been published (Needham 2013) and should be consulted for the fuller, more technical story.

23.6.1 PHASE 1 PROSTATE CANCER

The first Phase 1 human trials were actually in prostate cancer—a perfect place to start testing Thermodox^®. The prostate cancer trial (Celsion 2003–2009) was initiated in 2003 in order to determine the maximum tolerated dose of doxorubicin released from Thermodox^® via thermal microwave therapy in patients with adenocarcinoma of the prostate. However, the trial was terminated in 2009. I asked why, and did not get a real answer for a while until someone said, “business reasons.” The suggestion was that there already were lots of treatments for prostate cancer, and so there was no money to be made in it. Really?

The reason I say this was a perfect place to start is that Celsion already had a tried and tested prostate heating system—their own Prolieve^® Thermal Dilatation System and so the pairing of their own device with our drug seemed like a match made in heaven. A paper was actually published in Urology (Larson et al. 2006) using interstitial temperature mapping during Prolieve^® transurethral microwave treatment to show that the temperatures we required for Thermodox^® could actually be achieved in the prostate capsule. Other data on clinical efficacy obtained in the Phase 1, as far as I know, was never released. According to their current web site at MediFocus Inc. (2016), “The Prolieve^® System is an in-office technology, a medical device that both heats the prostate and dilates the prostatic urethra” using a balloon catheter, as shown schematically in Figure 23.9. However, even back then when they were naturally considering also using it to heat prostate cancer, they had already done a significant amount of characterization of what this technology could do for benign prostate hyperplasia and had an FDA application to test it (FDA 2004).

The Larsen study was performed using the Prolieve^® Thermodilatation System “funded by an unrestricted educational grant from Boston Scientific Corporation, Marlborough, Massachusetts” (Larson and Robertson 2006) that simultaneously compressed the prostate with a 46°C balloon circulating heated fluid and delivering microwave energy into the prostate. Results by actual interstitial temperature mapping showed that average peak temperatures of 51.8°C were attained at an average of 7 mm away from the prostatic urethra. These temperatures were greater near the bladder neck and midgland than toward the prostatic apex. Magnetic resonance imaging also revealed necrotic zones that were consistent with sustained temperatures greater than 45°C. Thus, as shown in Figures 23.9b and 23.10, Celsion were in possession of actual data showing that the Prolieve^® system heated the prostate right out to its boundaries, to a temperature that was more than enough to cause ThermoDox^® to release its drug, in less than 4 minutes. So, while BPH actually requires higher temperatures in the 50°C range, they could already attain temperatures that were perfect for ThermoDox^® in the prostate. And, moreover, unlike the trial they did choose to invest all their resources in (the primary liver cancer trial using RFA), here was a treatment modality where they could “heat and know” they were attaining the desired temperature, in contrast to the “heat-and-hope” strategy of the subsequent primary liver cancer trial. It was a system, completely owned and developed by Celsion, that was perfect for the ThermoDox^® technology they had licensed from Duke, and yet they abandoned the trial because of “business reasons.” Go figure! (Box 23.7).

FIGURE 23.9 The Prolieve^® system. (a) Schematic illustration of the Prolieve^® benign prostatic hyperplasia treatment device. (b) Intraprostatic temperature plot of representative subject showing temperature and time. All recorded temperatures measured are displayed, where each line represents temperature of individual sensor. (a: Courtesy of MediFocus Inc.; b: Adapted from Urology, 68(6), Larson, B.T. and Robertson, D.W., Interstitial temperature mapping during Prolieve^® transurethral microwave treatment: Imaging reveals thermotherapy temperatures resulting in tissue necrosis and patent prostatic urethra, 1206–1210. Copyright 2006, with permission from Elsevier.)

FIGURE 23.10 Schematic illustration of the Prolieve^® Thermodilatation System treatment, showing temperatures attainable throughout the prostate, as measured directly by thermal probes. Note: Even temperatures at the edge are at least 42°C, which is enough to release doxorubicin from ThermoDox^® in any prostate-encapsulated tumor. (Adapted from Urology, 68(6), Larson, B.T. and Robertson, D.W., Interstitial temperature mapping during Prolieve^® transurethral microwave treatment: Imaging reveals thermotherapy temperatures resulting in tissue necrosis and patent prostatic urethra, 1206–1210. Copyright 2006, with permission from Elsevier.)

BOX 23.7 LESSON AS WE GO #7: BE ON THE LOOKOUT FOR DECISIONS THAT ARE BASED ON “BUSINESS REASONS”

When CEOs and accountants do “analysis,” it’s not the same analysis that you or I might do as scientists, clinicians, chief medical officers, or patients. This Phase 1 trial was apparently already showing some efficacy in the limited dosing range (three patients per dose, 20–50 mg/m² dose escalation), even before it reached the expected MTD. And it was heatable using Celsion’s own Prolieve^® technology, then actually being used for benign prostate HT. Hmm, “business reasons” that probably ended what could have been an approvable procedure, the first for ThermoDox^®.

23.6.2 PHASE 2 RECURRENT CHEST WALL CANCER

As an advisor to Celsion, Mark Dewhirst was also very proactive in developing ThermoDox^®, within his (our) NIH-funded Hyperthermia Program. RCW was another ideal cancer for initial clinical testing on the way to FDA approval. But guess what? Despite Mark’s expert efforts and advice, it didn’t cut through the commercialization strategy either. This one wasn’t abandoned, but its progress was slowed considerably for a series of commercial reasons, including the fact that again, this was not a huge market. However, in Phase 1 trials, ThermoDox^® showed very promising results, at doses that were even 60% less than the expected MTD. This is where it gets really interesting and frustrating: interesting, in terms of ThermoDox’s outstanding results, and frustrating, because these positive results were in spite of what Celsion and their clinical investigators were deciding on, writing in the trial, and implementing, which was a protocol that went against everything we had been telling Celsion to do. What we had learned from our research suggested that their protocol almost completely missed the bioavailability of ThermoDox^®. It amounts to another series of “business decisions” (by definition, it’s a “company”) and “hospital decisions” (because they have budget rules), as opposed to “rational” decisions (because it’s the right scientific thing to do, and because it could actually benefit the patient), which probably set back any potential approval of ThermoDox^® by years.

This second human Phase 1/2 trial (called the “Duke study”) in breast cancer recurrence (Celsion 2012) was designed to evaluate the MTD, pharmacokinetics, safety, and efficacy of approved ThermoDox^® + HT, in patients with breast cancer recurrence at the chest wall. In the initial Phase 1 (which was actually started in 2001, but later became nonrecruiting (Celsion 2001), HT was administered with an eminently achievable temperature goal of 40°C–42°C, for 1 hour, using the BSD-500 PC System. However, the company supplying this HT equipment also seemed to be dragging its feet, because it was (historically) in competition with Celsion and (almost unbelievably) no one could get past the old bad blood. Surprisingly (but maybe not that surprising) given the preclinical data Manzoor et al. got later (2012) (see Figure 23.8), there were several instances of SD, PR, and two of the complete responses (CR), for a dose escalation of only 20–30 mg/m², compared to a final MTD of 50 mg/m². Several patients in this trial achieved either PR or CR (Vujaskovic 2007). As shown in Figure 23.11, for one patient, her widely disseminated chest wall tumor had completely disappeared after only four 1 hour mild HT treatment cycles with ThermoDox^® in her bloodstream.

This test dose (30 mg/m²) was thus only 60% of the expected MTD, and so there were also no side effects of the drug. A second patient had a similar CR at 30 mg/m² (Vujaskovic 2007). Again, here was a heating system where the temperatures required for release of Dox from ThermoDox^® were known, controlled, and eminently achievable, using this BSD machine (unlike the ensuing RFA trial that went horribly wrong).

FIGURE 23.11 (a) Treatment of chest wall recurrence of breast cancer, using a BSD-500 PC System to achieve a temperature goal of 40°C–42°C. (b) Same patient before treatment. (c) Precycle 5 given 30 mg/m². (Adapted from Zagar, T.M. et al., Int. J. Hyperther., 30(5), 285. Copyright 2014, Informa Healthcare.)

A recent update provided by Celsion (April 15, 2015) and published in an abstract and poster (Rugo et al. 2013) and peer-reviewed paper (Zagar et al. 2014), for this ongoing open-label Phase 2 DIGNITY trial of ThermoDox^® in RCW breast cancer, now shows that

• Of the 16 patients enrolled and treated, 12 were eligible for evaluation of efficacy

• 67% of patients experienced a clinical benefit of their highly refractory disease with a local response rate of 58% observed in the 12 evaluable patients

• Notably, there were five CR, two PR, and one patient with SD

When taken together with the previous (stalled) Duke study, of the 29 patients treated in the two trials (comprising 11 patients in this now-termed “DIGNITY” study, with 18 patients in the Duke study), 23 were eligible for evaluation of efficacy (Zagar et al. 2014). A local response rate of over 60% was reported in 14 of the 23 evaluable patients, with 5 CR and 9 PR. The simple Phase 1 study concluded that 50 mg/m² ThermoDox^® with mild HT is safe (the MTD of Dox itself) and produces objective responses in heavily pretreated RCW patients. It recommended that “future work should test thermally enhanced LTSL delivery in a less advanced patient population.”

Given the protocol, these really were/are very promising results—so what’s all about this protocol? Glad you asked, because here is another lesson to be learned.

23.6.2.1 RCW Protocol and ThermoDox^® Pharmacokinetics

There have been several measures of the plasma clearance of ThermoDox^®, its encapsulated Dox, free Dox, and even metabolites, over the years; for this discussion, it is important to review as many as we can find, some published in peer-reviewed papers, in investigator’s brochures, or in presentations made by the Celsion-hired clinicians carrying out the studies at international meetings. Although the data are mainly from the Phase 1 liver trial, the graphs serve the purpose of presenting the pharmacokinetics that the protocol should have taken into account. Figure 23.12 shows a graph of human plasma clearance of 50 mg/m² for ThermoDox^®, published in 2008 in the investigator’s brochure for ThermoDox^® + HT, for the treatment of solid tumors (Celsion 2008), and also shown in Poon and Borys’s (chief clinician and Celsion’s CMO, respectively) Expert Opinion paper (Poon and Borys 2009). It was for six liver cancer patients. In this study, patients were treated with a combination of RFA and ThermoDox^®. A 30 minute infusion of ThermoDox^® was given 15 minutes prior to ablation (Ravikumar et al. 2010). (Note: More details about ablation and RFA will be discussed next, but for now, let’s focus on the pharmakokinetics [PK].) It is very similar to the one produced by Brad Wood’s Phase 1 trial also in liver cancer using RFA (Wood et al. 2012) and in a preclinical animal study published by Banno et al. (2010). Assuming that all the analyses and assays are correct, the graph shows three main things*:

1. The peak in both ThermoDox^®-encapsulated Dox and free (unentrapped) Dox occurs just after the end of the 30 minute infusion.

2. Free Dox represents 43% of the total Dox area under the curve.

3. The half-life of the ThermoDox^®-entrapped “liposomal Dox” is only about 1 hour.

This means that as soon as the infusion is stopped, the formulation is already losing ground. As mentioned earlier and shown in Figure 23.3, it is well known that free Dox has a half-life itself after i.v. injection of only 2 minutes. Thus, the fact that the unencapsulated Dox tracks the encapsulated Dox in ThermoDox^® suggests that it is actually not as stable as the other more traditional liposomes and is, in fact, leaking from the formulation. So let’s call what it really is, leaked Dox, from ThermoDox^®. As is also well known and was, in fact, one of the major goals in its design, Doxil^®, on the other hand, retains its drug very well (maybe too well), and the total Dox and encapsulated Dox (i.e., the drug-to-lipid ratio) of Doxil^® tracks the liposomal half-life. Even though the stealth-like formulation of the ThermoDox^® liposome (containing, as it does, PEG) should probably circulate as long as Doxil^® does (half-life on the order of 24–48 hours) (Northfelt et al. 1996). ThermoDox^® does not keep its drug inside for too long, as also confirmed by Ravikumar et al. (2010). Preclinical animal data from other labs (Banno et al. 2010) have also confirmed that the Dox can slowly leak out of the Dox-LTSL when administered to mice in vivo.

FIGURE 23.12 Human plasma clearance of 50 mg/m² ThermoDox^® (Mean ± SE). (Courtesy of Celsion Corporation, Lawrenceville, NJ.)

To summarize, while the LTSL lipid itself has a plasma half-life of approximately 8 hour, consistent with PEGylation, the encapsulated drug half-life is only 1.3 hours. But looking at the preclinical data produced later by Manzoor et al. (2012), it should be pointed out that Dox doesn’t need to remain encapsulated for long periods; drug comes out in a few seconds when heated to the right 41°C–42°C mild HT temperature and fills the whole tumor. So, if this is the PK of ThermoDox^®, a somewhat leaky liposome (compared to the more drug-retaining traditional ones) that nevertheless, if given the chance, can completely fill the tumor and all its cells with one of the most deadly drugs on the planet in 20 minutes, how did Celsion and Duke set up their protocol?

Here’s how they describe ThermoDox^® at Clincaltrials.gov, for the trial called “Phase 1/2 Study of ThermoDox^® With Approved Hyperthermia in Treatment of Breast Cancer Recurrence at the Chest Wall (DIGNITY)” NIH identifier NCT00826085 (ClinicalTrials 2010):

ThermoDox^® is a 30-minute intravenous infusion followed by hyperthermia within 60 minutes of infusion completion.

WHAT?! “Within 60 minutes of infusion completion”? After all, we have published on the order of how to use our LTSL—“heat first, infuse ThermoDox^®, keep heating, and kill cancer”—and told Celsion countless times, and this is the protocol they come up with? Some of you may think this next figure, Figure 23.13, is somewhat flippant. It is not; it is deadly serious. You can imagine, as in Figure 23.13, the medical oncology doctor coming in to see and sign off on the patient, who as just had her infusion of ThermoDox^® in medical oncology. She is waiting for the orderly to come and wheel her down to the radiation oncology suite, where the radiation oncologists are waiting to heat the patient, saying to her and her family (because they are just adhering to the written Celsion protocol):

Sure, have another cup o’ tea, we got plenty of time

No! We do not have plenty of time. Look at the PK profile. If you heat the patient “within 60 minutes of infusion completion,” you will miss almost all of the drug availability. It’s not just a bottle or bag of red liquid, it’s ThermoDox^®, which has been designed to be given only when the tumor has been heated to 41°C–42°C. As to why the protocol was written this way in the first place—it seems it wasn’t Celsion’s fault. The anecdotal story was that the hospital administration would not let a medical oncology nurse work in a radiation oncology suite, because of “budgetary reasons.” And so, ThermoDox^® had to be administered first in medical oncology, and the patient wheeled all the way down to radiation oncology (which is always in the basement, with all their nasty radiation) to get the heating. Unbelievable. We did discuss the idea of Celsion hiring their own nurse, in order to make sure the correct protocol was strictly adhered to. And Celsion, correct me if I am wrong, but as far as I know, this never happened. In fairness, I understand that a company such as Celsion is bound by and must follow the rules of the particular hospital administration, including departmental boundaries and concomitant budgetary constraints. Furthermore, one could imagine that if Celsion were to pay for their own oncology nurse, it might compromise their impartiality with respect to their own clinical trial (Box 23.8).

FIGURE 23.13 Plasma pharmacokinetics for the maximum-tolerated dose cohort (50 mg/m²) with superimposed median radio-frequency ablation (RFA) time, showing times that the area under the curve (AUC) was exposed to RFA. (Graph reprinted from J. Vasc. Interv. Radiol., 23, Wood, B.J., Poon, R.T., and Lockin, J., A phase 1 study of heat-deployed liposomal doxorubicin during radiofrequency ablation for hepatic malignancies, 248–255. Copyright 2012, with permission from Elsevier.)

BOX 23.8 LESSON AS WE GO #8: BE ON THE LOOKOUT FOR PROTOCOL DECISIONS THAT ARE BASED ENTIRELY ON A “HOSPITAL ADMINISTRATION’S BUDGET SHEET”

When a company takes your invention and then tries to implement a protocol at one of its medical sites (like Duke), that requires two clinical departments, and therefore two different paid staff to cooperate; make sure that it is not the hospital administration’s budget sheet that determines your protocol; in our case this meant, essentially, that the patient missed almost all of the ThermoDox^® bioavailability.

23.6.3 PHASE 1 FOR LIVER CANCER

In 2006–2007, all this RCW was in the background when Celsion decided to focus almost exclusively on primary liver cancer (also known as hepatocellular carcinoma [HCC]). Clearly, this was a cancer that needed clinical treatment options. There is a much greater population with primary liver cancer than RCW, and so patient accrual could be achieved in a more reasonable amount of time. But also, with an eye on “business reasons,” for patent lifetime, the clock is always ticking. Many patients die quickly from primary liver cancer, taking the inverse of “survival” statistics from Cancer UK for primary liver cancer in general; the rates are 70% dead at 1 year and 90% dead at 5 years. Focusing on such a high mortality rate, a pharmaceutical company can determine much more quickly if their newly developed commercial product is working, compared to say, the situation for prostate cancer, which is characterized by both a slow progression of the disease and a low mortality rate. Given these considerations, the reasoning behind the “business decisions” to abandon the prostate treatment (even though it was showing to be relatively safe and actually seemed to be working) becomes a little clearer (although obviously still not necessarily good, from the perspective of a prostate patient’s health and well-being). And as scientists (who deal in the tens to hundreds of thousands of dollars that last us about 1–5 years in our small research lab budgets with lowly paid professors and even cheaper postdocs and graduate students), remember this all costs money, lots of money, in millions. Almost by definition, “business decisions” have to start and end with cost, risk, and benefit to the company. If the company goes under, no patient can benefit. And it wasn’t as though Celsion had buckets of cash; they were budgeting and rebudgeting on a monthly basis, with quite a high burn rate given the cost of setting up and managing clinical trials. So while you want to keep them on their toes, do cut your licensee some, but not too much, slack.*

Now that Tardugno^† was in charge (from January 3, 2007), the primary liver cancer Phase 1 trial was initiated in February 2007 and completed in good time by December 2009, with a primary completion date of October 2008 (final data collection date for primary outcome measure) (Celsion 2009). The results looked so good that it advanced to a Phase 3 without going through a Phase 2.

Primary liver cancer is one of the deadliest forms of cancer and ranks as the fifth most common solid tumor cancer. The incidence of primary liver cancer today is approximately 26,000 cases per year in the United States and approximately 40,000 cases per year in Europe. It is rapidly growing worldwide, at approximately 750,000 cases per year, 55% of which are in China, due to the high prevalence of hepatitis B and C in developing countries. The World Health Organization estimates that primary liver cancer may become the number one cancer worldwide, surpassing lung cancer, by 2020. The standard first-line treatment for liver cancer is surgical resection of the tumor; however, 90% of patients are ineligible for surgery. RFA has increasingly become the standard of care for nonresectable liver tumors, but the treatment cannot adequately ablate larger tumors. There are few nonsurgical therapeutic treatment options available, since radiation therapy and chemotherapy are largely ineffective in the treatment of primary liver cancer.

While single-agent Dox has been found to be effective, it has not become a standard treatment for primary liver cancer, due to its relatively high incidence of severe toxicity, including congestive heart failure and neutropenia. Hence, the new initiative was to attempt to increase primary liver cancer cure rate by combining two approaches: ThermoDox^® + RFA. So a drug that is known to be effective can now be delivered directly to the tumor site in excess quantities. Couple this with a heating system that is widely used and can heat tumors to ablate their centers, while peripheral tumor heating could also release the drug. What could go wrong?

As shown in Figure 23.14, placement of an RFA electrode in a liver tumor can produce temperatures in the ablation zone upward of 60°C. Clinically, RFA induces in situ thermal coagulation necrosis, through the delivery of high-frequency alternating current to the tissues. However, RFA still has its limits (Choi et al. 2001). With currently available devices, the largest focus of necrosis that can be induced with a single application is approximately 4–5 cm in greatest diameter, and lesions that size have a high frequency of marginal recurrences. Thus, the diameter of suitable lesions must be less than 3–4 cm. Furthermore, tumors located near large vessels may not be effectively ablated because the heat-sink effect (i.e., blood flow cooling) of these vessels prevents ablation temperatures from being reached. Brad Wood and colleagues found that the “drug works no matter how you heat” (Locklin et al. 2008). Drug release was independent of the heat source; equivalent cytotoxicity could be obtained via heating using RFA or a warm water bath.

As we saw in studies that established its in vitro performance (Mills and Needham 2005), ThermoDox^® releases drug at significant rates with a peak at 41°C–42°C. However, the ablation temperature at the tumor center is >55°C. As the ablation temperature drops off from the center out to the periphery, there has to be a zone (the “thermal zone”) where temperatures are in the range 50°C–39°C (Figure 23.14). And so, if temperatures are in this range in the periphery, then, when ThermoDox^® is infused into the bloodstream of an already warmed tumor, it will deposit high concentrations of Dox throughout this heated tumor interstitium and perhaps kill the dangerous micrometastases and the growing periphery of these deadly tumors.

FIGURE 23.14 Radio-frequency ablation (RFA) of liver tumors + ThermoDox^®. By itself, RFA does not target micrometastases outside the so-called ablation zone. However, the lower temperatures in the outer “thermal zone” can facilitate the release of doxorubicin from ThermoDox^®, thereby expanding the treatment area. (Courtesy of Celsion Corporation, Lawrenceville, NJ.)

Before this idea could be tested in an efficacy study, it had to be approved for safety. The ThermoDox^® primary liver cancer Phase 1 study (Celsion 2009) was a multicenter, open-label, single-dose, dose-escalation study. The objective of this Phase 1 study was to determine the MTD of ThermoDox^® when used in combination with RFA, in the treatment of primary and metastatic tumors of the liver (Celsion 2009). The protocol was that “patients with unresectable liver cancers underwent RFA with a 30-minute i.v. infusion of ThermoDox^® starting 15 minutes before percutaneous or surgical RFA.” (There’s that “give-ThermoDox^®-first, heat-later” problem, creeping in again.)

As reported by Poon et al. (2008), a total of 24 patients (9 with primary liver cancer and 15 with metastatic liver cancer were treated (3, 6, 6, 6, 3 patients at 20, 30, 40, 50, and 60 mg/m², respectively). Median tumor size was 3.7 cm (the range, however, was 1.7–6.5 cm). In total, 28 tumors were treated. The important toxicity findings were as follows:

• RFA + LTSL is safe and feasible.

• Neutropenia is an important toxicity.

• It has similar toxicity profile to Dox.

Even as a Phase 1, the trial did provide some interesting data on clinical efficacy. After treatment, 20 (83%) of the patients had no evidence of local tumor failure. Despite this only being a Phase 1 dose-escalation toxicity study, as shown in Figure 23.15, there was actually a dose–response relationship in terms of time to tumor progression (of 32, 53, 135, 185 days, respectively), giving ≈500% increase in PFS for the MTD (50 mg/m²) compared to the lowest starting dose.

Encouragingly, then, there appeared to be a preliminary dose–response relationship in terms of time to tumor progression as the study reached its MTD. A 2011 Future Medicine paper predicted that “Lyso-thermosensitive liposomal doxorubicin could be an adjuvant to increase the cure rate of radiofrequency ablation in liver cancer” (Poon and Borys 2011).

FIGURE 23.15 Results for Phase 1 dose-escalation study to determine maximally tolerated dose for ThermoDox^® in conjunction with radio-frequency ablation, in the treatment of primary liver cancer. Dose and response (mg/m²) vs time for tumors to progress (days). (Courtesy of Celsion Corporation, Lawrenceville, NJ.)

23.6.4 PHASE 3 FOR LIVER CANCER HEAT STUDY (HEPATOCELLULAR CARCINOMA STUDY OF RFA AND THERMODOX^®)

Uncertainty at so many levels clouded this trial.

Given the efficacy seen in the Phase 1 trial, clinical testing was moved rapidly to a Phase 3 trial, bypassing Phase 2. Phase 3 was a randomized, double-blind, placebo-controlled study of the efficacy and safety of ThermoDox^® + RFA, compared to RFA alone, in the treatment of nonresectable primary liver cancer (Celsion 2012). Let’s start with the results.

23.6.4.1 Phase 3 Trial Fails to Meet 33% PFS

Given all the encouraging preclinical data and the clinical responses seen in Phase 1 trials in prostate, RCW, and primary liver cancer itself, it was therefore somewhat surprising for all concerned when the top-line data, announced January 31, 2013, showed that

ThermoDox^® in combination with radiofrequency ablation (RFA) did not meet the primary endpoint of the Phase 3 HEAT Study in patients with hepatocellular carcinoma (HCC), also known as primary liver cancer. Specifically, Celsion has determined, after conferring with its independent Data Monitoring Committee (DMC) that the HEAT Study did not meet the goal of demonstrating persuasive evidence of clinical effectiveness that could form the basis for regulatory approval in the population chosen for study. The HEAT Study was designed to show a 33% improvement in PFS with 80% power and a p-value = 0.05. In the trial, ThermoDox^® was well-tolerated with no unexpected serious adverse events. The HEAT Study was conducted under a Special Protocol Assessment agreed to with the U.S. Food and Drug Administration (FDA).

Celsion (2013)

23.6.4.2 The Trial Itself

This Phase 3 trial engaged 75 different sites in 11 different countries and was the largest study ever conducted in the treatment of unresectable primary liver cancer (i.e., HCC) (Celsion 2014). It looked to treat the usually untreatable large 3–7 cm primary liver tumors. It was conducted under FDA Special Protocol Assessment, received FDA Fast Track Designation, and had been designated as a Priority Trial for liver cancer by the NIH. ThermoDox^® was granted orphan drug designation in both the United States and Europe for this indication. The European Medicines Agency (EMA) had confirmed the HEAT Study was acceptable as a basis for submission of a marketing authorization application. In addition to meeting the U.S. FDA and European EMA enrollment objectives, the HEAT Study also enrolled a sufficient number of patients to support registration filings in China, South Korea, and Taiwan, the three other large and important markets for ThermoDox^®.

The arms of the study were the following:

• Experimental 1. ThermoDox^® (50 mg/m² in 5% dextrose solution). Start 30 minutes infusion about 15 minutes before RFA begins.

• Sham comparator 2. Sham (5% dextrose solution). Start 30 minutes infusion about 15 minutes before RFA begins.

The primary outcome measures were as follows:

PFS was measured from the date of randomization to the first date on which one of the following occurred: (1) local recurrence, (2) any new distant intrahepatic HCC tumor, (3) any new extrahepatic HCC tumor, and (4) death from any cause (time frame: 3 years). A secondary confirmatory endpoint was overall survival (OS).

The main inclusion criteria were as follows:

• Diagnosed HCC.

• No more than four HCC lesions, with at least one ≥3.0 cm and none >7.0 cm in maximum diameter, based on diagnosis at screening.

• If a subject has a large lesion (5.0–7.0 cm), any other lesions must be <5.0 cm.

By May 2012, the HEAT study reached its enrollment objective of 701 patients. The primary endpoint for the study was to measure just a 33% improvement in PFS with a P value of 0.05. A total of 380 events of progression were required to reach the planned final analysis of the study. In the late 2012, 380 PFS events occurred.

23.6.4.3 Phase 3 Trial Results

As mentioned and reviewed previously, expectations were high, especially when considering a comparison between the dose and response seen in the Phase 1 study and the criteria for this Phase 3. The PFS in the Phase 3 was required to show only a 33% improvement compared to RFA alone. This comparison is very favorable with the increase in PFS in the Phase 1 seen for the dose escalation (from 20 to 50 mg/m²) of almost 500% (the RFA-alone control was not done). A caveat though is that in the Phase 1 trial, median tumor size was 3.7 cm (range 1.7–6.5 cm), and so a handful of patients in this Phase 1 study could have had tumors that were critically smaller than those that made up the 701 subsequent cases for the Phase 3: “No more than four HCC lesions, with at least one ≥3.0 cm and none >7.0 cm in maximum diameter.”

Here, in Figure 23.16, is the Kaplan Meier plot from the presentation made by Professor Riccardo Lencioni MD, at the European Conference on Interventional Oncology, entitled “New IO Approaches for HCC: An Update on Clinical Trials,” for the PFS analysis (Lencioni 2013). From the same presentation by Lencioni, the OS analysis (secondary endpoint) showed similar results, with both plots of ThermoDox^® + RFA treatment (Trt B) virtually coincident with the RFA control (Trt A).

We can all speculate as to why this trial failed at the first-line level (see the Reverse Engineering review paper [Needham 2013] for some suggestions). But basically, it would appear that the RFA-“recommended” protocol, as it was then, often failed to heat the margins, little or no drug was released there, and, in many cases, it was just like doing RFA with no drug. In fact, the control arm, just RFA, was 20% better than expected, because the protocol had the clinicians do a minimum of 12 minutes heating, when in usual practice they only do 6 minutes of intense RFA heating (that’s all that is needed to burn out the center of the tumors). So the Celsion trial actually made RFA better. And worse still, this 20% better for the “control” arm ate into any advantage ThermoDox^® + RFA might have had. But there was a silver lining to this thundercloud. Some clinical sites used a different RFA device, and some sites got some of it right, at least enough to show positive results. For example, it appeared that the OS improvements correlated with studies done in South Korea where they used a Covidien cool-tip RFA probe. This contrasted markedly with little or no improvement in the Chinese sites, where they predominantly used the Angio StarBurst probe. Who knew these pieces of equipment could be so different? Figure 23.17 shows the PK plot again (i.e., Figure 23.12) but with the RFA protocol overlaid.

FIGURE 23.16 HEAT trial progression-free survival analysis. Trt A, Treatment A, i.e., RFA control; Trt B, Treatment B, i.e., ThermoDox^® + RFA Treatment. (Courtesy of Celsion Corporation, Lawrenceville, NJ.)

FIGURE 23.17 Radio-frequency ablation (RFA) protocol overlaid on the PK profile. Injection is started 15 minutes before the initially intense RFA heating. Following completion of the 30 minutes infusion of the clinical dose of ThermoDox^® suspension, heating is then intermittent for the next 45 minutes, or up to 90 minutes in some cases. (Reprinted from J. Vasc. Interv. Radiol., 23, Wood, B.J., Poon, R.T., and Lockin, J., A Phase 1 study of heat-deployed liposomal doxorubicin during radiofrequency ablation for hepatic malignancies, 248–255. Copyright 2012, with permission from Elsevier.)

With regard to dosimetry, note that a mean peak plasma concentration of Dox of 2500 ng/mL = 4.3 μm and in the five cell lines studied in the paper (Yarmolenko et al. 2010), the IC₅₀ for Dox was in the range 129–168 nM. We therefore expect that as the liposomes do go through the warmed vasculature, they will be releasing all their drug and attaining even higher values in the tissue, perhaps as high as 50–100 μm, values in excess of the IC₅₀ and approaching an IC₉₀ or greater. Again, it is imperative to point out that ThermoDox^® has a 1 hour half-life for retaining its encapsulated Dox, and so even a 15 minutes delay is missing some of the area under the curve (AUC) for the available drug.

23.6.4.4 Second-Line Data

Once the trial failed, again to their credit, and I am sure at the speedy insistence of Messrs. Tardugno and Borys, Celsion wasted no time in analyzing the second-line data, digging deeper into the actual patient-by-patient and RFA-by-RFA details. Second-line data are now showing what went wrong and how to fix it, as presented recently in the Celsion corporate presentation of April 2015 (Celsion 2015). In this presentation, Celsion is showing how expanding the treatment zone addresses RFA limitations when using ThermoDox^® + RFA. This new protocol is to evaluate ThermoDox^® in combination with standardized RFA (sRFA), called ThermoDox^® + sRFA 45, after finding that heating for times greater than 45 minutes gives significant improvement. So now, although ThermoDox^® is still infused i.v. ≈15 minutes prior to sRFA, heating is continued, albeit intermittently, over a 45 minute period. They are really doing their best given all the constraints of the instrumentation and infrastructure. As shown in the earlier schematic in Figure 23.14, with this expanded protocol, ThermoDox^® is given a greater chance to concentrate in the “thermal zone,” and so Dox is released in the “thermal zone” expanding the treatment area.

And Celsion is now publicly offering their own take on lessons learned (Celsion 2015), so let me simply feature that, unedited (Box 23.9).

BOX 23.9 LESSON AS WE GO #9: “REVIEW DATA FROM (THE MOST POSITIVE) 285 PATIENT SUBGROUP”

• RFA must be used within its engineered design limitations.

• 3 cm or greater lesions require multiple overlapping ablations.

• Longer RFA time (>45 minutes) results in better outcomes.

• Heating duration directly affects clinical outcome, by allowing for high local perfusion of drug at the tumor site.

• High tissue concentration of ThermoDox^® prevents recurrence (supported by multivariate Cox regression analysis).

• PFS is not a reliable endpoint in HCC trials.

Evaluating all this with the FDA and their clinical partners and coming to these conclusions was, I am sure, a long and hard process. As is obvious from the previously mentioned, this post hoc analysis actually showed some very important and key aspects that are now moving the technology forward once again. If there is one thing Celsion is, it’s resilient. So, let me finish off this section with some very positive results that have shaped the new OPTIMA trial (Celsion 2014).

23.6.4.5 Second-Line Data Show Very Positive Results

As reported by Celsion, again in their Celsion Corporate presentation of April 2015, second line data is showing extremely positive results, even though the protocol, from my perspective, was not optimal in terms of the tumor being heated first, and only intermittent heating after the first 12 minutes of intense RFA. It is not clear what temperatures are attained during the protracted intermittent heating period. In any event, in the second-line data from a significant number (285) of patients in the trials, it was not surprising (to us) for them to report that the duration of heat from the RFA procedure is a key factor in a successful clinical outcome when combined with ThermoDox^® (Lencioni 2013). The data showed the following:

• ThermoDox^® + RFA where the intermittent heating was carried out for less than 45 minutes (37%) was no better than the RFA control arm.

• In ThermoDox^® + RFA where the intermittent heating was carried out for between 45 and 90 minutes (40%), the OS improved by 66% compared to control RFA.

• In ThermoDox^® + RFA where the intermittent heating was carried out for longer than 90 minutes (23%), the OS almost doubled compared to control RFA.

• When these results are combined, there was a 53% improvement in OS.

Thus, it is worth presenting the graph for ThermoDox^® + RFA, with intermittent heating >45 minutes. As shown in Figure 23.18, the subgroup analysis of the HEAT study data in 285 patients with standardized RFA (>45 minutes) showed a much-improved response, which is now over 80 months and still ongoing.

The survival probability has not yet reached the 50% mark, and so if we interpolate from the graph at the 80% mark, we see that the improvement for the RFA + ThermoDox^® is ≈13 months over RFA alone, and at 60% survival probability, it’s ≈24 months. Also in these time-to-event curves (and in the one shown later for Doxil^®), the HR is used as an expression of the hazard, or chance, of events occurring in the treatment arm, as a ratio of the hazard of the events occurring in the control arm. An HR of 1 means that there is no difference in survival between the two groups. Thus, the HR of 0.628 signifies that the TTP for the RFA arm is 0.628 of the average TTP in the treatment arm. Hence, the correlation with the simple interpolation of the data showing 26 vs 39 months at 80% survival (0.666) or 49 vs 73 months (0.671) for the 60% survival.

Celsion performed another comparison for these data, to evaluate the subgroup analysis (single lesion) of the HEAT study: 285 patients who received the standardized RFA for >45 minutes ± ThermoDox^® vs the 167 patients who received RFA for <45 minutes ± ThermoDox^®. Figure 23.19 shows the difference between the less-than-45-minute heating and the greater-than-45-minute heating, with ThermoDox^® given i.v. in the bloodstream, is now ≈39 months.

FIGURE 23.18 Subgroup analysis of HEAT study data for 285 patients with standardized radio-frequency ablation (>45 minutes); hazard ratio, 0.628 (95% CI 0.420–0.939); and P value, 0.02. (Courtesy of Celsion Corporation, Celsion Corporate Presentation, April 2015.)

FIGURE 23.19 Subgroup analysis (single lesion) of HEAT study for 285 patients standardized RFA > 45 minutes ± ThermoDox^® vs 167 patients RFA < 45 minutes. Presented are data for overall survival sRFA > 45 minutes ± ThermoDox^®; hazard ratio, 0.628 (95% CI 0.420–0.939); and P value, 0.02. (Courtesy of Celsion Corporation, Celsion Corporate Presentation, April 2015.)

23.6.5 THERMODOX^® VS. DOXIL^®

Without Doxil^®, there would be no ThermoDox^®. In fact, it was the (what seemed to be) only modest successes of this formulation that motivated the invention of the LTSL: “I need something I can heat and it releases drug, damn it.” Chezy, Alberto, Frank, Martin, Theresa, the late Dan Lasic, and Demitri, and the many, many more, too many to mention, are dedicated people that pioneered this whole field. In my opinion, they cannot get enough praise, thanks, and gratitude for all they achieved. And so this comparison is presented merely so that readers can put these two “nanomedicines” and later view their own nanomedicines, with some perspective.

Let’s briefly compare the ThermoDox^® results with those that got Doxil^® its approval, i.e., the Doxil^® vs topotecan study (Doxil^® 2015), and published by Gordon et al. (2001). This was a Phase 3, randomized, multicenter study of Doxil^® as a single agent, versus topotecan, in patients with recurrent epithelial ovarian cancer. As reported by Gordon and colleagues, “data show that PEGylated liposomal doxorubicin and topotecan, an established, efficacious agent for recurrent ovarian carcinoma, demonstrate equivalence in efficacy measures” (Gordon et al. 2001). Thus, Doxil^® was no better than the then current treatment. The data, as a table and just one of the graphs from the website and in Gordon’s paper, both in Figure 23.20, show overall clinical benefit assessed by the endpoints of overall response rates and OS* in the single-agent recurrent ovarian cancer study. The number of patients was 474, which represents the protocol-defined intent-to-treat population. The CR to Doxil^® given to treat ovarian cancer was just 3.8%, just 9 patients out of 474, while the standard of care, topotecan, showed 4.7% CR as 11 patients of the population are treated. Compare this with the Phase 1/2 for ThermoDox^® in RCW, where a local response rate of over 60% was reported in 14 of the 23 evaluable patients, with 5 CR and 9 PR, and some of these were doses that had not even reached the MTD. For example, the picture of the patient in Figure 23.11 is at only 30 mg/m², only 0.6 of the final MTD of 50 mg/m². And this was in a study where the investigators may have missed almost all the AUC.

FIGURE 23.20 (a) The table shows the overall response and median duration of the response for the two arms of the study. (b) The graph shows that the Doxil^® median overall survival was 14.4 months. The range was 1.7–258.3 weeks since the first dose. (Courtesy of Celsion Corporation, Celsion Corporate Presentation, April 2015.)

Comparing now to the Phase 3 liver trial, the Doxil^® graph in Figure 23.20 shows a similar analysis to how ThermoDox^® was analyzed in subgroup analysis, as OS.

Obviously, ovarian cancer is a very challenging disease, and the recurrent ovarian cancer population that Sequus chose to trial their Doxil^® formulation in was in very dire straits. In the Doxil^® arm, the median TTP was just 4.1 months (Doxil^® 2015), and as listed in the table of Figure 23.20, the median duration of response was only 6.9 months, compared to 5.9 months for Doxil^® and topotecan, respectively. And the HR is 0.822 for the 239 patients in the Doxil^® treatment arm for the previous median duration of the response. But it was 0.955 for the Doxil^® median TTP, which was 4.1 months, and so is as close to a value of 1 as makes no difference. This comparison could be even more striking when Celsion release the new data from the OPTIMA and RCW trials. Given all their lessons learned, Celsion have now stipulated “standardized” and not just “recommended” protocols and have (I am sure) done as much as they can to optimize the protocols given the constraints of a drug–device combination.

23.6.6 OUR RECOMMENDED PROTOCOL

No matter what heating system is being used, as shown in Figure 23.21, and repeated again here in the text (just for good measure), here is our recommended protocol:

• Establish steady-state temperature of 41°C–42°C prior to infusion of ThermoDox^®.

• Maintain temperature of tumor at 41°C–42°C throughout the whole infusion.

• And if possible for at least 30–60 minutes after the infusion is terminated, catch the majority of the circulating ThermoDox^®, and release the drug in the warmed tumor.

FIGURE 23.21 Our recommended protocol doxorubicin/lipid half-life is only 1 hour. Do not miss it. (Suit from online clothing store, my head from photo shoot by Malou Reedorf, photographer.)

For short, we call it “heat first, administer ThermoDox^®, keep heating for at least 1 hour, kill tumor.”

23.6.7 NEW AND ONGOING HUMAN CLINICAL TRIALS FOR THERMODOX^®

Celsion has made a huge commitment to a full clinical program that is underway for ThermoDox^®. As shown in Figure 23.22 (from their web page Celsion “Pipeline” 2015), in addition to the trials reviewed earlier (primary liver, OPTIMA; RCW, DIGNITY), new research and preclinical development has started in metastatic liver cancer, ovarian cancer, pancreatic cancer, breast cancer, and glioblastoma. One of the most exciting new inclusions here is the adaptation of HiFu as the source of targeted mild HT.

FIGURE 23.22 ThermoDox^® clinical programs at Celsion Corporation (July 2015).

23.7 LESSONS FROM THERMODOX^®

23.7.1 THE FASTEST WAY TO DO RESEARCH IS LARGELY INCOMPATIBLE WITH ITS DEVELOPMENT AND COMMERCIALIZATION

Before we get to specifically what I, personally, learned from ThermoDox^®, let me give some general advice about the process of research and development, sometimes called big R and little D and little R and big D, depending on who is doing the R and who is doing the D.

There’s an old saying that “the fastest way to herd cattle is slowly.” In my view, this also applies to research. The fastest way to do research is … sloooooowly, and surely. We spend so much time making sure we get it right by reviewing our current data, identifying the key observation, posing the right questions founded in scientific depth and rigor, and composing scientific hypotheses to be tested. Based on this, you try to carve out enough concentrated time away from teaching class, your ongoing research, one- and two-day trips to give invited seminars, conducting administration duties, and not to mention the social and domestic life that you do not have, in order to write the proposed Research Plan.

And this is a plan with full and referenced descriptions of its overall goal, specific aims, background and significance, preliminary studies, research design, methods, timetable, the budget, budget justification, resources and biographical sketches of key personnel, responding to the first rejection and resubmitting the proposal that has a 1-in-14 chance of being funded in the top 7 percentile; with 28 grants in the study section, yours has to be one of the top two. Say it does get selected for funding, and you are then busied by advertising, interviewing, and recruiting the best students and postdocs; training them; carrying out all the experimentation, modeling, and/or simulations; obtaining data in at least triplicate; analyzing and interpreting the data, writing up the papers; and responding to reviewers on the way to eventual publication, a publication that has to last forever, to be true. In philosophy, “truth by consensus” is the process of taking statements to be true simply because people generally agree upon them. In science, truth is the process of checking everything and getting the same result; it’s more like “truth by proof.”

The truth is something that we all agree upon. Obtaining that truth is a long and detailed process. As I say, in small posters I tape up around my lab and student offices, “CHECK EVEYRTHING,” just to remind them that mistakes can pop up where you least expect them, and we cannot make mistakes, not even typos. And if any of this research actually generates a useful idea, like a treatment for cancer, then we better be very sure we have a deep understanding of the laws, theories, and models that are consistent with its functions, the component design and mechanism of action, our choice of materials, and their CSP relationships, optimized to meet the requirements of the design; have made the formulation and tested its performance in in vitro and in vivo assays; and have convinced ourselves that what we think is happening is really happening.

You have done your job and done it well. You are excited; your family is proud of you, and all your hard work. Based on the animal studies, where 11/11 mice with implanted tumors were cured out to 60 days after just a single bolus i.v. injection and heating their tumor for just 1 hour, you have high expectations, even though you do not know what to expect. The University owns all this. You submit your invention disclosure as soon as you are sure you have something of utility, and the OLV logs it in with a number and date and evaluates it for potential patent application, before you submit for publication review. Okay, where is the disconnect?

It turns out that this academic, painstaking, yet very exciting journey—your life, is at almost complete odds with the development and commercialization of your invention and your collaborators’ proof of concept. It turns out that for all the reasons of pressure to return investment, a stock price that is under attack from every financial blogger, and a series of revolving-door CEOs who come and go without making a positive impact on your invention, the process of development and commercialization of your invention wants it done, quickly.

Thus, following Frank and George from earlier, in today’s commercialization, we might even say, “Those who do not listen and act on the advice of the inventor are condemned to fail!” So, for mistakes to still be happening in today’s commercialization would seem to be avoidable, but it would take some effort to assimilate all the lessons learned from the past that is actually made so much easier (and voluminous) by efficient search engines. It would take a licensee who can strike the right balance between the enormous tasks of clinical implementation, regulatory issues, hospital administrations, and return on investment, to bravely and smartly adhere to evidenced-based practice. And since the university even managed to obtain a licensee, you might want to check the terms of the license and make sure with monthly e-mails so that you do not forget to keep track of the license until it is all too late. Having said that and having seen Celsion go through this whole process, with, I must say, considerable effort, generated financing, and some real success, researchers need to respect the entrepreneurial process that follows their research-demonstrated feasibility. To the entrepreneurs, I would say, even though you licensed the invention and feel entitled, you would do well to listen to the inventors.

23.7.2 FINALLY! LESSONS LEARNED FROM THERMODOX^®

As I laid out in no uncertain terms in my presentation at the Peck Symposium (Needham 2015), here is a list of lessons I have learned and would offer, now a little more gently, as some of the area’s researchers, inventors, and, yes, even university offices of licenses and ventures and the companies they deal with might consider and be highly aware of (Box 23.10).

BOX 23.10 FINAL LESSONS FROM “THERMODOX^®”: YOU CAN’T LEAVE EVERYTHING UP TO PEOPLE WHOSE JOB IS TO DO IT RIGHT (YES, THIS IS THE SOFTER, GENTLER VERSION)

Your university administration

• Keep track of the changing rules by your university administration

• They may change the patent rules for the inventor’s share as they see fit.

Your Office of Licensing and Ventures (OLV)

• Have an ongoing and functional relationship with your OLV.

• They may not know how to create a license agreement, and if they do, they may not manage it with the expectations and enthusiasm that you would.

• They are probably very busy and have to make judgment calls as how to best use their limited resources, and you might not be top of their list

Your license agreement

• Review the license agreement.

• Make sure it is compatible with what the company is capable of actually delivering.

• If they license your technology for all drugs, all diseases and physiological conditions, for the lifetime of all the patents, for a pittance, make sure that they do due diligence on the value of competing technology (and adjust the “pittance” accordingly).

• Encourage them to actually keep track of the license agreement and all its milestones, even though it’s not their highest priority.

Meeting the milestones

• If (and I am sorry to add), or when, the company fails to meet the milestones, make sure your OLV renegotiates the license in an appropriate way.

• For example, do not let them give away all your sublicenses, and let the company that could not develop it keep the technology as is, only to find out that the company did manage to generate a sublicensee for $20 M and you, as the inventor, and your OLV and the University get nothing.

Your (inventor’s) relationship with the company

• Develop an ongoing and mutually respected relationship with the company that your, and the University’s, technology is licensed to.

• If they will communicate with you, listen to the company, and their issues, and the problems they face.

• Do as much as you can, up front, to make sure they listen to your expert advice with regard to what the technology is and how it should be used, and also listen to them.

• For example, “heat first, administer ThermoDox^®, keep heating for at least 1 hour, and kill tumor.”

• No matter how good the Phase 1 trial results appear to be, encourage them (and encourage your OLV to include it in the license agreement) to carry out a Phase 2 study before a Phase 3.

The distribution company (there is no way to sugarcoat this one)

• Encourage your university development office to show integrity and manage the stock themselves.

• Your university will select a distribution company to manage your stock at arm’s length, for fear of being accused of conflicts of interest. Do not trust the distribution company that your university has “selected” by way of the cheapest tender (and therefore likely to be one of the least competent outfits).

• The distribution company will invariably fail to do due diligence and will sell your (and your university’s) stock at the lowest value, without warning or consulting you (because of the arm’s length deal), just so that they can get their fee. You and your university could lose millions of dollars.

23.8 NEW DIRECTIONS: PUT THE DRUG IN THE CANCER’S FOOD

During the summer of 2011, I was forced to take time off to recover from a major train accident. I started to think about a problem that I might work on, which had been suggested to me earlier that year, by a colleague, Neil Spector. Neil was one of the lead clinicians who had carried out a clinical trial on a new hydrophobic, reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, lapatinib, invented by Stephen Frey when he was at GSK. Neil arranged a meeting with me and asked if I could help to reformulate lapatinib. I started to study the problem, and by early September, the result was a 50-page white paper I called “The Formulation of Hydrophobic Anti-Cancer Drugs Part I. Exploring Mechanisms of Endogenous Uptake of Drugs by Cancer Cells - that have the Potential to Deliver them to within Ångströms of their Target Molecule.” I never sought to publish it but used it to form the basis for a part of my Niels Bohr Professorship Application, which was successful and brought me to Denmark (Box 23.11).

BOX 23.11 NEW LESSON AS WE GO #11: CARVE OUT SOME TIME TO THINK!

We are all so busy that often we are not able to carve out some time to actually think (deeply). Give yourself time to search the literature, build your ideas, check eveyrthing (yes, typo on purpose). You might not want to go to the lengths I went to and get yourself in a train wreck (not recommended), but maybe just take that vacation you are owed and use it on something useful. Take the time to get back to where we started with Frank Szoka, read some literature, understand it, sleep on it, and give yourself time to have an original idea. I’m just sayin’.

Without going into too much detail, the overall idea was to see if we could utilize the nanoparticle CSP of the low-density lipoprotein (LDL) and its receptor-based natural targeting, as an endogenous mechanism to achieve greater uptake of anticancer drugs by tumor cells. Basically, it was to see if we could simply “put the drug in the cancer’s food.” By reverse engineering the LDL, we hope to make pure-drug nanoparticles that will bind to, and be taken in, by cancer cells. From a nanoparticle design’s point of view, rather than fill the particle up with cholesteryl ester and try to dissolve or partition a drug into that matrix, the thinking is that by making pure-drug nanoparticles, we can have 2000–3000 drug molecules per particle.

The choice of the LDL receptor (LDLR) or other receptors like folate (and even passive endocytic pinocytosis) as the cell entry point is motivated by several observations already in the literature: rapidly growing cancer cells have high numbers of these receptors; and numerous malignancies are known to overexpress LDLR, including brain, colon, prostate, adrenal, breast, lung, leukemias, and kidney tumors, where LDLR can be some four hundred times greater on cancer cells than on normal cells (Ho et al. 1978). As a result, cancers are known to take in more LDL than normal cells, and in patients with cancer, their LDL count is even known to go down. An abundance of LDLR is also a prognostic indicator of metastatic potential (Rudling et al. 1986), and a propensity to store cholesteryl ester is a sign of the aggressiveness of a patient’s cancer. Thus, with 48 pages of text, 44 figures, and 221 references, this white paper sought to review the LDL—nature’s own hydrophobic delivery system—and establish a basis for new research and development of endogenous-inspired advanced drug delivery. I tried to answer basic questions such as the following:

• “What makes the LDL so effective at reaching its normal targets (adrenals, muscle, liver) and also cancer cells?”

• “How are its contents processed?”

• “Could it be that lapatinib, taken orally, enters this chylomicron–LDL pathway, and is it this endogenous ‘cholesteryl ester delivery system’ that delivers it to the tumor cells anyway?”

It was then not such a leap to ask the following:

• Rather than lose 99.3% of the administered drug via an oral tablet, can we go i.v. and use the cholesteryl ester/cholesterol pathway to deliver similarly hydrophobic anticancer drugs to tumors?

• Would such delivery bring the drugs into the cell in such a way that they could target growth, metabolism, and survival pathways?

• Could the same pathway also be used for diagnostics, especially PET imaging?

FIGURE 23.23 Could we make and test a pure-drug, lipid-coated nanoparticle that was inspired by reverse engineering the natural low-density lipoprotein? (Courtesy of Encyclopedia Britannica, Inc., 2007.)

This led to the idea, shown schematically in Figure 23.23: “Could we make and test a pure-drug, lipid-coated nanoparticle that was inspired by reverse engineering the natural LDL?”

23.9 TRANSLATING DRUG DELIVERY WITHOUT PROFIT: OPEN-SOURCE PHARMACEUTICS?

So, if we look at what we learned, the question I have to ask is, would I do it all again? And the answer is a resounding “No.” At least not in the way I just reported and certainly not within this academic–corporate–hospital complex. I cannot and will not, again, be part of a commercialization process that has to move from an academic or research setting to an eventual product for sale with profit in order to be available, requiring that it successfully crosses, what former NIH Director Elias Zerhouni has dubbed, the “valley of death.” An interesting perspective of this is given by Declan Butler (Butler 2008). In it, he discusses the problem from the NIH perspective, identifying the “chasm that has opened up between biomedical researchers and the patients who need their discoveries,” and considers “how the ground shifted and whether the US National Institutes of Health can bridge the gap.” But he does not mention the word “profit,” and so is probably missing one of the main problems in how “the ecosystems of basic and clinical research have diverged.” I personally see this as a major driver—whether direct profits or the all-important “stock price.” I will not put my ideas, hopes, and dreams into the hands of others, especially where profits and budgets dominate medical protocols, and “business decisions” made are not necessarily in the best interests of my invention and even the patient’s treatment. So what is the alternative? I asked myself:

What would happen if we just took profit completely out of the equation?

And others are certainly asking the same question, as the concept of “Nonprofits and the Valley of Death in Drug Discovery” is gaining some traction (Moos 2010). Moos states, quite categorically, “When we get mired in the economics or politics of the pharmaceutical industry, we can lose sight of some essential facts—the development of effective drugs saves lives.”

And here I am not just saying, “Pharma, open up on failed drugs,” like they did in 2010, where two large pharmaceutical companies participated in the unprecedented deposition of hundreds of thousands of potential leads for new malaria drugs into an open-source database (Strauss 2010). As explained by Strauss, in an article entitled, “Pharma embraces open source models”:

The proposition is for companies to de-emphasize intellectual property rights at least on early biology and be more open about sharing negative results so that knowledge advances faster in drug discovery research

I am not sure what to make of this except, in sharing negative results. Maybe they were fishing for some much-needed help, but for perhaps less profitable drugs that are targeting third-world diseases, rather than, for example, cardiovascular or cancer—but that’s just me being a little cautious in not totally trusting everything I read.

But the focus of all this is still on new drug entities. I am more concerned with effective formulations, and especially reformulations, of drugs we already have, but that have been poorly formulated in the first place.

What if we could develop and test the feasibility of this nonprofit idea in drug delivery and nanomedicines?

Could we scale up the nanoparticle production, and then offer it AT COST, NO PROFIT, and under a public license?

Thus, rather, I am saying, make the formulations open source so that more cancer patients actually get more of the drugs we already have (or develop in the future) to their cancers, in a much more efficient way than simply putting them in a cellulose tablet and settling for treating cancer as a chronic disease. There are strategies to help increase bioavailability of hydrophobic drugs taken orally—like a fat chaser. Just ask Burris and Spector and company (Burris et al. 2005) and their analysis of the lapatinib trial, where women who ate fatty meals seemed to have an increased response. This has since been investigated further and quantified, finding that “the AUC_0–24 increased following Lapatinib administration 1 hour after a low-fat meal by 1.80-fold and after a high-fat meal by 2.61-fold” (Devriese et al. 2014).

Before we get there though, i.v. formulations are required that actually stand a chance of delivering the appropriate dose (as we are investigating via more endogenous-inspired formulations). But if all we nanomedicine people do is create the next “nanowidget” company, hoping to make it big and then sue each other for having similar products (like what happened in the liposome field in the 1990s), it’s not going to work. There are lots of liposomologists around who have all been there, seen it, and done it, and the only people to profit are the lawyers and some of the independent expert witnesses (thank you very much).

I guess we will see, because I am determined to at least try it, and Denmark may just be the right place to do it. With 338 cancers per 100,000 people (WCRF International 2015), it is the so-called cancer capital of the world. To be fair, France is only 14 behind in second place, and the United States at 318 per 100,000 is sixth. Everyone in the Western world and Australia is in the low 300s or high 200s. They do smoke like factory chimneys here and eat a lot of red meat (and two vegetables), but is it the ubiquitous and accessible health care and available screening that makes Denmark’s numbers so high? Maybe Denmark is just the “cancer detection capital of the world.” If we can prove feasibility in my remaining 2.5 years on the job here as the Niels Bohr professor with my group and close collaborators, maybe we can make Denmark the “cancer treatment capital of the world.” I wouldn’t put it past us.

And to the young and restless researchers out there, join us. What have you got to lose? As you take your precious invention forward, with all the enthusiasm in the world, only to come up against a whole series of other agendas (some good for you, some not so good) outside your lab door, it is unlikely that you will make any significant money from your inventions anyway. So you might as well do some good. Join us. Peace.

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* After graduating in the summer of 1975, I was washing dishes in a hotel in Majorca, then working at a pigment factory 1976–1977 in the backstreets of Manchester, and then doing my PhD in gas-solid catalysis 1977–1980 with Professor Dan D. Eley, FRS, and all the while, Gregoriadis et al. were developing liposomes, who knew!

* Sterically stabilized liposomes are often described as “stealth” carriers (but the capitalized word “Stealth” is actually a registered trade name of Johnson & Johnson).

* Abbreviations: Dipalmitoylphosphatidylcholine, DPPC; dimyristoylphosphatidylcholine, DMPC; distearoylphosphatidylcholine, DSPC; monopalmitoylphosphatidylcholine, MPPC; mono-oleoyl phosphatidylcholine, MOPC; monostearoylphosphatidylcholine, MSPC; stearoyl-oleoyl phosphatidylcholine, SOPC; palmitoyl-oleoylphosphatidylcholine, POPC; distearoyl-phosphatidylethanolamine-poly(ethylene glycol)-2000, DSPE-PEG²⁰⁰⁰ (2000 molecular weight PEG).

* Note: You can only get these kinds of microscope-chamber temperatures on a winter day in Canada, where the humidity is so low; it would never happen in North Carolina.

^† 595 on Web of Science today.

* The abscopal effect is a phenomenon in the treatment of metastatic cancer where localized treatment of a tumor causes not only a shrinking of the treated tumor but also a shrinking of tumors in different compartments from the treated tumor.

* On one of my, too-infrequent, invited visits to Celsion, I noticed that this very same graph was also on a poster, I guess, proudly displayed on the wall of the CEO, who must have walked past it every day going in and out of his office.

* I can’t believe I said that; I must be getting soft in my old age.

^† And I really like Mike; his heart and mind are in the right place. He believes in our LTSL concept and always tries very hard to do the right thing. Thanks Mike. See http://celsion.com/docs/about_management for more details.

* The time to progression (TTP) is also available for viewing at https://www.doxil.com/hcp/for-recurrent-ovarian-cancer/efficacy, accessed March 17, 2016. Here Doxil^® median TTP was 4.1 months, range 1.3–106.9 weeks since first dose, and the hazard ratio was 0.955.