June 13, 2015

Reproducibility, the Hallmark of Western Science

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Provectus Biopharmaceuticals Chairman, CEO and co-founder Dr. Craig Dees, PhD frames outcomes of PV-10 use — preclinical or clinical, study or trial, generated by the company, a clinical investigator or a third-party medical researcher, affiliated or unaffiliated with Provectus — in the context of reproducibility, which he calls the hallmark of Western science.

He says (paraphrasing) if a scientist cannot repeat an experiment’s outcome and another cannot replicate the result, one should be skeptical about the veracity of the original work and claims made from it. PV-10’s track record of reproducible therapeutic features as well as preclinical and clinical results by multiple parties in multiple cancer indications in multiple species is noteworthy.

Wikipedia’s reproducibility[1] entry provides historical context to Craig’s hallmark of Western science remark:
“The first to stress the importance of reproducibility in science was the Irish chemist Robert Boyle, in England in the 17th century. Boyle's air pump was designed to generate and study vacuum, which at the time was a very controversial concept. Indeed, distinguished philosophers such as René Descartes and Thomas Hobbes denied the very possibility of vacuum existence. Historians of science Steven Shapin and Simon Schaffer, in their 1985 book Leviathan and the Air-Pump, describe the debate between Boyle and Hobbes, ostensibly over the nature of vacuum, as fundamentally an argument about how useful knowledge should be gained. Boyle, a pioneer of the experimental method, maintained that the foundations of knowledge should be constituted by experimentally produced facts, which can be made believable to a scientific community by their reproducibility. By repeating the same experiment over and over again, Boyle argued, the certainty of fact will emerge.” [The underlined emphasis is mine.]
December 2011 Wall Street Journal article Scientists' Elusive Goal: Reproducing Study Results[2] highlighted the lack of reproducibility in biomedical research, noting:
“This is one of medicine's dirty secrets: Most results, including those that appear in top-flight peer-reviewed journals, can't be reproduced.”
Related March 2012 Reuters story In cancer science, many "discoveries" don't hold up[3] expanded on two studies mentioned in the Journal artical, underscoring widespread lack of reproducibility in research:
“During a decade as head of global cancer research at Amgen, C. Glenn Begley identified 53 "landmark" publications – papers in top journals, from reputable labs – for his team to reproduce. Begley sought to double-check the findings before trying to build on them for drug development. Result: 47 of the 53 could not be replicated”[4]…Scientists at Bayer did not have much more success. In a 2011 paper titled, "Believe it or not," they analyzed in-house projects that built on "exciting published data" from basic science studies. "Often, key data could not be reproduced," wrote Khusru Asadullah, vice president and head of target discovery at Bayer HealthCare in Berlin, and colleagues. Of 47 cancer projects at Bayer during 2011, less than one-quarter could reproduce previously reported findings, despite the efforts of three or four scientists working full time for up to a year. Bayer dropped the projects.”[5]
The Wikipedia reproducibility entry presents a corollary to Boyle’s argument:
“The philosopher of science Karl Popper noted briefly in his famous 1934 book The Logic of Scientific Discovery that “non-reproducible single occurrences are of no significance to science.”
To be fair, however:
“…[I]t is worth remembering that irreproducible onetime events can still be a tremendously important source of scientific information. This is particularly true for observational sciences in which inferences are made from events and processes not under an observer's control.”[6]
Nevertheless, editors-in-chief Dr. Arturo Casadevall, MD, PhD (mBio[7]) and Dr. Ferric Fang, MD (Infection and Immunity[8]) wrote:
There may be no more important issue for authors and reviewers than the question of reproducibility, a bedrock principle in the conduct and validation of experimental science…“Reproducibility” is defined by the Oxford English Dictionary as “the extent to which consistent results are obtained when produced repeatedly…” Given the requirement for reproducibility in experimental science, we face two apparent contradictions. First, published science is expected to be reproducible, yet most scientists are not interested in replicating published experiments or reading about them…This leads to a second paradox that published science is assumed to be reproducible, yet only rarely is the reproducibility of such work tested or known. In fact, the emphasis on reproducing experimental results becomes important only when work becomes controversial or called into doubt…The assumption that science must be reproducible is implicit yet seldom tested, and in many systems the true reproducibility of experimental data is unknown or has not been rigorously investigated in a systematic fashion. Hence, the solidity of this bedrock assumption of experimental science lies largely in the realm of belief and trust in the integrity of the authors.” [The underlined emphasis is mine.]
Craig and his fellow co-founders, President Dr. Timothy Scott, PhD and CTO Dr. Eric Wachter, PhD, are outsiders to the biopharmaceutical industry. Hailing from Tennessee’s Oak Ridge National Laboratory, a U.S. Department of Energy (“DOE”) multi-program science and technology facility with a rich history of discovery and innovation, they are accomplished technology inventors and R&D 100 Award[9] winners[10],[11]. If the bedrock assumption of experimental science does indeed lies on the belief and trust in the integrity of authors of such work, then outsiders like Craig, Tim and Eric historically faced — and may still face — an uphill battle to convince industry constituents like the FDA, Big Pharma, the medical community, healthcare-focused Wall Street, the medical media and biotechnology investors of the veracity of their claims about Rose Bengal/PV-10 and its therapeutic benefits for cancer.

In Rose Bengal, PV-10’s active pharmaceutical ingredient (“API”) Provectus cofounders identified a small molecule lying around in plain sight for more than 60 years:
  • From American doctor G.D. Delprat’s anecdotal patient-reported outcome of “clinical benefit” in the late-1920s: “The patient, a Chinaman with a carcinoma of the pancreas with complete biliary obstruction, showed no toxic effects after the intravenous injection of 100 mg. of the dye [Rose Bengal]. In fact, while observing with interest the injection of this beautifully colored “medicine” he stated that he felt much better and wanted more”[12],
  • To Japanese researchers Ito et al.’s observation in the mid-1980s of dose-dependent increases in survival of mice receiving oral Rose Bengal[13],
  • To the rediscovery of the compound’s antineoplastic properties in the late-1990s by Craig, Tim and Eric.[14] Antineoplastic means acting to prevent, inhibit or halt the development of a tumor.[15]
Explaining how Rose Bengal/PV-10 works appears to have been a historically secondary consideration for Craig because I think his fellow cofounders and he believed they had a ready-made product that already worked, belying the arduous, not-so-straightforward and very lengthy process of bringing a drug to market, which some investors regularly underestimate. Seemingly making the task that much more daunting yet potentially more rewarding was the cofounders’ reality of bringing a mostly unknown small molecule to market that had at least two heretofore-unrecognized mechanisms of action (one local and one systemic) and an unappreciated treatment delivery route (intratumoral).

Provectus presented preliminary full study data for the company’s Phase 2 trial of PV-10 for advanced melanoma (Stage III and IV patients) in November at the Melanoma 2010 Congress.[16] Eric (who leads all aspects of the company’s clinical development program), however, met with the FDA in April for an end-of-Phase 2 or EOP2 meeting to seek accelerated approval (“AA”) for the drug, where and when he learned that it was not a suitable candidate for AA and a consensus Phase 3 trial randomized against a control was required.[17] In addition to designing a Phase 3 trial protocol, he began developing a parallel clinical development sub-program with the goals of fully validating the so-called bystander effect, whereby non-injected, distant tumors shrink or are completely destroyed as a result of injecting tumors elsewhere, and assessing immune markers in peripheral blood and tumor tissue.[18] During an ASCO 2010 investor presentation Eric noted in regards to the bystander effect: “Response in untreated proximal and visceral lesions consistent with immunologic process. PV-10 chemoablation yields immediate reduction in tumor burden. Ablation appears to recruit immune cells to exposed tumor antigens.”

Craig repeatedly has said mechanism of action data was not required for PV-10’s approval; a pivotal Phase 3 trial was and is, and agreeing to a suitable protocol with the FDA took Eric quite a long time. Eric, however, sought to have a medical research organization reproduce and validate Craig’s murine model work and also what was being observed in humans —thus, independently establish PV-10’s clinical value proposition. The undertaking began in 2011 when Provectus agreed on using Moffitt Cancer Center (“Moffitt”) to do the necessary work (and after also considering at least MD Anderson Cancer Center).

As early as March 2002 Craig, Eric and others, while at Photogen (the precursor to Provectus), when they believed Rose Bengal required photoactivation to prompt its therapeutic benefit (some short time thereafter the cofounders had their Eureka moment when they realized it did not), published their position that injection of Rose Bengal into cancer tumors generated both local and systemic immune responses[19]:
“Treatment of cutaneous melanoma with rapidly-scanned, tightly-focused NIR light appears to elicit selective hyperthermic and photodisruptive destruction of tumor tissue. Potential non-linear effects due to multiphoton excitation of melanin precursors appear to be of minimal significance. Since the effects are localized to the treated lesion, suppression of the host’s immune system (such as is associated with conventional radiation therapy and chemotherapy) is avoided, enabling the host to achieve maximal immune response to released tumor antigens and residual tumor tissues. Accordingly, such treatment is capable of stimulating an acute, localized inflammatory response leading to long-lasting systemic anti-tumor immunity. Stimulation of such immunity may, in humans, lead to improved outcome at both the treatment site and at sites of distant metastasis.” [The underlined emphasis is mine.]
I describe PV-10’s clinical value proposition as:
  • Safe, and sparing of tissue,
  • Locally and systemically effective, the drug’s two-prong approach to fighting cancer,
  • Multi-indication viable,
  • Synergistic in combination with other therapies and therapeutic agents,
  • Supportive of patient compliance,
  • Easy to use, re-use, ship, store and handle, and
  • Globally affordable.[20]
Fast forward to this year, when I recently discovered more third-party reproduction of key foundational pillars of PV-10’s proposition: local and systemic efficacy, and multi-indication viability. Thus, two medical research organizations — Moffitt in 2013 and the University of Illinois at Chicago (“UIC”) in 2015 — have reproduced key aspects of Craig’s and each other’s work.

According to Eric, the organizations received unrestricted research grants from Provectus to undertake their work. They received PV-10 from the company for their experiments and presented their results at medical conferences. For now only Moffitt has published its work in a peer-reviewed journal. I assumed Provectus had no undue influence on either Moffitt or UIC: i.e., no influence on designing and writing their respective study protocols, collecting data, performing analyses of study results, and writing respective manuscripts. Eric confirmed my assumption: "The work to date with UIC fits that description completely. Our initial work with Moffitt did as well..."

Biopharmaceutical companies produce and publish non-clinical work exclusively of their employees. Sometimes these companies produce and publish work that combines the involvement and contributions of their employees and affiliated third parties. Moffitt and UIC’s respective preclinical work appears to be theirs, and theirs alone. I have not found examples of multiple and separate cancer researchers reproducing the experimental results of other researchers for a compound with which they have no direct association (i.e., they did not discover it).

At the 2012 annual meeting of the Society for Immunotherapy of Cancer (“SITC”), Craig, Tim, Eric and others wrote on a poster titled Generation of an Antitumor Response and Immunity Using a Small Molecule Drug (PV-10):
“PV-10 (10% Rose Bengal in 0.9% saline) has been used to chemoablate a wide variety of tumors in human clinical trials and in animal patients when delivered by intralesional (IL) injection. PV-10 exhibits direct antitumoral activity targeted only on tumor tissue while sparing normal tissue. Additionally, PV-10 has stimulated the resolution of remote untreated tumors by an apparent immune-mediated antitumor response, including cutaneous and lung metastases. Therefore, we investigated the mechanism by which this remote “bystander” effect is produced using immunocompetent and incompetent mice. Tumor models examined include: hepatocellular carcinoma (HCC), melanoma, pancreatic and colon adenocarcinomas. PV-10 was found to chemoablate all tumors tested with no apparent side effects. No response was achieved in control tumors injected with vehicle only. Durable immunity was produced to the cell line that was ablated, however tumors of different origin could be established in treated mice. Immunity to establishment of a tumor could be transferred using spleen cells from mice whose tumors had been previously ablated. Untreated tumors in the opposite flank of mice completely regressed or were reduced in size in immunocompetent mice (i.e., 8/8 untreated HCC tumors), but no remote tumors were reduced in size in immunocompetent mice when a second tumor was treated by PV-10 chemoablation. In conclusion, resolution of untreated tumors is dependent on an immune mechanism that appears to involve immune cell mediation. We hypothesize the production of a vaccine-like immune response using a small molecule drug is possible and requires: 1) an intralesional route of injection that generates rapid, durable tumor destruction via autolysis; 2) rapid clearance of drug from normal tissue; and 3) antitumor effects targeted only to tumor tissue. PV-10 appears to meet each of these requirements. Co-administration of PV-10 immunochemoablation with other systemic therapy can yield potent synergy in uninjected tumors.” The underlined emphasis is mine. [The underlined emphasis is mine. Provectus previously referred to PV-10’s rapid destruction of injected tumors as chemoablation, but now refers to it as ablation.]
At the 2013 annual meeting of the American Association for Cancer Research (“AACR”), Moffitt wrote in an abstract titled Intralesional injection with PV-10 induces a systemic anti-tumor immune response in murine models of breast cancer and melanoma[21]:
“PV-10 is a 10% solution of Rose Bengal that is currently being examined as a novel cancer therapeutic. In melanoma patients, intralesional injection (IL) of PV-10 has led to regression of injected lesions as well as distant metastases. In this study, we examined the efficacy and potential immune mechanism of PV-10 treatment in murine models of breast cancer and melanoma. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions (p<0.05 compared to IL-PBS-treated mice). A significant increase in survival was observed in mice treated with PV-10. To examine immune response, MT901-specific IFN-gamma production and cytotoxicity were measured in splenocytes collected from mice treated with IL-PBS or IL-PV-10. MT901-bearing mice treated with IL-PV-10 demonstrated enhanced IFN-gamma production (992 ± 453 pg/ml) compared to splenocytes from PBS-treated mice (174 ± 105, p<0.05). In addition, a significant increase in lysis of MT-901 cells by T cells after PV-10 treatment was observed (p<0.01 compared to PBS-treated mice). No lysis of irrelevant CT-26 cells was detected. In a murine model of melanoma, B16-F10 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL-PV-10 led to regression of the injected lesion as well as distant B16 melanoma lung metastases. In B16-bearing mice, treatment with IL-PV-10 led to the induction of T cells that produced IFN-gamma in response to B16 tumors but not irrelevant tumor (p<0.05) and demonstrated specific lysis of B16 (p<0.01 compared to T cells isolated from PBS-treated mice). In total, these studies support the induction of tumor-specific T cell-mediated immunity after single treatment with IL-PV-10 in multiple histologic subtypes. The immune mechanism of PV-10 ablation in cutaneous melanoma patients is currently under investigation at our institution.” The underlined emphasis is mine. The above Moffitt work was published as a paper in 2013 in PLoS ONE.[22] [The underlined emphasis is mine.]
Moffitt observed that PV-10-injected melanoma and breast cancer tumors regressed — the drug’s local affect of tumor ablation, the first prong of its dual mechanisms of action. Moffitt then observed that distant or non-injected melanoma and breast cancer tumors regressed too, and that IFN-γ (referred to by one of the Moffitt study team members as the “quintessential antitumor cytokine”[23]) was produced in both indications following PV-10 injection — the compound’s systemic effect of a tumor-specific immune response, the second prong. Cytokines are the messengers of the immune system.[24] In cancer therapy cytokines are generally used to enhance immunity.

At the 2015 annual meeting of the Society of Surgical Oncology (“SSO”) UIC wrote in an abstract titled Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer[25]:
“INTRODUCTION: Early phase studies using intratumoral injection of PV-10 (10% Rose Bengal) have shown regression of in-transit melanoma deposits and non-treated bystander lesions. The effects of PV-10 on colorectal cancer (CRC) cells and established tumors is unknown. METHODS: Murine and human colorectal cancer cells were treated in vitro with varying concentrations of PV-10. Cell viability was determined by the MTS assay, trypan blue, DAF-FM, and SNARF-1 staining. To determine an underlying immune mechanism, a murine CT26 syngeneic CRC model was utilized. Bilateral subcutaneous CRC cell tumors were established in Balb/C mice and one tumor in each mouse was injected with PV-10 at a dose equal to half the calculated tumor volume. Tumors were measured daily. Splenocytes from treated animals were co-cultured with irradiated CT26 cells and supernatants analyzed for INF-γ production by ELISA. RESULTS: PV-10 induced near total cell death, corresponding increases in nitric oxide production, and decreased intracellular pH in both CT26 murine and HT29 human CRC cells within hours of exposure compared to controls (p<0.01), and at levels similar to 5FU. Treatment of subcutaneous tumors with a single injection of intralesional PV-10 led to near complete responses in all animals within days of exposure and significant regression of the injected lesions compared to controls (n=6 per group, p=0.027). PV-10 treatment was associated with occasional bystander responses in contralateral untreated tumors and trended towards a decreased rate of growth in these lesions. Splenocytes isolated from tumor bearing mice treated with PV-10 displayed enhanced tumor-specific IFN-γ production compared to splenocytes from PBS-treated mice (p = 0.025). CONCLUSION: Rose Bengal induced potent cell death in human and murine colon cancer cells in vitro. Intralesional injection in established tumors induced an anti-tumor immune response and significant tumor regressions in vivo. These studies establish that intralesional PV-10 therapy warrants further study as a potential immunotherapeutic agent in colorectal cancer and metastases.” [The underlined emphasis is mine.]
Separately, UIC also observed that PV-10-injected tumors, this time colorectal cancer, regressed — the local affect of tumor ablation, the first prong. UIC then observed that distant colorectal tumors regressed, and that IFN-γ was produced following PV-10 injection — the systemic effect of a tumor-specific immune response, the second prong.

Thus, Moffitt and UIC reproduced Craig’s original work, which first demonstrated PV-10’s two-prong approach and ability to successfully fight cancer in multiple indications:
  • Tumor ablation (the local effect): destruction of injected tumors,
  • A tumor-specific immune response: destruction of non-injected tumors, and
  • Tumor-specific IFN-γ production,
  • In melanoma, breast cancer and colorectal cancer.
There also are unaffiliated researchers who have noted the cancer-fighting benefits of Rose Bengal (or a derivative of it) that are consistent with Craig, Moffitt and UIC’s observations and in addition to Delprat and Ito’s.[26],[27],[28]

The Wikipedia reproducibility entry opens with:
“Reproducibility is the ability of an entire experiment or study to be duplicated, either by the same researcher or by someone else working independently. Reproducing an experiment is called replicating it. Reproducibility is one of the main principles of the scientific method.”[29]
The company's principals have some way to go in their stewardship of Provectus, as public company managers, to converge its extrinsic value (i.e., market capitalization) with the company's intrinsic value. There should be no disagreement, however, regarding the veracity of the principals’ claims about Rose Bengal/PV-10 and its therapeutic benefits for cancer. After all, Craig, Tim and Eric’s work achieved reproducibility, the hallmark of Western science.

Updated: This letter was revised on June 14, 2015.

[9] R&D 100 Awards History: see http://www.rd100awards.com/node/318
[10] 1996: Craig (Gencell 101, bacterial cellulase for textile finishing and other uses) and Eric (noncontact micromechanical thermometer and microcantilever mercury vapor sensor); the source URL is http://web.ornl.gov/info/ornlreview/rev30-12/text/awards.htm
[11] I cannot find a source URL for Tim’s award.
[17] ASCO 2010 investor briefing presentation, Clinical Program Overview (Eric), slide no. 39 of 55
[18] ASCO 2010 investor briefing presentation, Clinical Program Overview (Eric), slide no. 42 of 55
[20] From Provectus’ corporate website presentation slide titled An Opportunity to Make a Global Impact, which I created but that draws from Moffitt’s Dr. Vernon Sondak, MD’s PV-10-related presentation at the 4th European Post-Chicago Melanoma Meeting
[26] Rose Bengal acetate photodynamic therapy (RBAc-PDT) induces exposure and release of Damage-Associated Molecular Patterns (DAMPs) in human HeLa cells (2014); the source URL is http://www.ncbi.nlm.nih.gov/pubmed/25140900
[27] Rose Bengal suppresses gastric cancer cell proliferation via apoptosis and inhibits nitric oxide formation in macrophages (2014); the source URL is http://www.researchgate.net/publication/260429114_Rose_Bengal_suppresses_gastric_cancer_cell_proliferation_via_apoptosis_and_inhibits_nitric_oxide_formation_in_macrophages
[28] Selective toxicity of rose bengal to ovarian cancer cells in vitro (2012); the source URL is http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403562/

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