"Provectus Biopharmaceuticals Announces Terms Of Definitive Financing Commitment"

♢♢ Note: I and others entered into a securities transaction with Provectus in March 2017. Please see the Blog's Disclosures page and/or refer Provectus' Securities and Exchange Commission filings for further, future disclosures.

The source link of Provectus' March 23rd press release related to my and others' securities transaction and involvement with the Company (reproduced below) is here. Also see the Disclaimer to the right, and the blog's Disclosures page.

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KNOXVILLE, Tenn., March 23, 2017 /PRNewswire/ -- Provectus Biopharmaceuticals, Inc. (OTCQB: PVCT, www.provectusbio.com) ("Provectus" or the "Company"), a clinical-stage oncology and dermatology biopharmaceutical company, today disclosed terms of the previously announced Definitive Financing Commitment Term Sheet (the "Definitive Financing") it entered into on March 19 with a group of the Company's stockholders, who are referred to in the Definitive Financing as "PRH Group" in a Form 8-K filed with the Securities and Exchange Commission.

The members of PRH Group include Edward Pershing (serving exclusively in an advisory capacity only); Dominic Rodrigues and Bruce Horowitz, who were previously named as special advisers to the Board of Directors of Provectus ("the Board") on February 23; and additional members as the PRH Group may determine at its sole discretion.

Under the Definitive Financing, PRH Group would undertake best efforts to arrange for a financing of a minimum of $10 million and a maximum of $20 million, which would be provided to Provectus in several tranches.  The structure of the Definitive Financing takes the form of a secured loan that would convert into a new series of preferred stock with a liquidation preference upon the sale, dissolution or liquidation of the Company, a conversion into common stock that prices one common share at approximately $0.29, and customary voting rights on an as-converted basis.

Within the next 30 days or sooner, a total of $5 million will be made available to the Company, comprised of the acceleration of the remainder of the $2.5 million from the previously announced promissory note issued to Eric Wachter, Ph.D., co-founder and Chief Technology Officer and an additional $2.5 million from PRH Group. Once funded, the $5 million will become secured convertible promissory notes from the Company to each Investor.

Three current members of the Board will resign simultaneously upon the PRH Group's funding of the $2.5 million tranche into escrow, and PRH Group will nominate three new directors for approval by and appointment to the Board.  Additionally, the Company has agreed that, as soon as practicable after the funding of the second tranche of the financing, in which PRH Group intends to raise an additional $5 million by June 30, 2017, the two remaining current Board members will resign and be replaced by two new directors appointed to and approved by the Board.

PRH Group is joining with the Company to expand its current search for additional executives with biotechnology and pharmaceutical industry experience, including but not limited to a new Chief Executive Officer.

Dominic Rodrigues said, "PRH Group believes the fundamentally strong value of Provectus' asset base and intellectual property has attracted the initial interest of highly qualified managerial talent.  As the Company reestablishes a sound financial foundation, PRH Group is confident that Provectus will be well positioned to hire talented and experienced executives and managers. We are delighted to be a part of Provectus' evolution as a biopharmaceutical company by guiding its advancement of clinical development programs to develop vitally needed new treatments to help cancer patients."

About Provectus

Provectus is a clinical-stage biopharmaceutical company developing new therapies for the treatment of solid tumor cancers and dermatologic diseases. Provectus' investigational oncology drug, PV-10, is an oncolytic immunotherapy currently enrolling patients in Phase 3 clinical trials for metastatic melanoma. The Company has received orphan drug designations from the FDA for its melanoma and hepatocellular carcinoma indications. PH-10, its topical investigational drug, has completed Phase 2 clinical trials as a treatment for atopic dermatitis and psoriasis. Information about these and the Company's other clinical trials can be found at the NIH registry, www.clinicaltrials.gov. For additional information about Provectus, please visit the Company's website at www.provectusbio.com or contact Allison + Partners.

FORWARD-LOOKING STATEMENTS: This release contains "forward-looking statements" as defined under U.S. federal securities laws. These statements reflect management's current knowledge, assumptions, beliefs, estimates, and expectations and express management's current views of future performance, results, and trends and may be identified by their use of terms such as "anticipate," "believe," "could," "estimate," "expect," "intend," "may," "plan," "predict," "project," "will," and other similar terms. Forward-looking statements are subject to a number of risks and uncertainties that could cause our actual results to materially differ from those described in the forward-looking statements. Readers should not place undue reliance on forward-looking statements. Such statements are made as of the date hereof, and we undertake no obligation to update such statements after this date.

Risks and uncertainties that could cause our actual results to materially differ from those described in forward-looking statements include those discussed in our filings with the Securities and Exchange Commission (including those described in Item 1A of our Annual Report on Form 10-K for the year ended December 31, 2015, as supplemented by those described in Part II, Item 1A of our Quarterly Report on Form 10-Q for the quarter ended September 30, 2016) and the following:

• Provectus' potential receipt of sales from PV-10 and PH-10, transaction fees, licensing and royalty payments; payments in connection with the Company's liquidation, dissolution or winding up, or any sale, lease, conveyance or other disposition of any intellectual property relating to PV-10 or PH-10;

• our ability to raise additional capital if we determine to commercialize PV-10 and/or PH-10 on our own;

• our ability to close on an equity financing from PRH; and

• whether our securities remain listed on the NYSE MKT.

"Colon cancer cell treatment with rose bengal generates a protective immune response via immunogenic cell death"

Article source link is here.

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"Abstract:

Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer."

"Discussion:

...In the current study, we have provided several lines of evidence demonstrating that RB exhibited pronounced direct cytotoxicity in colorectal cancer cells both in vivo and in vitro. Our data also confirmed RB-induced prominent cell growth arrest at the G2/M phase and predominant necrotic cell death that was partially dependent on lysosome function. Furthermore, the findings from the current study identified that RB promoted expression of hallmarks related to ICD in colon cancer cell lines. Vaccination with RB-treated colon cancer cells and intralesional tumor injection resulted in retardation in tumor growth or prevention of subsequent tumor formation following challenge with the same tumor cells. These findings show that RB may serve as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer. Additional studies are warranted to elucidate the therapeutic potential of RB-induced ICD."

Rose Bengal (PV-10) + Oncology + Pediatrics

Updated below: 12/8/16 and 12/15/16.

Provectus issued a press release and filed an associated 8-K today regarding a collaboration (currently, an "agreement to establish a framework for collaborative pre-clinical research projects") to explore the use of Rose Bengal/PV-10 in pediatric cancer, Announces Agreement with POETIC (Pediatric Oncology Experimental Therapeutics Investigators Consortium) to Study Potential of PV-10 for Pediatric Cancer.

Image source

POETIC's website is here. POETIC co-founder Dr. Tanya Trippett, MD (Memorial Sloan Kettering Cancer Center) attended April healthcare conference at the Vatican, where Australia's Peter MacCallum Cancer Centre's Dr. Grant McArthur discussed PV-10.

See Infantile (July 26, 2016) on the blog's Archived News VI page:

"There also is a robust library of biomedical literature experimenting on/with, describing and discussing Rose Bengal's diagnostic applications in adults, and notably in children. See, for example October 15, 2015 blog post Still Standing, or Rose Bengal in children (hepatoblastoma, radiopharmaceutical) (May 6, 2016) on the blog's Archived News V page. In medicine, children are not small adults when it comes to safety, efficacy, dosing, etc. Most of the pediatric literature related to Rose Bengal refers to the API as liver function diagnostic 131I-Rose Bengal. From a safety and PK perspective, it is interesting, available for review, and dates back to at least the 1960s."

See also October 15, 2015 blog post Still Standing:

"Rose Bengal’s medical properties have been established in the clinic, adults and children[5], and the literature, as well as with the FDA. The compound was noted as a stain for visualizing corneal ulcers in 1919[6] and a marker for impaired liver function in 1923[7]. Currently there are more than 3,800 Rose Bengal references in the U.S. National Institutes of Health's National Library of Medicine’s PubMed Central database.[8] The compound has [non-therapeutic] FDA safety profiles as an intravenous hepatic diagnostic called Robengatope® and a topical ophthalmic diagnostic called Rosettes® or Minims®."

Updated (12/8/16).1: Additional Information & Takeaways

• I'm led to believe MSKCC (Memorial Sloan Kettering Cancer Center) [on the pediatric oncology side] began using reagent grade Rose Bengal (i.e., drug substance, or the active pharmaceutical ingredient [API] in PV-10; e.g., available from Sigma-Aldrich) -- presumably in in vitro models -- in the spring (following the above mentioned Vatican healthcare conference). Apparently, PV-10 (i.e., pharmaceutical grade drug product) was shipped to MSKCC and other POETIC partners (specifically, I would imagine to Alberta Children's Hospital, which will lead the pre-clinical development of another POETIC collaboration; see CorMedix below).

• I imagine one of Dr. Trippett and others' pediatric oncology focus areas may be sarcomas, which are tumors that occur in the bones and soft tissues.

• I also imagine POETIC and others' treatment approach to pediatric cancer patients may mirror the approach being taken with adult cancer patients in the current"age," "era" or time of immuno-oncology; that is, combination of PV-10 with checkpoint inhibition.

• See also CorMedix's collaboration with POETIC -- the parties' press release from September 2016, and Grammatical error (September 21, 2016) on the blog's Archived News VI page.

Updated (12/15/16).2: "Copyright infringement?" Yuck, yuck...

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Provectus' attendance link is here:

Turning [more anti-PD-1] non-responders into responders

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Updated below: 10/19/16.

What is PV-10's clinical value proposition to Merck & Co. (pembrolizumab, Keytruda®) and Bristol-Myers (nivolumab, Opdivo®), among other Big Pharma in the oncology space? In no particular order, is it, among other things:

• As a primer, front-end, turner-on-of-the-engine, stepper-on-the-gas pedal, [insert your favorite over-, weakly- or wrongly-used analogy or metaphor],

• Synergism, where from an efficacy perspective 1 + 1 >> 2,

• Agnosticism to tumor type/cancer indication,

• Safety profile, and/or

• Turning cold tumors hot, and hot tumors hotter?

Industry discussion appears to recognize immune checkpoint inhibitors work — when/where they do work — in a portion of cancer patients. Is the summary clinical value proposition of PV-10 in combination with Keytruda/Opdivo to make the latter (i.e., these anti-PD-1 drugs) work better when and where they work? Or is PV-10's proposition, the more powerful one, to show it can make Keytruda/Opdivo work better where they do not work?

PV-10's clinical value proposition to Merck & Co. (pembrolizumab, Keytruda®) and Bristol-Myers (nivolumab, Opdivo®) is that it (PV-10) can turn more anti-PD-1 non-responders into responders than any other partner drug or investigational compound.

For this blog post, consider, among other things, two combinations with pembrolizumab (for advanced melanoma):

• Intralesional* agent electroporation with plasmid interleukin-12 (epIL-12) (ImmunoPulse, OncoSec), the combination of a medical device and an investigational agent, and

• Intratumoral* agent toll-like receptor 9 (TLR9) agonist SD-101 (Dynavax), an investigational agent too.

* Intralesional = intratumoral

OncoSec. OncoSec announced in November 2014 it would combine ImmunoPulse and pembrolizumab, UC San Francisco and OncoSec Medical Collaborate to Evaluate Investigational Combination of ImmunoPulse and Anti-PD-1 Treatment. Data from this investigator-initiated study were presented at AACR 2016 (April), "Positive Melanoma Clinical Data at American Association for Cancer Research (AACR) Annual Meeting 2016," where patients initially were treated with ImmunoPulse and, then, some went to receive systemic anti-PD-1/PD-L1 therapy. Notably, however, OncoSec announced this month data from the same study would be presented at SITC 2016 (November), "Acceptance of Late Breaking Abstract at Upcoming Society for Immunotherapy of Cancer (SITC) Annual Meeting 2016," where the focus would be on [clinical data from] patients with a low likelihood of response to an anti-PD-1 alone (i.e., anti-PD-1 failures).

Dynavax. Dynavax and Merck & Co. announced a collaboration in June 2015, Investigating the Combination of Immuno-Oncology Therapies. Initial clinical data of the combination of SD-101 and pembrolizumab in patients with metastatic melanoma was presented at ESMO 2016, "Phase 1b/2, Open-Label, Multicenter, Dose-Escalation and Expansion Trial of Intratumoral SD-101 in Combination With Pembrolizumab in Patients with Metastatic Melanoma." Preclinical work on SD-101 was presented at AACR 2016 by Dynavax observed, "These data provide a strong rationale for the clinical assessment of SD-101 in combination with agents blocking the PD-1/PD-L1 pathway in patients unresponsive to PD-1 blockade alone." Dynavax and Merck jointly observed on their ESMO 2016 poster, "Preclinical studies suggest that the immunostimulatory effects of SD-101 might also boost the activity of PD-1 checkpoint inhibitor therapy. In mouse models, SD-101 converted anti-PD-1 non-responders into responders by increasing the quantity and quality of tumor-specific T cells." {my underlined emphasis}

In order for Provectus CTO Dr. Eric Wachter, PhD to put Provectus in a position to garner a collaboration with a Big Pharma and its immune checkpoint inhibitor, he has to provide a compelling demonstration of the features of PV-10 in combination with an anti-PD-1 drug like pembrolizumab (e.g., clinical trial PV-10 in Combination With Pembrolizumab for Treatment of Metastatic Melanoma). The features of this demonstration would include (a) preliminary safety and efficacy results, (b) immune biomarkers to facilitate appropriate patient selection if and when the combination is approved, and, presumably, (c) the ability of PV-10 to better turn anti-PD-1 non-responders into responders.

Contesting anti-PD-1 non-responders into responders should be a big deal for Merck and Bristol-Myers because such contestation is all about eating more of the rest of the pie, much more so than fighting over the same sliver of it.

Updated (10/19/16): OncoSec. ref. "OncoSec (ONCS) Q4 2016 Earnings Call Transcript," Seeking Alpha

I referenced epIL-12 (and OncoSec) above because of the useful information regarding anti-PD-1 failures or non-responders. The oncology playing field continues to evolve, and combination therapy approaches clearly are evolving as well across multiple dimensions, like (i) determining which patients when and how [immune biomarkers], and (ii) expanding the addressable market from responders to non-responders.

Among other aspects of an analysis of epIL-12 (and OncoSec), which historically has been mentioned together with Amgen's T-Vec and Provectus' PV-10, like at ASCO 2014 (see "Expert Point of View: Axel Hauschild, MD," The ASCO Post, Caroline Helwick, July 25, 2014), (a) there does not appear to be an initial pathway to approval yet (if at all) for epIL-12 as a monotherapy and (b) the investigator-initiated study was neither designed nor powered to transition to a pivotal trial as a combination therapy. OncoSec hopes to secure agreement with (acquiescence by) the FDA on a pivotal/registration trial design by the end of the year. Initial pathways to approval, like what Provectus has with PV-10 as a monotherapy for locally advanced cutaneous melanoma, as with valuable beachfront property, is valuable drug treatment "real estate." Nevertheless, it is a good strategy for OncoSec to focus on PD-1 failures; using emerging biomarker data to select "likely" PD-1 failures, however, is likely to prove somewhat more challenging. It will be interesting to see how this plays out.

Dynavax. This is a true treatment combination and company collaboration (compared to the OncoSec treatment combination, for which the clinical trial protocol is here). The results are interesting, if not very preliminary (e.g., efficacy from 5 patients, measurement [for purposes of the ESMO 2016 abstract] was made after only 12 weeks). One would have hoped they could have provided a few more details (e.g, the number of injections of SD-101 [presumably 11 in total], more details on patient stage [particularly Stage IV, like M1a, M1b and/or M1c], what the grade 4 SAE was, etc.).

A 25% serious adverse event (SAE) rate seems a bit high, and this is kind of an odd way to report safety data; usually this is reported as CTCAE Grade 3 or higher events since this includes both severe AEs and the subset of those that qualify as SAEs. Robert et al. reported 10.1-13.3% rate of Grade 3 or higher AEs for pembrolizumab alone (NEJM 2015;372:26).

Notably, investigators/clinical sites on the poster included Agarwala/St. Luke's. The trial itself also is recruiting at Huntsman Cancer Institute (Andtbacka).

Since this is a collaboration with Merck, it would appear the Big Pharma is not requiring rigorous safety testing before the project moves to Phase 2 (a Merck staffer is a co-author on the ESMO 2016 poster). The poster's Methods section notes the trial is a dose-escalation and dose expansion study. It shows data from dose-escalation (i.e., 2 mg, 4 mg, 8 mg), but does appear to refer to the dose expansion portion, which normally would be additional patients at the highest tolerated dose. The N = 6 at 8 mg is dose expansion but, again, if Merck wants to green light this work to a Phase 2 trial, [as a Big Pharma] they probably are not going to get significant push-back from the FDA or institutional review boards (IRBs). If a small biotechnology company has a major player backing it, it is possible to do things that are not plausible for outsiders (i.e., the golden rule). Finally, this study might give Amgen pause, since SD-101 appears to function similarly to T-Vec, may produce a more robust effect than T-Vec and, most importantly, is not a live virus.

Immunity

Edited (by me) image above; original image source

Let us take as given, finally in and by 2016, that PV-10 (chemical small [but heavy] molecule and halogenated xanthene Rose Bengal) is an immunotherapy

That is, the investigational compound is potentially capable of inducing the human body's immune system to mount a possibly worthy defense against solid tumor cancer after first being injected into lesions and tumors of said cancer.

That PV-10 has been independently shown to be that immunotherapy, at arms length from Provectus separately by both Moffitt Cancer Center (Moffitt) and the University of Illinois at Chicago (UIC) — reproducibility, the hallmark of Western Science— should make the claims about and data on PV-10 that much more veracious. See footnotes 1-5 below.

But let's now examine the notion, and growing preclinical and clinical evidence, that PV-10 treatment — singularly or in combination — potentially may lead to something greater and more profound than just the inducement or generation of an immune response....immunity.

AACR 2016 (April), Moffitt: T cell mediated immunity after combination therapy with intralesional PV-10 and co-inhibitory blockade in a melanoma model

2016 (May) peer-reviewed publication, Moffitt: Intralesional rose bengal in melanoma elicits tumor immunity via activation of dendritic cells by the release of high mobility group box 1

SITC 2016 (November), Moffitt: Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer



Footnotes of non-Provectus biomedical research for "PV-10 is an immunotherapy:"

1. SSO 2012, Moffitt: Intralesional Injection of Melanoma with Rose Bengal Induces Regression of Untreated Synchronous Melanoma In a Murine Model,
2. 2013 peer-reviewed publication, Moffitt: Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer,
3. SSO 2015, UIC: Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer,
4. 2015 peer-reviewed publication, UIC: The Potential of Intralesional Rose Bengal to Stimulate T-Cell Mediated Anti-Tumor Responses, and
5. ASO 2016, UIC: PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells,

April 2016: Grant McArthur Discusses the Memory of Our Immune System

Shapeshifter

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Rose Bengal is a molecule that has been around for a long time and used in several very different ways. In addition to being an industrial dye and a food dye (i.e., Red Food Dye No. 105), the molecule has a long history as a diagnostic agent with an established safety history. Use as a diagnostic was Rose Bengal's original and first medicinal use (i.e., an intravenous hepatic diagnostic: 131I-radiolabeled Rose Bengal/Robengatope®; a topical ophthalmic diagnostic: Rosettes®, Minims®).

Rose Bengal's second medicinal use is as a therapeutic, which is being advanced by Provectus in both oncology (PV-10) and dermatology (PH-10). There are other halogenated xanthenes the company owns.

Note the concentrations: intralesionally (intratumorally) injected PV-10 for oncology is a 10% solution of Rose Bengal, and topically applied PH-10 for dermatology is a 0.001% to 0.01% gel of the active pharmaceutical ingredient (API) Rose Bengal. PV-10 up-regulates or stimulates, while PH-10 appears to locally down-regulate.

This is a blog page I update from time to time as information is gathered about Rose Bengal's shape changing (transformation) abilities; h/t a shareholder hatter.

For now, first consider Xu et al., "Aggregation of Rose Bengal Molecules in Solution," Journal of Photochemistry and Photobiology, A: Chemistry, 40 (1987) 361-370.

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The authors note (my underlined emphasis):

"Rose bengal is a bis anionic dye and it has been suggested that it also has a tendency to aggregate in solution. It does not follow Beer’s law at concentrations above 10e5 M and the spectra are both of different shape and shifted at higher concentrations. Thus the aggregation phenomena of rose bengal and the other xanthenes have been previously studied in solution by means of absorption spectroscopy 171. The absorption spectrum of rose bengal in dilute solution consists of two peaks separated by about 30 nm. In more concentrated solution the shorter wavelength of these peaks grows and shifts several nanometers toward the blue. We can now assign the longer wavelength band exclusively to the monomer while the shorter-wavelength band derives from a combination of the monomer and the dimer respectively and its size as well as its shape depend on concentration."

There has been some discussion of the pH environment in a tumor cell, and surrounding healthy tissue. There are differences in the nutritional and metabolic environment of cancerous and healthy tissues.

Pathobiology of Cell Membranes, Volume 2, edited by Benjamin F. Trump, Antti U. Arstila (1980), discuss, among other things, the effect of pH, and mechanism:

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Red Cell Shape: Physiology, Pathology, Ultrastructure, edited by M. Bessis, R. I. Weed, P. F. Leblond (1973), note the factors that facilitate shape transformation (sphering) to varying degrees include pH, temperature, ionic composition, and concentration:

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Intralesional PV-10 for In-Transit Melanoma—A Single-Center Experience

Article: Lippey et al., "Intralesional PV-10 for in-transit melanoma-A single-center experience," J Surg Oncol, 2016 May 30.

H/t a shareholder and regular hatter for access to the above paper. Bolding-in-paragraph and underlined emphasis below is mine.

[From the end of the paper] "Synopsis: This paper reviews a single metropolitan cancer hospitals experience with PV-10 for the treatment of in-transit melanoma. Over a 4-year period, we have treated 19 patients with PV-10 for melanoma and achieved disease control in 68% of patients. We find it a helpful tool in our armory for local control of this often-difficult clinical scenario."

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"Toxicity: The majority of treatments (73%, 24/33) were well tolerated without any reported side effects. Edema, pain and erythema were the most common side effects, although these were minor in severity, limited in duration, and easily managed by simple analgesia. Five patients required opiate analgesia for pain associated with PV-10 injection.

One patient was readmitted to hospital 1 week following treatment with lower limb cellulitis requiring intravenous antibiotics for 2 days. This patient was obese and elderly and the cellulitis arose in the area of recent injection."

"Treatment Response: After a median follow up of 11.7 months, disease control was achieved in 63% of patients. Five patients (26%) achieved a complete response, another five (26%) patients achieved a partial response, and two patients had stable disease (11%) at the time of last follow-up. Seventy-four percent (14/19) of patients had a clinical response at time of first follow-up (median time 21 days); range 8–91 days. Younger patients and those with smaller lesions were more likely to respond to treatment (Table III). The number of injected lesions and the time from primary diagnosis to treatment were not predictive of response.

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Ten patients did not have all lesions injected, primarily due to the number of lesions present. A bystander response was noted in un-injected lesions in 50% of patients who did not have all their lesions directly injected (Table II). After a median follow up of 11.7 months, eight patients had died

from metastatic melanoma."

"DISCUSSION: This single-center retrospective review demonstrates that intralesional PV-10 is an effective, safe, and well-tolerated treatment option for patients with ITMs and loco-regional recurrence of melanoma. Treatment was delivered to a group of patients who were elderly (median age 82 years) and in many cases considered inappropriate for more aggressive and potentially toxic therapies such as ipilimumab or isolated limb infusion.

There have been several previous reports of success with intratumoral injection of PV-10, providing local control in this group of patients with an acceptable toxicity profile. There is still a lack of data about durability of PV-10 as well as a lack of long-term survival data. The largest published study assessing the use of PV-10 in the setting of refractory melanoma published in 2014 analyzed 80 patients from seven international sites. In this study, a 52% overall response rate and 26% complete response rate were described which is comparable to the overall response rate of 63% and complete response rate of 26% in the current series as well as in another previously published single-center series (Table IV).

The effective treatment options for metastatic and locoregional inoperable disease melanoma have rapidly improved over the last 5 years. With the introduction of novel systemic agents targeting immune checkpoints (ipilimumab, pembrolizumab, nivolumab), and mutations in the MAP kinase

pathway (dabrafenib and trametinib), the treatment options for patients with unresectable metastases have increased and the prognosis for these patients has significantly improved. However, these agents may be associated with significant toxicity and in the case of PD-1 targeted therapy require frequent hospital visits for infusions. Intralesional PV-10 compares favorably with other

intralesional therapies including talimogene laherparepvec (T-VEC) which in the recently published OPTiM study demonstrated on overall response rate of 26.1%. It is important to note that the

patient population in the OPTiM study had more advanced disease than the population in the current study, and the response rate for patients with all lesions injected lesions with T-VEC was 33%. For an

elderly patient with ITM, a simple and effective local therapy with minimal side effects is an attractive option. In our center, the use of ILI has steeply decreased with the availability of PV-10.

In the modern era of effective systemic therapies, patient selection for intralesional therapy is critical. Previously described factors predictive of response include the presence of ulceration, blistering, eschar, or pain following injection. In the current study, lesion size was also found to be predictive. Of the five patients who achieved a complete response, the average lesion diameter was 3 mm compared to the cohort average size of 6.3 mm. We did not specifically collect data on eschar formation but anecdotally have seen significant ulceration and eschar in most responders which may represent a brisk immune response to treatment or a direct toxic

effect.

Aside from the local toxicity of pain and edema and an isolated report of photosensitivity, PV-10 remains a very safe treatment option. As compared to radiotherapy, PV-10 has the advantages of allowing a wider field of treatment which may be repeated if necessary. A successful combination of radiotherapy and intralesional PV-10 has been reported and may warrant further

investigation.

The limitations to this study are the retrospective nature and the variable treatment regimens which were tailored to patients according to social, geographic, and oncological factors. As our cohort consisted of elderly and comorbid patients, we often limited their required visits to hospital which is reflected in the short follow-up intervals.

There are a growing number of options for the treatment of unresectable in-transit disease and choice depends on many factors including availability of treatment, patient suitability, and disease factors. Intralesional PV-10 compares favorably in that it is well tolerated especially in an elderly patient or one with significant comorbidities.

Could PV-10 (Rose Bengal) be implicated in different kinds of cell death?

Image source

Reference article: Garg et al., Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation, Cell Death and Differentiation (2016) 23, 938–951.

N.B. There is no reference to Rose Bengal (generic name) or PV-10 (proprietary name) in the February 2015 Garg et al. article.

Article Abstract: Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of ‘eat me’ signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.

Provectus says PV-10/Rose Bengal:

• Does not rely on a single pathway to work [I assume 'signalling pathway'],

• Does not focus on a single receptor to work [I assume 'cell receptor'], and

• Has no known resistance [I assume little no cancer drug resistance].

A highly specific compound in its targeting of only diseased (cancerous) tumors/lesions, tissue and cells, sparing healthy ones in the process, might the veracity of PV-10/Rose Bengal's "multiplicity" be based in its lack of "specificity" in regards to cell death?

That is, might Provectus' investigational compound's apparent implication in different kinds of cell death help explain why PV-10/Rose Bengal does not rely on a single pathway or focus on a single receptor to work, and has no known resistance?

Of note in Garg et al.'s article are (a) a table describing "major cell death pathways and their immunobiological" profiles and (b) a figure illustrating "therapeutic exploitation of phagocytosis of dying cancer cells for T-cell-mediated cancer cell elimination."

The table of major cell death pathways includes (i) apoptosis, (ii) autophagy (autophagic cell death) and (iii) immunogenic cell death.

Click to enlarge. Image source

The illustration appears to draw three paths to anti-tumor immunity; one that is direct (e.g., DAMPs like HMGB1), and two that additionally employ co-stimulatory signals such as TLR agonists or co-inhibitory signals such as immune checkpoint therapy.

Click to enlarge. Image source

A sampling of Provectus and independent medical researcher work implicates PV-10 in apoptosis, autophagy, necrosis, and immunogenic cell death:

• Provectus: "The patient who had chemotherapy radioresistant recurrent disease in the head and neck area had rapid necrosis of the injected metastasis and this as well as 3 uninjected sc metastases underwent complete remission over a period of several months," "Rose Bengal induces dual modes of cell death in melanoma cells and has clinical activity against melanoma," Melanoma Research 16(Suppl 1):S8, Sep 2006

• Provectus: "PV-10 accumulation elicits acute autophagy of cancer cells," "Chemoablation of Metastatic Melanoma with PV-10," Presentation at 4th Interdisciplinary Melanoma & Skin Cancer Centres Meeting, Sydney, AU, Nov 2010

• University of Illinois at Chicago: "Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis," "PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells," ASC 2016

• Quote by Provectus about Moffitt Cancer Center work: "This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells." "Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity Via Activation of Dendritic Cells by the Release of High Mobility Group Box 1," Oncotarget 2016.

Garg et al. observe "The mechanisms of cancer cell death elicited by anticancer therapy and the type of phagocytes (e.g., tumour-resident versus therapy-recruited) interacting with dying cells are decisive factors in making a difference between anti-inflammatory or pro-inflammatory responses."

Some cell death via PV-10 occurs in the injected lesion or tumor (i.e., tumor-resident), which is the upstream trigger of subsequent cell death via a tumor-specific immune response (i.e., therapy-recruited).

Rose Bengal (PV-10) at ASCO 2016

[Colored emphasis below is mine.]

A phase 2 study of intralesional PV-10 followed by radiotherapy for localized in transit or recurrent metastatic melanoma.

Author(s): Matthew C Foote, Bryan H Burmeister, Janine Thomas, Tavis Read, Bernard Mark Smithers; Princess Alexandra Hospital and University of Queensland, Brisbane, Australia; Princess Alexandra Hospital, Brisbane, Australia

Background: Intralesional rose bengal (IL PV-10) can elicit ablation of injected tumors and a T-cell mediated abscopal effect in untreated lesions. Phase 2 testing in patients with Stage III-IV melanoma yielded a 51% objective response rate (ORR) with 50% complete response (CR) when all disease was injected. Three patients who progressed received external beam radiotherapy (XRT) to their recurrent lesions with an impressive response without an increased radiation reaction. Methods: An open-label, single-arm phase 2 study was performed to assess efficacy and safety of IL PV-10 followed by XRT. Eligibility included recurrent localized dermal, subcutaneous, in-transit or metastatic malignant melanoma (stage IIIb / IIIc) suitable for intralesional therapy and XRT. Patients received a single course of PV-10 into lesions treatable within a localized radiotherapy field. If CR was not achieved patients received 30 Gy (6 fractions of 5 Gy twice weekly over 3 weeks) 3D conformal radiotherapy (photons or electrons) commencing 6-10 weeks after PV-10. Outcome assessments included ORR and clinical benefit (CR+PR+SD) of in-field target lesions by RECIST criteria, toxicity using CTCAE V3.0, and progression free survival (PFS). Results: There were 15 patients enrolled with 13 completing the radiotherapy component. Two patients had rapidly progressive distant disease following PV-10 injection. The mean age of patients was 69 years. With a median follow up duration of 19.3 months the overall response rate was 87% (CR 33%, PR 53%) with 93% clinical benefit on an intent-to-treat basis. The mean time to best response was 3.8 months, mean duration of complete response (PFS) 12.2 months, overall loco regional progression rate 80% and melanoma specific survival 65.5 months. Size of metastases ( < 10mm) predicted potential for lesion complete response. Treatments were well tolerated with no treatment associated grade 4 or 5 adverse events. Conclusions: The combination of IL PV-10 and radiotherapy resulted in lesion specific, normal tissue sparing, ablation of melanoma tumors with minimal local or systemic adverse effects. The study results justify expanded evaluation in a randomized trial.

Intralesional rose bengal for treatment of melanoma.

Author(s): Sanjiv S. Agarwala, Robert Hans Ingemar Andtbacka, Kristen N. Rice, Merrick I. Ross, Charles Raben Scoggins, Bernard Mark Smithers, Eric D. Whitman, Eric Andrew Wachter; St. Luke's Hospital and Health Network and Temple University, Bethlehem, PA; Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; Medcl Onc Assoc of San Diego, San Diego, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; University of Louisville, Louisville, KY; Princess Alexandra Hospital and University of Queensland, Brisbane, Australia; Atlantic Melanoma Ctr, Morristown, NJ; Provectus Biopharmaceuticals, Inc, Knoxville, TN

Background: Intralesional rose bengal (PV-10) is an investigational small molecule ablative immunotherapy that can elicit primary ablation of injected tumors and secondary T-cell activation. Phase 2 testing in Stage III-IV melanoma yielded a 51% objective response rate (ORR) with 50% complete response (CR) when all disease was injected. PV-10 is currently undergoing phase 3 testing as a single agent in patients with locally advanced cutaneous melanoma and phase 1b testing in combination with immune checkpoint inhibition for more advanced disease. Methods: Study PV-10-MM-31 (NCT02288897) is an international multicenter, open-label, randomized controlled trial of PV-10 versus investigator’s choice of chemotherapy (dacarbazine or temozolomide) or oncolytic viral therapy (talimogene laherparepvec). A total of 225 subjects with locally advanced cutaneous melanoma (Stage IIIB or Stage IIIC recurrent, satellite or in-transit melanoma) randomized 2:1 will be assessed for progression free survival (PFS) by RECIST 1.1 (using blinded Independent Review Committee assessment of study photography and radiology data). Comprehensive disease assessments, including review of photography and radiology data, are performed at 12 week intervals; clinical assessments of progression status are performed at 28-day intervals. Study PV-10-MM-1201 (NCT02557321) is an international multicenter, open-label, sequential phase study of PV-10 in combination with pembrolizumab. Stage IV metastatic melanoma patients with at least one injectable cutaneous or subcutaneous lesion who are candidates for pembrolizumab are eligible. In the current phase 1b portion of the study, up to 24 subjects will receive the combination of PV-10 and pembrolizumab (PV-10 + standard of care). In phase 2 an estimated 120 participants will be randomized 1:1 to receive either PV-10 and pembrolizumab or pembrolizumab alone. The primary endpoint for phase 1b is safety and tolerability with PFS a key secondary endpoint; PFS is the primary endpoint for phase 2. Clinical trial information: NCT02288897

Moffitt: IL RB in melanoma elicits tumor immunity via activation of DCs by the release of HMGB1

Updated below, again.

Article link: Intralesional rose bengal in melanoma elicits tumor immunity via activation of dendritic cells by the release of high mobility group box 1

H. Lee Moffitt Cancer Center and Research Institute departments & facilities: Immunology, Flow Cytometry, Translational Science, Cutaneous Oncology, Pathology, and Cutaneous Data Management

Click to enlarge.

Takeaways:

These data/results:

• "...support the role of IL [intralesional] RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response,"

• "...suggest that IL PV-10 can induce tumor-specific T cells with memory characteristics in M05 melanoma-bearing mice,"

• "...show that CD8+ T cells are crucial for the tumor-specific immune response induced by IL injection of PV-10,"

• "...support that IL injection of PV-10 can boost T cell infiltration in tumors,"

• "...support a role for IL PV-10 to induce DCs [dendritic cells] to take up antigens at the tumor site, infiltrate into the DLN [draining lymph node], and become functionally mature,"

• "...suggest that PV-10-treated tumors may release factors that activate DCs,"

• "...suggest that PV-10 can kill tumor cells at a dose that is not toxic to non-tumor cells,"

• "...support the role of IL PV-10 treatment to induce a systemic anti-tumor immune response in patients with metastatic melanoma," and

• "...support the design of additional clinical studies to measure anti-tumor immune responses after IL injection of PV-10 in patients with melanoma."

The Cancer Immunity Cycle & PV-10

Click to enlarge.

ABSTRACT

Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose Bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. IL injection of RB has been shown to induce regression of injected and uninjected tumors in murine models and clinical trials. In this study, we have shown a mechanism of tumor-specific immune response induced by IL RB. In melanoma-bearing mice, IL RB induced regression of injected tumor and inhibited the growth of bystander lesions mediated by CD8+ T cells. IL RB resulted in necrosis of tumor cells and the release of High Mobility Group Box 1 (HMGB1), with increased dendritic cell (DC) infiltration into draining lymph nodes and the activation of tumor-specific T cells. Treatment of DC with tumor supernatants increased the ability of DCs to stimulate T cell proliferation, and blockade of HMGB1 in the supernatants suppressed DC activity. Additionally, increased HMGB1 levels were measured in the sera of melanoma patients treated with IL RB. These results support the role of IL RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response.

RESULTS, summary of article subtitles

• IL PV-10 elicits a tumor-specific immune response
• IL PV-10 leads to DC activation
• PV-10 treatment increases DC activation via HMGB1
• IL PV-10 leads to HMGB1 increase in the sera of melanoma patients

DISCUSSION

Melanoma incidence rates have increased rapidly in the United States over the past 30 years and is the fifth most common cancer in men and the seventh most common cancer in women [38]. IL therapy is a promising treatment modality for patients with dermal and/or subcutaneous metastatic melanoma. Importantly, it may induce not only local tumor regression but also a systemic anti-tumor immune response. In a recent clinical trial in metastatic melanoma patients, IL PV-10 led to a
50% objective response rate with mild to moderate side effects [17]. In treated patients, 8% had no evidence of disease after 52 weeks and 26% experienced complete regression in bystander lesions. However, the mechanism by which IL PV-10 leads to systemic anti-tumor immunity is unknown.

In this study, we showed that IL PV-10 led to the necrosis of melanoma cells and the release of HMGB1. These data are consistent with the observation that HMGB1 was passively released from photosensitized HeLa cells treated with a Rose Bengal analog [39]. Pretreatment with Rose Bengal acetate led to apoptosis and autophagy and the secretion of HSP70, HSP90 and HMGB1. In contrast, our results showed that PV-10 treatment induced necrosis in melanoma cells and the secretion of HMGB1, but not HSP70, while the amount of HSP90 was unchanged. This discrepancy may be explained by differences in response to RB and its acetate analog, dose of test article, differences in the cell lines used, or mechanisms of ablative and photodynamic therapies. Moreover, HMGB1 levels in the sera of patients were increased after IL PV-10. This is in line with another study that showed increased HMGB1 levels in the serum of cancer patients after chemoradiation; notably, HMGB1
levels were increased in patients with antigen-specific T cell responses and higher expression of HMGB1 in resected tumor samples was correlated with better survival [40].

Maturation of DCs is crucial for priming CD8+ T cells [41]. HMGB1 has been shown to be important for activation of myeloid and plasmacytoid DCs [25, 31, 42–46]. In our model, DC maturation with up-regulation of CD40, CD80 and CD86 was measured in tumor draining LN after IL PV-10. Furthermore, our study showed that HMGB1 in the supernatant of tumor cells treated with PV-10 was responsible for the up-regulation of CD40 expression on BM-derived DCs and for the increased ability of DC to stimulate T cell activation. It has been shown that short-term CD40 signaling augments DC migration to tumor-draining LNs and induced protective immunity. Moreover, HMGB1 has been shown to enhance DC responses to CCL9 and CXCL12 [47]. Interactions between HMGB1 and RAGE can induce the migration of s.c. injected DCs into DLNs [48]. In our study, IL PV-10
increased the number of DCs migrating from the tumor site into the draining LNs.

In this study, we have shown a mechanism of tumor-specific immune response induced by IL PV-10.
In melanoma-bearing mice, IL PV-10 induced necrosis of tumor cells leading to the release of HMGB1, which is crucial for DC activation. This resulted in DC maturation and infiltration into draining LNs for the activation of tumor-specific T cells. Additionally, increased HMGB1 levels measured in sera of patients treated with IL PV10 suggests that HMGB1 may be involved in eliciting a systemic immune response in patients. We have shown that circulating T cell populations and tumor-specific CD8+ T cells are increased in melanoma patients after IL PV-10 therapy. Together these results support the design of additional clinical studies to measure anti-tumor immune responses after IL injection of PV-10 in patients with melanoma.

MATERIALS AND METHODS, Incl. Human subjects

Fifteen patients with dermal and/or subcutaneous metastatic melanoma were enrolled in a pilot study
(NCT01760499). Peripheral blood and serum were collected prior to biopsy, 7-14 days after IL PV-10 injection into a single melanoma tumor, and 21-28 days after IL PV-10 injection. PBMCs were isolated by Ficoll–Paque Plus (GE healthcare). Blood samples were sent for HLA typing to determinate HLA-matched tumor and HLA mismatched tumor for each patient. Serum was prepared by collecting the supernatant after incubation of blood at room temperature for 1 hour and centrifugation at 1,000 g. Two tumor lesions in each patient were sampled by biopsy pre-treatment; one of the two lesions was injected with IL PV-10 7 days after biopsy, then both residual sites were completely excised 7-14 days later. Biopsy specimens were fixed in formalin and embed in paraffin. The specimens were stained with hematoxylin and eosin stains for determination of pathologic complete response. Immunohistochemistry for melanin A (mel A) was performed. Flow cytometry was performed to detect CD3, CD4, CD8, and CD56 staining on PBMC.

ACKNOWLEDGMENTS

We thank Dr. Dmitry Gabrilovich for valuable comments during the preparation of this manuscript. This work was supported in part by the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the Moffitt Cancer Center, and in part by the Cancer Center Support Grant P30 CA076292 from the National Cancer Institute. This work was also supported by NCI-5K23CA178083-02 (AAS). PV-10 was provided by Provectus Biopharmaceuticals.

Updated (5/13/16): Provectus issued a press release and made an associated 8-K filing today related to Moffitt's PV-10 mechanism of action paper, "Announces Publication of Article in Oncotarget Detailing PV-10's Immuno-Ablative Mechanism of Action" -- with the company's CTO Dr. Eric Wachter, PhD calling the paper's publication "a a watershed event in the development of PV-10."

I really liked the press release, which is rare praise for an aspect of the company — corporate communications — that has been woeful and woefully lacking dating back to when I began due diligence on Provectus. I found the PR crisp, cogent, insightful and nuanced.

Key takeaway: In my view, the upshot of the release, and more importantly the Oncotarget paper, stemming from Moffitt's initial mouse work first presented at AACR in April 2013 — "Intralesional Injection with PV-10 Induces a Systemic Anti-tumor Immune Response in Murine Models of Breast Cancer and Melanoma" — is that PV-10 is an immunotherapy, or an immuno-ablative as Provectus has labelled its lead, advanced, investigational oncology drug that should focus attention on PV-10's physical chemistry properties (i.e., ablative, and e.g., ablation, chemoablation, etc.) rather than the biological chemistry properties of immune checkpoint inhibitors, oncolytic viruses, and certain other classes of immunotherapies. Keep in mind that folks more recently are wondering about the potential immunotherapeutic properties of chemotherapy and radiotherapy, which are "non-biologics."

Mouse-to-man-to-mouse: I'd venture, in my limited experience as a biotechnology or pharmaceutical industry investor, that Moffitt's work might be the epitome of a translational study, going from mouse to human, and back to mice before returning to human, as the cancer center team confirmed and/or discovered new things in their work. As Eric said in the PR {underlined emphasis below is mine}:

"The Moffitt researchers have systematically documented each of the key steps in the immuno-oncology cycle described by Chen and Mellman in their landmark review article (Oncology Meets Immunology: the Cancer-Immunity Cycle. Immunity 2013; 39: 1-10). In an exemplary demonstration of translational medicine, this team identified important immunologic markers in model systems and verified key facets of these in clinical trial participants, and similarly identified other markers in clinical trial participants and substantiated these in mouse models. While a number of their main observations were previously reported at scientific meetings, these are presented here in detailed, integrated fashion for the first time."

Moffitt team leader Dr. Shari Pilon-Thomas also broached this mouse-to-man-to mouse approach:

"Concordance of tumor-specific T cells in peripheral blood of clinical trial participants and mice led us to look for triggers of T cell activation. Working back from these observations, we found that HMGB1 release was common in mouse and man after tumor ablation with PV-10. These results support PV-10 ablation and the resulting tumor necrosis as the upstream trigger for systemic anti-tumor response." {concordance = agreement}

PV-10 is an immunotherapy: With Moffitt's work, presentation as Eric noted in a "detailed, integrated fashion for the first time," I'm hard pressed to understand anyone saying, in an intellectually honest fashion of course, that PV-10 is anything but an immunotherapy. As Eric further noted:

"This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells."

Additionally, Eric underscored the immunotherapeutic role PV-10 plays as a single agent or monotherapy, and in combination with other therapies and therapeutics {bolded emphasis is mine, too}:

"This mechanism of action informed the design of the two active PV-10 clinical trials: NCT02288897 in patients with locally advanced cutaneous melanoma (melanoma limited to the skin) to test the hypothesis that PV-10 alone can produce a systemic immune response that translates to longer progression free survival (PFS); and NCT02557321 in patients with later stage melanoma to test whether combination of PV-10 with the recently approved systemic immunotherapy, pembrolizumab, can 'induce and boost' an immune response against melanoma."

Updated (5/13/16): I discussed choice of medical journal with Eric. He said New England Journal of Medicine (NEJM), Journal of Clinical Oncology (JCO) and the like focus on relatively large clinical trials. Moffitt's topic and study were not a good match for NEJM, JCO, etc. Oncotarget is a high-impact journal specializing in oncology mechanism and therapeutics (i.e., translational medicine), having a 2014 impact factor of 6.4. For comparison*, for example:

• NEJM's 2014 impact factor was 55.9,
• Lancet, 45.2,
• JCO, 18.4,
• Cancer Research 9.3,
• Clinical Cancer Research, 8.7,
• Oncotarget, 6.4,
• Cancer, 4.9,
• Journal of Immunotherapy, 4.0
• Immunology 3.8, and
• Melanoma Research, 2.2.

The NEJM and the Lancet cover all diseases, while Melanoma Research only covers melanoma. As such, readership naturally is very different, as are resulting citations.

* The information above can be downloaded from this file.

A new article, a new clinical trial

Last week Reuters' Bill Berkrot wrote an article on Provectus, its drug PV-10 and the drug's active pharmaceutical ingredient Rose Bengal entitled Old red dye shows promise as new cancer foe. Provectus also initiated a new clinical trial last week entitled A Phase 1 Study of PV-10 Chemoablation of Neuroendocrine Tumours (NET) Metastatic to the Liver. Some thoughts of mine regarding my takeaways from and questions about them are below.

Article. (1) By far the biggest takeaway for me from Berkrot's article was the association, finally, between PV-10/Rose Bengal and an immune system response.

Click to enlarge. Purple emphasis is mine.
Screenshot of Old red dye shows promise as new cancer foe

The article effectively labeled or categorized PV-10 as an immunotherapy (at least a potential one), like Bristol-Myers' ipilimumab (Yervoy) and nivolumab (Opdivo), Merck & Co.'s pembrolizumab (Keytruda) and Amgen's talimogene laherparepvec (Imylgic), among others.

PV-10's consideration as an immunotherapy, or potential one, is nothing new given Moffitt Cancer Center's long-time work (presented and published since 2012; first pre-clinical, then clinical) and the University of Illinois at Chicago's more recent work (presented and published since 2015; pre-clinical to date). But, PV-10 hasn't been readily recognized as an immunotherapy or a potential one, despite both pre-clinical and clinical, company and third party work that should have at least encouraged such thinking. I believe this is changing, such as PV-10 and Rose Bengal's treatment in Garbe et al.'s 2016 review paper Intralesional immunotherapy as a strategy to treat melanoma. See T cells (February 24, 2016) on the blog's Current News page for more information.

(2) Another major takeaway for me was the presentation of the article's content, which was came across as a down-the-middle-of-the-fairway, opening journalistic piece, delivering facts and perspective with little or no opinion, and providing information about Rose Bengal's long, unique history.

Rose Bengal is an industrial chemical that has been around for well over a century. It's been used as a dye. It's been used as food coloring. It's been used as a diagnostic. It's inexpensive to manufacture. Berkrot and/or his editors could have colored some of that perspective, but I think wisely elected not to. In doing so, the article can be a foundational piece of information for those new to or unaware of Rose Bengal, PV-10 and Provectus' stories.

For example, the author wrote:

"While some doctors are encouraged by the research, government approval is years off and not guaranteed. The company must replicate its early results on a bigger scale, and a U.S. Food and Drug Administration decision is not expected before 2019."

Factually true. Provectus' pivotal melanoma Phase 3 trial, per its ClinicalTrials.gov webpage, has an estimated study completion date of October 2017. Add to this some time for preparation of a new drug application (NDA), a 60-day NDA filing review period and normally a 10-month period for the FDA to review new drugs, and one can easily understand Berkrot's 2019 timeframe. Nowhere in a down-the-middle-of-the-fairway article could there be room for, say, management's guidance of a mid-year interim assessment of efficacy and safety or pursuing accelerated approval on the basis of such data.

In another example, Berkrot wrote:

"In a study of 80 people with advanced melanoma, half of the patients who had all of their lesions injected appeared cancer free after an average of two months. A year later, 11 percent continued to show no signs of cancer, according to a report published the Annals of Surgical Oncology. The lesions were destroyed from the inside with no apparent harm to healthy tissue, researchers said. Reported side effects included injection site pain and blistering."

Again, factually true. PV-10 treatment is safe and potentially effective.

The author does not broach the detail that patients who had all of their lesions injected in the above Phase 2 trial received a second injection of PV-10 into all of their melanoma tumors two months after the first injection. In Provectus' ongoing Phase 3 trial, patients will have all of their lesions injected once a month until their lesions go away.

In a third example Berkrot factually and crisply summarizes the goal of this Phase 3 trial, which is to demonstrate PV-10 can prevent or forestall the progression of Stage III melanoma to Stage IV (underlined emphasis below is mine):

"Final results from an ongoing 225-patient melanoma trial of the experimental drug compared to chemotherapy are expected in early 2018. The hope is that the drug, known as PV-10, will prevent melanoma from progressing beyond Stage III, in which the disease has spread but not yet to other organs, and allow patients with more advanced cancer to live longer."

(3) Another minor takeaway would be Berkrot's take on the discovery of Rose Bengal's therapeutic benefit: an accident, but what was the real accident?

That Japanese researchers investigating Rose Bengal's food dye version in the 1980s first observed its therapeutic benefit (i.e., dose-dependent survival) but did nothing, or that Big Pharma researchers in their global, decades-long attempts to boil the oceans in search of new drug compounds did not find this work?

(4) A final and minor takeaway is the author not mentioning anything or making a "big deal" about the local agent's administration (i.e., no specific mention of intralesional (IL) delivery by name; rather, simply the observation PV-10 is injected).

Trial. Takeaways for and questions from me about the initiation of this new clinical trial include:

(1) It's a liver trial.

Specifically, it's a cancer metastatic to the liver or secondary liver cancer trial, rather than a hepatocellular carcinoma (HCC) or primary liver cancer trial.

The company currently is running a Phase 1 liver trial, A Study to Assess PV-10 Chemoablation of Cancer of the Liver, which has explored and is exploring HCC and cancer metastatic to the liver. Preliminary results from this work noted the treatment of several different types or kids of liver mets including colorectal, non-small cell lung, melanoma, and ovarian. I also previously noted a NET metastatic to the liver had been treated with PV-10.  See New clinical study (February 26, 2016) on the blog's Current News page for more information.

(2) Where is the previously management-guided progression of the original Phase 1 liver trial?

In July 2015 presentations of Provectus' preliminary liver cancer data, both HCC and metastatic, the company's CTO Dr. Eric Wachter, PhD indicated the next step in this clinical program would be an Asia-Pacific Phase 1b/2 combination study of HCC (i.e., a single arm trial of regional standard of care + PV-10, followed by a randomized control trial of regional standard of care + PV-10).

Why was a NET metastatic liver Phase 1 trial initiated before the Asia-Pacific Phase 1b trial? What is the significance of NET mets?

(3) Dosing and the number of lesions that can be treated have increased.

The original Phase 1 liver trial permitted treatment of a single lesion up to a maximum PV-10 dose of 7.5 mL. As the trial expanded, an expansion cohort (Expansion Cohort 1, or EC1) was established where a single lesion was treated with up to a maximum PV-10 dose of 15 mL.

In addition, the same two-step dosing approach (two cohorts, low and high PV-10 doses: Expansion Cohort 2.1 or EC2.1 and Expansion Cohort 2.1 or EC2.1, respectively) was provided to patients already receiving sorafenib.

The new Phase 1 liver trial will permit treatment of, first, a single lesion up to a maximum PV-10 dose of 15 mL, and second, if safety is established, all amenable lesions to a maximum dose of 15 mL.

(4) The new liver trial will collect biomarker, symptom and quality of life data.

The original trial collected changes in markers of hepatic function, pharmacokinetics of PV-10 in the bloodstream following IL injection, and pharmacokinetics of sorafenib in the bloodstream following IL injection.

The full title of the new trial is A Phase 1 Study to Assess the Safety, Tolerability and Effectiveness of PV-10 Chemoablation of Neuroendocrine Tumours (NET) Metastatic to the Liver in the Reduction of Biochemical Markers and Symptoms Caused by Secretory Products. Information to be collected includes:

• Change in NET biomarkers (chromogranin A or CgA, and/or 5-Hydroxyindole Acetic Acid or 5-HIAA),
• Reduction in major symptoms (diarrhea and flushing) using EORTC QLQ-C30 and GI.NET21 symptom scores vs. baseline values,
• Reduction in other symptoms (including bronchoconstriction and abdominal cramping) using the same approach immediately above, and 
• Change in peripheral blood mononuclear cells (PBMC), which was measured by Moffitt Cancer Center's Phase 1 feasibility study.

See NET symptoms below:

Click to enlarge. Image source

(5) Initial efficacy data to be collected in the new liver trial will be objective response rates.

On February 26th the FDA approved Novartis' everolimus (Afinitor) for the treatment of adult patients with progressive, well-differentiated non-functional, NET of gastrointestinal (GI) or lung origin with unresectable, locally advanced or metastatic disease.

From Novartis' pivotal trial "...overall response rates were 2% in the everolimus arm and 1% in the placebo arm. At the planned interim analysis, there was no statistically significant difference in overall survival between arms...Everolimus was discontinued for adverse reactions in 29% of patients and dose reduction or delay was required in 70% of everolimus-treated patients. Serious adverse reactions occurred in 42% of everolimus-treated patients and included 3 fatal events (cardiac failure, respiratory failure, and septic shock)." {Underlined emphasis is mine}

Click to enlarge. Image source

One obviously cannot compare the following for many reasons, but I believe it is worthwhile to note that in Provectus' preliminary liver cancer data, a tumor-specific objective response rate of 50% was achieved -- in 4 patients, however.

Click to enlarge. Image source

(6) I imagine Provectus will press release more information about the new liver trial this coming week.