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European Biopharmaceutical Review

Peptide Potential

p28 is a synthetic peptide originally derived from secreted protein azurin that is showing promise in Phase 1 clinical trials as an anti-cancer agent. This could represent the first of a new generation of cell-penetrating peptides with broad therapeutic and diagnostic imaging potential.

Azurin, a 128-amino acid member of the cupredoxin family of copper-containing redox proteins, demonstrates preferential entry into malignant cells versus their normal counterparts (1). Azurin also exhibits significant anti-cancer activity in xenograft models of human cancer, prompting an effort to identify and isolate the region within azurin responsible for preferential entry and anti-cancer activity (2,3). One 28 amino acid fragment of azurin, assigned the name ‘p28’, is essentially responsible for the preferential entry of azurin into cancer cells and the anti-cancer activity. p28 binds to the tumour suppressor protein, p53, and stabilises it, subsequently inhibiting cancer cell proliferation and inducing apoptosis. Rapid and efficient solid state cGMP synthesis of p28 formed the basis for a recently completed accelerated dose, Phase 1 clinical trial in late-stage patients with solid tumours that had failed multiple, post-surgical systemic chemoand immunotherapies.

Study Results

Results from the completed Phase 1 trial of first-in-class, first-in-human, peptide inhibitor of p53 metabolism in patients with metastatic refractory solid tumours were presented at the 2011 American Society of Clinical Oncology (ASCO) Annual Meeting last June in Chicago (4). Although preclinical animal testing on rodents and non-human primates had suggested minimal toxicity to recipients, none of the 15 patients – seven of which had melanoma, four colon, one pancreatic, one prostate and two sarcoma – with Eastern Cooperative Oncology Group (ECOG) performance status of zero to two and exposed to p28 (three times per week for four weeks as an intravenous bolus) exhibited an immune (IgG) response or toxicity grade more than one. Consequently, the no observed adverse effect level (NOAEL) and maximum tolerated dose (MTD) were above the highest dose studied. Strikingly, objective responses were observed with stable disease observed in 47 per cent of this group, partial regression in 20 per cent and complete regression in 13 per cent. Objective responses were observed in the target lesions of 10 of 15 patients and the group’s survival paralleled the objective responses witnessed in target tumours.

As of the time of writing this article, the median overall survival is 29 weeks, with a range of 42 to 87 weeks and median of 69 weeks. Twenty per cent of patients are living with disease (survival ranging from 16 to 87 weeks since their first trial infusions), with one patient living with no evidence of disease (NED) greater than 80 weeks. The remaining 80 per cent have succumbed to disease, with the survival periods ranging from eight to 72 weeks.

While these observations are encouraging in the context of a safety trial of a potential single agent chemotherapeutic, and remarkable for a peptide administered intravenously, the most noteworthy observations came in the form of apparent patient benefit. When administered as a single agent to late-stage cancer patients whose tumours had failed other therapies, p28 appeared to have a direct, quantifiable and sustained effect upon tumours based on response criteria in solid tumours (RECIST) 1.1. In addition, patients in the trial exhibited survival well beyond what might be expected in individuals with such advanced disease. In one noteworthy case, a patient who entered the Phase 1 clinical trial with stage 4 melanoma remains alive, with no evidence of disease more than 80 weeks after entering the trial. The apparent patient benefit exhibited by this and other trial patients, when accompanied by a lack of toxicity and immunogenicity, suggests that p28 represents a promising new therapeutic compound (4).

Effects of p28

p28 distributes rapidly (t˝, 0.1hr), and has a prolonged terminal half-life (t˝ γ more than 1.7hr above 2.5mg/ kg), where plasma clearance and total distribution (Vdss) is maximal over the five dose range (0.83hr to 4.16mg/ kg) of the trial. When coupled with the prolonged retention of intact p28 in the nucleus of cancer cells, this suggests that prolonged exposure rather than higher doses of p28 are associated with a maximal response. Interestingly, the expression of p53, measured as equal or more than 10 per cent of cancer cells exhibiting immunohistochemical staining for p53, was unrelated to patient response or survival. This suggests that p28, and similar agents, exert their anti-proliferative, anti-cancer effects in cells presenting with either ‘wild type’ or mutated p53.

Recent preclinical studies have demonstrated that p53 forms a common thread underlying the cytostatic and cytotoxic activity of p28 in cancer cells. p28 exerts its anti-proliferative activity by reducing the proteasomal degradation of p53 through a formation of p28:p53 complex within the hydrophobic DNA-binding domain (aa 90-160) of p53, where it inhibits the binding of the E3 ligase COP-1 increasing p53 levels and DNA-binding activity (5). By binding to a virtually nonmutable region or cold spot within p53, p28 significantly increases the level and activity of p53 in multiple solid tumour cell lines expressing widely different amounts of wild type and mutated p53 without altering the level or function of p53 in normal cells. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 in cancer cells reduces CDK2 and cyclin A levels inhibiting the cell cycle at G2/M in a time-dependent manner leading to apoptosis. As such, p28 represents a first-in-class inhibitor of p53 degradation whose mechanism of action is unrelated to binding to and inhibiting the action of HDM2 – a major protein in the p53 degradation pathway and the current focus of numerous pharmacologic investigations and clinical trials.

The unique pharmacology of p28 is not confined to its activity as a single agent. Preclinical studies indicate that p28 enhances the activity of DNA-damaging agents such as doxorubicin (adriamycin) and mitotic inhibitors such as paclitaxel in several cancer cell lines and xenografts. In addition, p28 has recently been shown to have potent antiangiogenic activity, penetrating human umbilical vein endothelial cells (HUVEC) co-localised with caveolin-1 and VEGFR-2. p28 binds with high affinity to VEGFR-2 and FGFR-1, inducing a non-competitive inhibition of VEGFR2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAKmediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells that is completely unrelated to its ability to increase intracellular levels of p53 in cancer cells, may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumour cell proliferation.

Other Findings

In addition to demonstrating the possible utility of p28 as an anti-cancer compound when administered as a single agent, it appears that p28’s preferential entry into malignancies could provide a targeted means of delivering adjunct chemotherapeutic agents in a more localised fashion by means of linking those agents to p28 prior to administration (6). Furthermore, the first 18 amino acids of p28 is the region responsible for p28 and azurin’s preferential entry into malignancies versus normal cells. It is azurin’s protein transport domain (PTD), but does not exert any therapeutic activity. This shorter (and similarly easy to synthesise) peptide (p18), may have utility as a means of enhancing the specificity of diagnostic imaging during surgery and to identify metastatic disease. By linking imaging/ contrast agents (metallic, functional and so on) to p18, it may be possible to detect aggregations of ‘labelled’ p18 within metastases. Preclinical studies in animal models suggest this is feasible, and could significantly enhance the utility of diagnostic imaging related to metastatic cancers (7).

In addition to p28’s potential role as a first-in-class anti-cancer agent, the novel peptide has exhibited properties which might make it useful as a therapy directed against disease indications other than cancer in humans. For example, the canine oncology market represents a massive yet poorly served market. Preclinical studies are underway to investigate whether p28 might be useful in treating certain canine cancers that metastasise, including melanoma and osteosarcoma.

Conclusion

The origin of p28 as a peptide within a redox protein suggests that the numerous members of the cupredoxin famly of redox proteins could yield similar easy-to-synthesise peptides possessing cell-penetrating characteristics and therapeutic activity against an array of cancers and other diseases. As p28 advances toward initiation of a Phase 2 clinical trial and onward, preclinical studies of other redox protein family members have produced several peptides representing candidate compounds worthy of further investigation and advancement toward the clinic.

References

  1. Taylor BN et al, Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells, Cancer Research 69: pp537-546, 2009
  2. Yamada T et al, Peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells, Molecular Cancer Therapeutics 8: pp2,947-2,958, 2009
  3. Jia L et al, Cancer Chemother Pharmacol, 2011 [Epub ahead of print]
  4. Richards JM et al, A first-in-class, first in human, Phase 1 trial of p28, a non-HDM2 mediated peptide inhibitor of p53 ubiquitination in patients with metastatic refractory solid tumours, Oral abstract presentation to 2011 American Society of Clinical Oncology Annual Meeting, 6 June 2011
  5. Mehta RR et al, Angiogenesis, 11 June 2011 [Epub ahead of print]
  6. Mehta RR et al, A 28-amino-acid peptide fragment of the cupredoxin azurin prevents carcinogeninduced mouse mammary lesions, Cancer Prevention Research 3: pp1,351-1,360, 2010
  7. NIH, NCI Small Business Innovation research (SBIR) 1 R43 CA159771-01, Multifunctional Peptides for (MRI) Imaging

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Tapas K Das Gupta is an internationally recognised surgical oncologist and Department Head of Surgical Oncology at the University of Illinois, Chicago. As a Founder and Chairman Emeritus of CDG Therapeutics, Inc, he continues to serve as the guiding light in clinical and basic science R&D. Tapas received his MD degree from the University of Calcutta (India), and his PhD and DSc from London University, UK. He is board certified in surgery and has authored more than 440 publications, served on FDA advisory panels, and has taken part in numerous clinical trials. Email: tkdg@cdgti.com

David R Volk is President and Chief Executive Officer of CDG Therapeutics, Inc. Previously, David was a Founder and Principal of White Oak Advisors, a consulting firm providing financial and strategic advisory services to start-up and emerging growth life sciences companies. Prior to White Oak Advisors, he was Managing Director and Head of Life Sciences Investment Banking for RBC Capital Markets. David received his BA in Economics from the University of California at Berkeley, and his MBA from the Kellogg School at Northwestern University. Email: drvolk@cdgti.com

Craig W Beattie is the Chief Science Officer of CDG Therapeutics, Inc. Craig served five years as a Professor in the Department of Animal Biotechnology at the University of Nevada, Reno, as well as working as Chair of the Department and Director of the Nevada Genome Center. Prior to the University of Nevada, he served as a Professor in the Department of Veterinary PathoBiology at the University of Minnesota. Craig received his BS and MS in Biology from Fairleigh Dickinson University, and his PhD in Biological Sciences from the University of Delaware. Email: cbeattie@uic.edu

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Tapas K Das Gupta
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David R Volk
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Craig W Beattie
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