Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Rayes, B. F.
Right arrow Articles by Philip, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Rayes, B. F.
Right arrow Articles by Philip, P. A.
Clinical Cancer Research Vol. 12, 7059-7062, December 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

Phase I Study of Bryostatin 1 and Gemcitabine

Basil F. El-Rayes, Shirish Gadgeel, Anthony F. Shields, Stephanie Manza, Patricia Lorusso and Philip A. Philip

Authors' Affiliation: Karmanos Cancer Institute, Wayne State University, Detroit, Michigan

Requests for reprints: Basil F. El-Rayes, Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University, 4100 John R Street, Detroit, MI 48201. Phone: 313-576-8723; Fax: 313-576-8729; E-mail: elrayesb{at}karmanos.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Bryostatin 1 is a macrocyclic lactone with protein kinase C inhibitory activity. Gemcitabine is a nucleotide analogue with a broad spectrum of anticancer activity. Bryostatin 1 enhanced the activity of antitumor agents including gemcitabine in preclinical models. The primary objective of this phase I study was to determine the recommended doses for phase II trials of bryostatin 1 and gemcitabine.

Experimental Design: Eligible patients had histologic or cytologic diagnosis of nonhematologic cancer refractory to conventional treatment; life expectancy of >3 months; normal renal, hepatic, and bone marrow function; and a Southwest Oncology Group performance status of 0 to 2. Gemcitabine was administered i.v. over 30 minutes and was followed by bryostatin 1 by i.v. infusion over 24 hours on days 1, 8, and 15 of a 28-day cycle. Bryostatin 1 (µg/m2) and gemcitabine (mg/m2) doses were escalated as follows: 25/600, 25/800, 25/1,000, 30/1,000, 35/1,000, and 45/1,000, respectively.

Results: Thirty-six patients (mean age, 57 years; male/female 15:21) were treated. The median number of treatment cycles per patient was 3 (range, 0-24). Four patients developed dose limiting toxicities: myalgia, 2; myelosuppression, 1; and elevation of serum alanine aminotransferase levels, 1. Ten grade 3 toxicities were observed (anemia, 2; neutropenia, 5; thrombocytopenia, 3). No treatment-related death was seen. The recommended doses for phase II trials for bryostatin 1 and gemcitabine were 35 µg/m2 and 1,000 mg/m2, respectively. Two heavily pretreated patients with breast and colon cancer experienced partial responses lasting 22 and 8 months, respectively. Eight patients had stable disease.

Conclusion: The combination of bryostatin 1 and gemcitabine seemed to be well tolerated with limited grade 3 toxicity. The recommended dose of bryostatin 1 in combination with full doses of gemcitabine was 35 µg/m2.

Protein kinase C (PKC) is a family of serine/threonine kinases composed of 12 isoforms subdivided into three major groups based on their cofactor requirement for activation (1). The PKC family has a central role in signal transduction and is linked to cell growth, differentiation, apoptosis, and angiogenesis (2, 3). Aberrant regulation of the PKC enzymes activity was shown in a number of malignancies including breast (4, 5), pancreatic (6), and non–small-cell lung cancers. Modulation of PKC activity in vitro promotes apoptosis and sensitizes cancer cells to the effects of cytotoxic agents (7). Consequently, PKC represents a rational target for drug development.

Bryostatin 1 is a macrocyclic lactone that has been shown to regulate PKC activity (8). In preclinical models, bryostatin 1 inhibited cell growth and angiogenesis, promoted apoptosis, and induced differentiation of cancer cells (9). In phase I trials, bryostatin 1 had minimal toxicity that included myalgia (10, 11). Myelosuppression was rarely observed with bryostatin 1. Single-agent activity of bryostatin 1 has been investigated in colorectal (12), renal (13), melanoma (14), and head and neck (15) cancers. Results indicated minimal or no appreciable clinical activity. The combinations of bryostatin 1 and cytotoxic agents were tested because PKC activation contributed to chemoresistance. Bryostatin 1 was shown to potentiate the proapoptotic effects of gemcitabine in human breast (16) and pancreatic cancer cell lines (6).

Gemcitabine (2,2'-difluorodeoxcytidine) is a potent and specific deoxycytidine analogue. In phase I trials, the maximal tolerated dose of gemcitabine administered weekly as an i.v. infusion over 30 minutes was between 790 and 1,370 mg/m2 (17, 18). The dose limiting toxicity was myelosuppression. Gemcitabine showed a broad-spectrum of antitumor activity in patients with pancreas (19), breast (20), ovary (21), and lung (22) cancers.

Based on the preclinical data of potentiation of gemcitabine proapoptotic effects by bryostatin 1 and the nonoverlapping dose-limiting toxicities of the two agents, we conducted a phase I trial to define the recommended dose for phase II trials of the bryostatin 1 and gemcitabine combination.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient eligibility. Patients were eligible for the study if they had a confirmed pathologic diagnosis of a nonhematologic malignancy for which no standard curative or palliative therapy was available. Patients were also required to have a Southwest Oncology Group performance status of ≤2, a life expectancy of at least 3 months, and adequate hematologic, renal, and hepatic function defined by the following variables: hemoglobin ≥8 g/dL, neutrophil count ≥1,500/µL, platelet count ≥100,000/µL, serum creatinine ≤1.5 mg/dL, total serum bilirubin within the institution's 1.5 times upper normal limit and serum aminotransferases <2.5 times upper normal limit. Patients were also required to have bidimensionally measurable disease. Irradiated tumors with no evidence of progression after radiation therapy were not considered measurable. Prior chemotherapy, surgery, or radiation therapy was permitted as long as the patient had recovered adequately from these procedures. Female patients of childbearing potential must have had a negative serum pregnancy test before enrollment, and all fertile patients must have agreed to use contraception during the study. The study was approved by the Wayne State University Human Investigations Committee and all subjects provided a signed informed consent in accordance with the Wayne State University Human Investigation Committee guidelines before participation on the study.

Patients were excluded from study participation if they had uncontrolled intercurrent illness such as active infection, symptomatic congestive heart failure, or unstable angina.

Drug administration. Cycles were repeated every 28 days. Gemcitabine (Gemzar, Eli-Lily Pharmaceuticals, Indianapolis, IN) was infused i.v. over 30 minutes on days 1, 8, and 15. Bryostatin 1 (provided by Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD) was administered by a 24-hour i.v. infusion on days 1, 8, and 15 via an indwelling central venous catheter. Bryostatin 1 infusion was started after the completion of gemcitabine administration.

Dose escalation. The starting doses for gemcitabine and bryostatin were 600 mg/m2 and 25 µg/m2, respectively. Table 1 summarizes the dose escalation schedule. No dose escalations were allowed within an individual patient. A minimum of three toxicity-evaluable patients was entered onto each dose level. Patients who received less than one cycle of therapy without toxicity were considered inevaluable for toxicity evaluation. If one dose limiting toxicity occurred, three additional patients were entered on that dose level. Dose escalation stopped when two or more dose limiting toxicities were observed. The recommended dose of bryostatin and gemcitabine for phase II trials was defined as the dose level at which one or less of six patients developed a dose limiting toxicity. At least 10 patients would be treated on the recommended dose for phase II trials to confirm its safety.


View this table:
[in this window]
[in a new window]

  		Table 1. Dose escalation schema of gemcitabine and bryostatin 1

 
Dose limiting toxicity was defined as the occurrence of (a) grade 3 or 4 nonhematologic toxicity excluding alopecia, nausea, and vomiting; (b) grade 4 thrombocytopenia; or (3) grade 4 neutropenia complicated by either fever or treatment delay of >1 week during any cycle of therapy.

On-study evaluation. Baseline evaluations included a complete history and physical examination, complete blood and differential counts, serum electrolytes, total serum bilirubin, serum alanine aminotransferase, serum aspartate aminotransferase, and urinalysis. Toxicity was graded by the National Cancer Institute Common Toxicity Criteria version 2. Evaluation for toxicity was undertaken after each treatment cycle and whenever clinically indicated. Radiological evaluation for response was done every two cycles and at the time of clinical disease progression. Objective response assessments were based on the WHO criteria.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patient characteristics. A total of 36 patients were enrolled on the study. Two patients were nonevaluable for toxicity: one patient on dose level 1 had treatment interruption due to infection and the second patient on dose level 4 had early disease progression. All patients had received prior cytotoxic therapy. Table 2 summarizes the patient characteristics. A total of 101 cycles of chemotherapy were administered with a median of three cycles per patient (range, 0-24).


View this table:
[in this window]
[in a new window]

  		Table 2. Characteristics of the 36 patients treated with gemcitabine and bryostatin 1

 
Dose escalation and toxicity. Tables 3 and 4 summarize all the observed toxicities. No dose limiting toxicities were observed on dose level 1. Six patients were enrolled on dose level 2 because of one dose limiting toxicity. A patient developed grade 3 neutropenia requiring a 2-week delay in treatment and dose reduction. No dose limiting toxicities were observed on dose levels 3 and 4. One dose limiting toxicity was observed on dose level 5. A patient developed hepatotoxicity consisting of grade 3 and grade 2 elevations of serum aspartate aminotransferase and alanine aminotransferase, respectively. Two of the six patients enrolled on dose level 6 experienced grade 3 myalgia. Subsequently, additional seven patients were enrolled on dose level 5 to verify the maximum tolerated dose of the combination. No additional dose limiting toxicities were observed. Overall, 10 episodes of grade 3 toxicities were observed (anemia, 2; neutropenia, 5; and thrombocytopenia, 3). No treatment-related deaths were seen.


View this table:
[in this window]
[in a new window]

  		Table 3. The occurrence of treatment-related hematologic toxicity in 36 patients treated with gemcitabine and bryostatin 1

 

View this table:
[in this window]
[in a new window]

  		Table 4. The occurrence of treatment-related nonhematologic toxicity in 36 patients treated with gemcitabine and bryostatin

 
Objective response. Two heavily pretreated patients had a partial response. The first patient had breast cancer and experienced a partial response lasting 22 months. She had previously been treated with doxorubicin, 5-fluorourcil, and paclitaxel, followed by high-dose chemotherapy with stem cell support. Subsequent to that, she had received trastuzumab and vinoralbine. The second patient had colon cancer and experienced a partial response lasting 8 months. This patient received two prior treatment regimens, oxaliplatin and capecitabine, and then irinotecan, 5-fluorouracil, and leucovorin with an investigational agent. Eight patients had stable disease. Three patients with non–small-cell lung cancer had disease stabilization lasting longer than 4 months.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
PKC promotes cellular proliferation and inhibits apoptosis through the activation of downstream signaling pathways that include Akt/nuclear factor {kappa}B, mitogen-activated protein kinase, and Ras. PKC also has a central role in angiogenesis (9). The binding of the vascular endothelial growth factor to its receptor leads to the activation of PKC resulting in proliferation and growth of endothelial cells. Inhibition of PKC can potentially sensitize cancer cells to the proapoptotic effects of cytotoxic agents and inhibit angiogenesis.

Bryostatin 1 is a modulator of PKC activation (8). Short-term exposure of tumor cells to bryostatin 1 results in PKC activation and translocation of the enzyme to the nuclear membrane. Prolonged exposure to bryostatin 1 leads to the depletion of PKC and inhibition of downstream signaling (23). Based on the mechanism of action of bryostatin 1, phase I trials of single agent bryostatin 1 were conducted using a prolonged continuous i.v. infusion schedule (11). The most frequent toxicity associated with bryostatin 1 was myalgia. The incidence and severity of myalgia was dose dependent and was cumulative. Therefore, the schedule of administration was changed to weekly infusions administered 3 out of 4 weeks. This decreased the incidence of myalgia and delays in treatment.

Phase II trials showed no significant clinical activity for single agent bryostatin 1 was observed in melanoma (14), colorectal (12), or renal (13) cancers by traditional tumor shrinkage criteria. In preclinical models, bryostatin 1 sensitized breast and pancreatic cancer cells to the effects of gemcitabine (6, 16). The potentiation of gemcitabine by bryostatin 1 was sequence dependent with maximal apoptosis observed when gemcitabine preceded bryostatin 1. A similar sequence-dependent potentiation was observed in gastric cancer cells when bryostatin 1 was used with mitomycin C and paclitaxel (9). The recognition of PKC overexpression and activation in most human cancers and the role of PKC modulation in potentiating conventional cytotoxic agents were the rationale to develop combinations of bryostatin 1 and broad-spectrum anticancer cytotoxic agents, such as gemcitabine.

In this phase I trial, the combination of gemcitabine followed by bryostatin 1 was well tolerated at doses of 1,000 mg/m2 and 35 µg/m2, respectively. This dose of gemcitabine was the recommended full dose in single-agent therapy. The dose limiting toxicities to gemcitabine and bryostatin 1 included myalgia and hepatotoxicity. The hematologic toxicity of the gemcitabine was not altered by the addition of bryostatin 1. Similarly, no potentiation of toxicity of either arabinofuranosylcytosine (24) or vincristine (25) by bryostatin 1 was observed in phase I trials. The lack of toxicity potentiation with bryostatin 1 may be explained by its mechanism of action that selectively targets activated PKC in the tumor cells (6, 16).

Clinical trials of bryostatin 1 have used different i.v. infusion schedules ranging from 1 to 72 hours. The optimal dose rate delivery based on pharmacokinetics of bryostatin 1 is unknown because of lack of reliable assays to quantify its serum levels. The choice of the 24-hour infusion was to produce a prolonged inhibition of PKC within tumor cells. Bryostatin 1 infusion was started after the completion of the gemcitabine delivery to mimic the preclinical findings with respect to the optimum sequence that resulted in maximal tumor cell kill (6, 16). Nevertheless, the optimal interval between gemcitabine and bryostatin 1 in vivo may be influenced by the half-life of gemcitabine. In this respect, gemcitabine has a relatively short plasma half-life ranging from 32 to 94 minutes (26). All patients in this study received prior cytotoxic therapies for their advanced cancers. Two patients with breast and colon cancer experienced partial responses lasting 22 and 8 months, respectively. The occurrences of those partial responses were of interest. Although responses to gemcitabine were expected in breast cancer, the duration of the response was longer than normally seen in this situation. Likewise, gemcitabine was considered an inactive drug in colorectal cancer. Eight additional patients had stable disease, with significant stability in patients with non–small-cell lung cancer, a disease that may respond to gemcitabine. In future studies, it will be necessary to conduct trials of bryostatin 1 or other PKC modulators in patients selected for their tumoral high expression of PKC isoenzymes. However, at this time, no information is available on the predictive value of individual PKC isoenzymes on bryostatin 1 antitumor effects.

In conclusion, the gemcitabine and bryostatin 1 combination was well tolerated and the preliminary results indicate promising activity. Based on the preclinical data and the tolerability of bryostatin 1 and gemcitabine, phase II trials in breast and pancreatic cancer represent a rational approach for further development of this regimen.


   Footnotes
 
Grant support: Cancer Center Core Grant CA-22453 and the UO-1 CA62487 from the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/13/06; revised 8/14/06; accepted 9/13/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Deacon EM, Pongracz J, Griffiths G, Lord JM. Isoenzymes of protein kinase C: differential involvement in apoptosis and pathogenesis. Mol Pathol 1997;50:124–31.[Free Full Text]
  2. Philip PA, Harris AL. Potential for protein kinase C inhibitors in cancer therapy. Cancer Treat Res 1995;78:3–27.[Medline]
  3. Azzi A, Boscoboinik D, Hensey C. The protein kinase C family. Eur J Biochem 1992;208:547–57.[Medline]
  4. Gordge PC, Hulme MJ, Clegg RA, Miller WR. Elevation of protein kinase A and protein kinase C activities in malignant as compared with normal human breast tissue. Eur J Cancer 1996;32A:2120–6.[CrossRef]
  5. O'Brian C, Vogel VG, Singletary SE, Ward NE. Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue. Cancer Res 1989;49:3215–7.[Abstract/Free Full Text]
  6. El-Rayes BF, Ali S, Sarkar F, Philip PA. Protein kinase C: a target for therapy in pancreatic cancer. ASCO proceedings, 2003.
  7. Jarvis WD, Grant S. Protein kinase C targeting in antineoplastic targeting in antineoplastic treatment strategies. Invest New Drugs 1999;17:227–40.[CrossRef][Medline]
  8. Pettit GR, Gao F, Blumberg PM, et al. Antineoplastic agents. 340. Isolation and structural eludication of bryostatins 16-18. J Nat Prod 1996;59:286–9.[CrossRef][Medline]
  9. Kortmansky J, Schwartz GK. Bryostatin 1: a novel PKC inhibitor in clinical development. Cancer Invest 2003;21:924–36.[CrossRef][Medline]
  10. Jayson GC, Crowther D, Prendiville J, et al. A phase I trial of bryostatin 1 in patients with advanced malignancy using a 24 hour intravenous infusion. Br J Cancer 1995;72:461–8.[Medline]
  11. Philip PA, Rea D, Thavsau P, et al. A phase I study of bryostatin 1: assessment of interleukin 6 and tumor necrosis factor {alpha} induction in vivo. The Cancer Research Campaign Phase I Committee. J Natl Cancer Inst 1993;85:1812–8.[Abstract/Free Full Text]
  12. Zonder JA, Shields AF, Zalupski M, et al. A phase II trial of bryostatin I in the treatment of metastatic colorectal cancer. Clin Cancer Res 2001;7:38–42.[Abstract/Free Full Text]
  13. Pagliaro L, Daliani D, Amato R, et al. A phase II trial of bryostatin 1 for patients with metastatic renal call carcinoma. Cancer 2000;89:615–8.[CrossRef][Medline]
  14. Propper DJ, Macaulay C, O'Byrne KJ, et al. A phase II study of bryostatin 1 in metastatic malignant melanoma. Br J Cancer 1998;78:1337–41.[Medline]
  15. Brockstein B, Samuels B, Humerickhouse R, et al. Phase II studies of bryostatin 1 in patients with advanced sarcoma and advanced head and neck cancer. Invest New Drugs 2001;19:249–54.[CrossRef][Medline]
  16. Ali S, Aranha O, Li Y, Pettit GR, Sarkar FH, Philip PA. Sensitization of human breast cancer cells to gemcitabine by the protein kinase C modulator bryostatin 1. Cancer Chemother Pharmacol 2003;52:235–46.[CrossRef][Medline]
  17. Abbruzzese JL, Grunewald R, Weeks EA, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol 1991;9:491–8.[Abstract]
  18. Martin C, Pollera CF. Gemcitabine: safety profile unaffected by starting dose. Int J Clin Pharmacol Res 1996;16:9–18.[Medline]
  19. Burris HA III, Moore MJ, Anderson J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer; a randomized trial. J Clin Oncol 1997;15:2403–13.[Abstract/Free Full Text]
  20. Carmichael J, Possinger K, Phillip P, et al. Advanced breast cancer: a phase II trial with gemcitabine. J Clin Oncol 1995;13:2431–6.[Abstract/Free Full Text]
  21. Toschi L, Finocchiaro G, Bartolini S, Gioia V, Cappusso F. Role of gemcitabine in cancer therapy. Future Oncol 2005;1:7–17.
  22. Anderson H, Hopwood P, Stephens RJ, et al. Gemcitabine plus best supportive care (BSC) versus BSC in inoperable non-small cell lung cancer-a randomized trial with quality of life as the primary outcome. UK NSCLC Gemcitabine. Non-Small Cell Lung Cancer. Br J Cancer 2000;83:447–53.[CrossRef][Medline]
  23. Hennings H, Blumberg PM, Pettit GR, Herald CL, Shores R, Yuspa SH. Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin. Carcinogenesis 1987;8:1343–6.[Abstract/Free Full Text]
  24. Cragg LH, Andreeff M, Feldman E, et al. Phase I trial and correlative larobatory studies of bryostatin 1 (NSC 339555) and high-dose 1-ß-D-arabionfuranosycytosine in patients with refractory acute leukemia. Clin Cancer Res 2002;8:2123–33.[Abstract/Free Full Text]
  25. Dowlati A, Lazarus HM, Hartman P, et al. Phase I and correlative study of combination bryostatin 1 and vincristine in relapsed B-cell malignancies. Clin Cancer Res 2003;9:5929–35.[Abstract/Free Full Text]
  26. Heinemann V, Hertel LW, Grindley GB, Plunkett W. Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-ß-D-arabinofuranosylcytosine. Cancer Res 1988;48:4024–31.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Rayes, B. F.
Right arrow Articles by Philip, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Rayes, B. F.
Right arrow Articles by Philip, P. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online