Biomedical subjects

G R Pettit

Publications and source records attributed to G R Pettit.

At least 127 records · Page 7Linked to original sources

PubMed1995

Antineoplastic agents 337. Synthesis of dolastatin 10 structural modifications.

New structural modifications of the marine shell-less mollusk peptide constituent dolastatin 10 (1) have been synthesized, and evaluated against a variety of cancer cell lines and for their ability to inhibit tubulin polymerization. A number of useful structure-activity relationships were uncovered. The most important observation was that the dolaphenine unit of dolastatin 10 could be satisfactorily replaced with a phenethylamine. Peptide 11C, designated auristatin PE, was found to exhibit inhibition of cancer cell growth and tubulin assembly comparable to that of dolastatin 10.

Amino Acid Sequence↗

PubMed1995

Interaction of dolastatin 10 with tubulin: induction of aggregation and binding and dissociation reactions.

We have prepared [3H]dolastatin 10 and examined its interactions with tubulin. Binding kinetics appeared to be biphasic, with a rapid initial reaction that could not be accurately measured, followed by a slower second reaction. Bound drug was stable in centrifugal gel filtration, column gel filtration, and high performance liquid chromatography gel filtration, but the bound drug could be displaced by an active isomer of dolastatin 10. Scatchard analysis of binding data was consistent with two classes of binding sites. However, dolastatin 10 induced an aggregation reaction upon binding to tubulin, complicating analysis of the data, and incorporation of [3H]dolastatin 10 into large aggregates was readily demonstrated. The chromatographic properties of the smallest radiolabeled species that could be documented were most consistent with a complex consisting of two molecules of alpha/beta-tubulin dimer and two molecules of [3H]dolastatin 10. The coexistence of an aggregation reaction with a binding reaction at a single site probably underlies the biphasic binding kinetics and the biphasic Scatchard plot. Of peptides that strongly inhibit tubulin polymerization (dolastatin 10, dolastatin 10 isomers, segments, and analogs, dolastatin 15, and phomopsin A), only those previously shown to be strong inhibitors of vinblastine binding and nucleotide exchange also strongly inhibited [3H]dolastatin 10 binding and induced tubulin aggregation (dolastatin 10 itself, two chiral isomers of dolastatin 10, and phomopsin A). The morphology of dolastatin 10-induced aggregates was compared with that of vinblastine-induced aggregates under a variety of reaction conditions. With both drugs the aggregates had a more organized appearance when microtubule-associated proteins were included in the reaction.

Animals↗

PubMed1995

Antineoplastic agents 320: synthesis of a practical pancratistatin prodrug.

Owing to its sparingly soluble properties, the potential anticancer drug pancratistatin (1) resisted conventional drug formulation procedures and the synthesis of a water-soluble prodrug became necessary. That important objective for further pre-clinical development was met by devising a route to a disodium phosphate derivative (5). The key step in the synthesis of the phenolic phosphate was phosphorylation of 1,2,3,4-tetraacetoxy-pancratistatin (2) with dibenzyloxy(N,N-diisopropylamido)-phosphine. Subsequent oxidation with m-chloroperbenzoic acid afforded phosphate 4a. Hydrogenolysis of the benzyl esters followed by base-catalysed hydrolysis of the acetate groups led to the water-soluble prodrug 5 in high yield.

Amaryllidaceae Alkaloids↗

PubMed1995

Antineoplastic agents 322. synthesis of combretastatin A-4 prodrugs.

Combretastatin A-4 (1a), the principal cancer cell growth-inhibitory constituent of the Zulu medicinal plant Combretum caffrum, has been undergoing preclinical development. However, the very limited water solubility of this phenol has complicated drug formation. Hence, derivatives of the combretastatin A-4 (1a) 3'-phenol group were prepared for evaluation as possible water-soluble prodrugs. As observed for combretastatin A-4, the sodium salt (1b), potassium salt (1c) and hemisuccinic acid ester (1e) derivatives of phenol 1a were essentially insoluble in water. Indeed, these substances regenerated combretastatin A-4 upon reaction with water. A series of other simple derivatives (1d, 1f-j) proved unsatisfactory in terms of water solubility or stability, or both. The most soluble derivatives evaluated included the ammonium (1l), potassium (1m) and sodium (1n) phosphate salts, where the latter two proved most stable and suitable. Both the potassium (1m) and sodium (1n) phosphate derivatives of combretastatin A-4 were also found to exhibit the requisite biological properties necessary for a useful prodrug. Sodium salt 1n was selected for drug formulation and further pre-clinical development.

Animals↗

PubMed1994

Antineoplastic agents. 278. Isolation and structure of axinastatins 2 and 3 from a western Caroline Island marine sponge.

The Republic of Palau marine sponge Axinella sp. was found to be an exceptionally productive source of cell growth inhibitory substances. The strongly antineoplastic polyether macrocyclic lactones halichondrin B (1) and homohalichondrin B (2) were isolated in 1.2 x 10(-6)% and 5.4 x 10(-7)% yields, respectively. In addition to axinastatin 1 (3), two new and cytostatic (GI50 values of 0.35 to 0.0072 microgram/mL against six human cancer cell lines) cycloheptapeptides designated axinastatins 2 (4) and 3 (5) were discovered in 1.4 x 10(-6)% and 1.25 x 10(-6)% yields. Structures were elucidated by high-resolution FABMS and tandem MS/MS techniques augmented by high-field (400 and 500 MHz) 2D-NMR spectral analyses. The absolute configurations were established by a combination of hydrolysis, derivatization, and chiral gas chromatographic methods.

Amino Acid Sequence↗

PubMed1994

Zinc finger domains and phorbol ester pharmacophore. Analysis of binding to mutated form of protein kinase C zeta and the vav and c-raf proto-oncogene products.

The phorbol ester binding domain consists of a cysteine-rich region with a postulated consensus sequence for binding that includes 15 amino acids (Ahmed, S., Kozma, R., Lee, J., Monfries, C., Harden, N., and Lim, L. (1991) Biochem. J. 280, 233-241). In PKC zeta, the only PKC isoform lacking phorbol ester binding, this region differs in a single residue from the consensus (proline in position 11 of the motif). Restoration of this proline by site-directed mutagenesis of PKC zeta does not restore binding of either [3H]phorbol 12,13-dibutyrate or of the ultrapotent ligand [3H]bryostatin 1, suggesting that even a low affinity ligand interaction is absent. In addition, the vav and c-raf proto-oncogene products, proteins that possess cysteine-rich regions with high homology to PKC isozymes and other phorbol ester receptors, are unable to bind any of these ligands. Instead, all of these cysteine-rich regions bind zinc. Our results suggest that other amino acids besides those postulated for the consensus must be necessary for ligand binding and argue against direct modulation of PKC zeta, Vav, and c-Raf by phorbol esters.

Amino Acid Sequence↗

PubMed1994

Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells.

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗

PubMed1994

The role of protein kinase C isoenzymes in the growth inhibition caused by bryostatin 1 in human A549 lung and MCF-7 breast carcinoma cells.

Bryostatin I is a natural product currently under clinical evaluation as an antitumor agent. Like the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) it activates protein kinase C (PKC). Bryostatin I inhibits the growth of the human-derived A549 lung and MCF-7 adenocarcinoma cell lines, but much more weakly than TPA. The hypotheses were tested that differences between cell lines in their response to bryostatin I are related to cellular PKC isotype content, and that differences between TPA and bryostatin I in their effects on cell growth are associated with differential abilities to modulate specific PKC isoenzymes. PKC isozyme profiles were studied by Western-blot analysis in the cytosol, particulate and nuclear fractions of A549 and MCF-7 cells. PKCs-alpha, -epsilon and -zeta were detected in both cell types with predominant location in the cytosol. Separation of cytosolic PKC isoenzymes in A549 cells by hydroxylapatite column chromatography and determination of PKC activity in fractions yielded a major peak which contained PKC-alpha. Exposure of cells to bryostatin I or TPA for 30 min caused the redistribution of PKCs-alpha and -epsilon from the cytosol to the particulate and nuclear fractions in a concentration-dependent fashion. PKC-epsilon was completely down-regulated by exposure to 10 nM bryostatin I for 18 hr or to TPA for 24 hr. Down-regulation of PKC-alpha was partial at 10 nM and complete at 1 microM of either agent. Bryostatin I inhibited incorporation of [3H]-labelled thymidine into cells only transiently, whereas TPA arrested growth for several days in A549 cells and irreversibly in MCF-7 cells. A549 cells, in which PKC was depleted by exposure to phorbol ester for 9 weeks, were resistant towards bryostatin-induced inhibition of DNA synthesis. The results suggest that the susceptibility of adenocarcinoma cells towards bryostatin-induced growth delay are determined by cellular levels of PKCs-alpha and/or -epsilon. However, differences between bryostatin I and TPA in their abilities to inhibit cell growth do not seem to be intrinsically related to differences in redistribution or down-regulation of specific PKC isoenzymes.

Antineoplastic Agents↗

PubMed1994

The macrocyclic lactone protein kinase C activator, bryostatin 1, either alone, or in conjunction with recombinant murine granulocyte-macrophage colony-stimulating factor, protects Balb/c and C3H/HeN mice from the lethal in vivo effects of ionizing radiation.

We have examined the in vivo radioprotective effects of the macrocyclic lactone protein kinase C (PK-C) activator, bryostatin 1, administered either alone or in conjunction with recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF), in Balb/c and C3H/HeN mice subjected to lethal total body irradiation (TBI). When administered alone on a divided dose schedule (24 hours and 30 minutes before TBI), rmGM-CSF (20 micrograms/kg) was ineffective in increasing survival in either strain. However, in Balb/c mice, bryostatin 1 alone (1 microgram) permitted the long-term survival (60 days) of 70% of the animals following TBI, and 80% when administered in conjunction with rmGM-CSF. Bryostatin 1 administered alone according to this schedule exerted minimal radioprotective effects in C3H/HeN mice, but, when combined with a subeffective dose of rmGM-CSF, allowed 50% of the animals to survive. Treatment of Balb/c mice with bryostatin 1 administered as a single dose 4 hours before TBI resulted in a 20% survival rate, and 45% when administered with rmGM-CSF; corresponding values for the C3H/HeN strain were 60% and 40%, respectively. Lastly, the survival rates of Balb/c mice treated with bryostatin 1 administered as a single dose 4 hours following TBI was 20%, and 25% with rmGM-CSF; corresponding values were 50% and 25% for C3H/HeN mice. These findings indicate that the PK-C activator bryostatin 1 exhibits intrinsic in vivo radioprotective effects in lethally irradiated Balb/c and C3H/HeN mice, and may, under some circumstances, augment the radioprotective capacity of rmGM-CSF. They also underscore the critical role that strain differences and scheduling considerations play in determining the in vivo radioprotective capacity of bryostatin 1, as well as its interactions with rmGM-CSF.

Animals↗

PubMed1994

Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts.

Bryostatin 1 and phorbol 12-myristate 13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in many systems bryostatin 1 induces only a subset of the responses to PMA and blocks those which it does not induce. We report here that in NIH 3T3 fibroblasts PMA showed similar potencies for translocating PKC isozymes alpha, delta, and epsilon to the Triton X-100-soluble and -insoluble fractions and for the down-regulation of the three isozymes. Bryostatin 1 was slightly was more potent than PMA for down-regulating it. Bryostatin 1 was markedly more potent than PMA for translocating PKC delta but showed a biphasic dose-response curve for down-regulating this isozyme. 1-10 nM bryostatin 1 down-regulated PKC delta to a similar extent as PMA; lower (10-100 pM) or, unexpectedly, higher (100 nM to 1 microM) doses of bryostatin 1 caused either no or reduced down-regulation. Moreover, these high (100 nM to 1 microM) doses of bryostatin 1 inhibited the down-regulation of PKC delta by 1 microM PMA when coapplied. Bryostatin 1 caused translocation of PKC epsilon with slightly higher potency than PKC delta, but there was no protection of this isozyme at any of the doses examined. Bryostatin 1 induced a long-term increase in c-Jun level. The dose-response curve for bryostatin 1 was biphasic, with maximal induction at 1-10 nM bryostatin 1, coincident with the maximal down-regulation of PKC delta. We conclude that bryostatin 1 showed substantially different regulation for PKC alpha, PKC delta, and PKC epsilon, whereas PMA distinguished only weakly between these isozymes.

3T3 Cells↗

PubMed1994

Successful treatment of human Waldenström's macroglobulinemia with combination biological and chemotherapy agents.

The immunomodulating effects and antitumor activity of two biological agents, bryostatin 1 (Bryo1) and alpha-interferon, were tested in vitro and in vivo either alone or prior to chemotherapy agents, against a Waldenström's macroglobulinemia tumor line (WSU-WM). Bryol caused a decrease in the expression of CD10, CD19, IgM, Leu10, and CD22 and a temporary growth inhibition as measured by cell cycle analysis. alpha-Interferon did not show any major effects. In vivo, severe combined immunodeficient mice were used to test the activity of the agents against WSU-WM. Bryo1 (i.p.) was given either alone or sequentially with doxorubicin (i.v.), vincristine (i.v.), melphalan (i.v.), and alpha-interferon (i.v.). Bryo1 given 24 h before vincristine or melphalan resulted in the highest tumor growth inhibition, tumor growth delay, and tumor cell kill. Two of five mice receiving Bryo1/vincristine combination were free of tumors > 200 days after treatment and were considered cured. In light of our findings, we recommend that Bryo1 be considered for clinical investigation in human B-cell tumors and might best be given combined with other chemotherapy agents used in the treatment of that disease. Whether Bryo1 is acting as a differentiating agent or as a direct anti-Waldenström's macroglobulinemia tumor agent, remains unclear.

Animals↗

PubMed1994

Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro.

Bryostatin 1 is a protein kinase C activator that inhibits growth of tumour cells and activates lymphocytes in vitro, properties that have encouraged its use in phase 1 clinical studies as an anticancer agent. We investigated interleukin-2(IL-2)-induced proliferation and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC) from cancer patients receiving Bryostatin intravenously. After Bryostatin administration both LAK generation and proliferation were enhanced when patients' PBMC were stimulated with IL-2 in vitro. However, when normal donors' PBMC were cultured in vitro in the presence Bryostatin and IL-2, LAK induction was inhibited while IL-2-driven proliferation was increased. These effects were also seen following only 2 h exposure to Bryostatin and could be elicited by conditioned medium from Bryostatin-pretreated cells. Neither IL-4 nor interferon gamma was detected in the conditioned medium. Bryostatin in vitro was found to increase expression of IL-2 receptors on CD4+, CD8+ and CD56+ cells and augment the proportion of CD8+ cells in conjunction with IL-2. We conclude that Bryostatin in combination with IL-2 in vitro enhances proliferation and IL-2 receptor expression on lymphocytes, favouring CD8+ cells while suppressing the generation of LAK activity. Intravenous administration of Bryostatin increases the potential of IL-2 to induce proliferation and LAK activity in lymphocytes which, taken together with its putative direct antitumour effect, makes Bryostatin an interesting candidate for clinical trials in combination with IL-2.

Adjuvants, Immunologic↗

PubMed1994

Isolation and structure of cephalostatins 10 and 11.

Further investigation of antineoplastic constituents from the marine worm Cephalodiscus gilchristi, employing a 450 kg re-collection from the Indian Ocean (Southeast Africa), has led to isolation and structural determination of two previously undetected members of the cephalostatin series, designated cephalostatins 10 [4] and 11 [5]. Structural analyses were conducted primarily employing high field 2D nmr and high resolution mass spectral techniques. All the stereochemical assignments were deduced using the original X-ray crystal structure of cephalostatin 1 and ROESY 2D nmr methods. Both cephalostatins 10 and 11 strongly inhibited growth of a series of important human cancer cell lines.

Animals↗

PubMed1994

Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1.

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.

Animals↗

PubMed1994

Bryostatin 1 protects protein kinase C-delta from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol ester-induced differentiation.

Bryostatin 1 and phorbol-12-myristate-13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in primary mouse keratinocytes bryostatin 1 does not induce differentiation and blocks PMA-induced differentiation. We report here that in primary mouse keratinocytes PMA caused translocation of PKC-epsilon to the Triton X-100-soluble fraction with an approximately 2-order of magnitude higher potency, compared with translocation of PKC-alpha and PKC-delta. The kinetics of translocation were fastest for PKC-epsilon, slower for PKC-alpha, and slowest for PKC-delta. At 5-20 min bryostatin 1 showed potency similar to that of PMA for translocating PKC-alpha, higher potency for translocating PKC-delta, and lower potency for translocating PKC-epsilon. At a later time (6 hr), bryostatin 1 was 1-2 orders magnitude more potent than PMA for causing loss of PKC-alpha, -delta, and -epsilon from the soluble fraction. Bryostatin 1 was 40-fold more potent than PMA for down-regulating PKC-alpha and showed a biphasic dose-response curve for down-regulating PKC-delta. Bryostatin 1 at 0.1-1 nM down-regulated PKC-delta to a similar extent as did PMA. Bryostatin 1 at 100 nM to 1 microM, on the other hand, failed to induce down-regulation, and these high (100 nM to 1 microM) doses of bryostatin 1 showed noncompetitive inhibition of PKC-delta down-regulation by 1 microM PMA after coapplication. This protected portion of PKC-delta retained kinase activity. The dose-response curve for bryostatin 1 protection of PKC-delta from down-regulation by PMA correlated with bryostatin 1 inhibition of the effects of PMA on cornified envelope formation (a marker of differentiation) and epidermal growth factor binding. Although PKC-epsilon was readily translocated by both PMA and bryostatin 1, the PKC-epsilon originally associated with the particulate fraction showed no down-regulation by either of these agents. We hypothesize that differential regulation of PKC isozymes by PMA and bryostatin 1 may contribute to the different patterns of biological responses that they induce.

Amino Acid Sequence↗

PubMed1994

Binding of [26-3H]bryostatin 1 and analogs to calcium-dependent and calcium-independent protein kinase C isozymes.

In this study we explored the pattern of protein kinase C (PKC) isozyme selectivity of the bryostatins, a unique class of PKC activators that induce only a subset of the typical phorbol ester responses and antagonize those phorbol ester-mediated responses that they themselves fail to induce. The binding properties of individual recombinant PKC isozymes that had been expressed in insect cells, isolated, and reconstituted in Triton X-100/phosphatidylserine mixed micelles were determined. [3H]Bryostatin 1 showed lower affinity for PKC-beta 1 and -gamma, compared with PKC-alpha, -delta, -epsilon, and -eta. This pattern contrasts with that observed for other PKC ligands. These latter assays were conducted with isozymes reconstituted in phosphatidylserine, conditions that unfortunately do not permit quantitation of bryostatin 1 binding under equilibrium conditions. Using delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10, we could distinguish the respective roles of ligand and lipid in the pattern of selectivity. When isozymes were reconstituted in phosphatidylserine vesicles, delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10 showed similar affinities for PKC-alpha and -gamma, similarly to the phorbol esters. However, in the mixed micellar system, PKC-gamma showed a significantly lower binding affinity, as had been observed for bryostatin 1. These results suggest that the unique pattern of biological responses to the bryostatins does not represent a unique pattern of isotype recognition. Furthermore, the lipid environment of PKC plays an important role in determining the binding selectivity for individual isozymes.

Animals↗

PubMed1993

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.

BACKGROUND: Many oncogenes have been shown to code for growth factor receptors that are involved in regulation of cell growth and proliferation and can activate transcription via protein kinase C. Bryostatin 1, a partial agonist of protein kinase C, has demonstrated potent antitumor activity in vitro and in vivo in human tumor xenografts. PURPOSE: The aim of this phase I study was to determine the optimal dosage and toxicity profile of bryostatin 1 and its influence on cytokine release in vivo. METHODS: Three successive cohorts consisting of 35 patients with various malignant tumors were treated with bryostatin 1 by intravenous infusion over 1 hour as follows: cohort A--35 micrograms/m2 (three patients) or 50 micrograms/m2 (eight patients) once every 2 weeks; cohort B--25 micrograms/m2 once a week (eight patients); and cohort C--25 micrograms/m2 once a week for 3 weeks, with no treatment during the 4th week (16 patients). Plasma levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) were measured by immunoradiometric assay and by radioimmunoassay, respectively. RESULTS: The dose-limiting toxicity was grade 3 or 4 myalgia in four of 11 patients in cohort A, in two of eight in cohort B, and in none of 16 in cohort C. Occurrence of myalgia was dose related. There was no significant myelosuppression, apart from a small and transient fall in platelet count. Six patients experienced acute but transient skin flushing, dyspnea, hypotension, and bradycardia, probably related to the bryostatin 1 vehicle. TNF-alpha and IL-6 were detected in plasma at 2 and 24 hours after treatment, respectively, and the levels were dose related (P = .02). Two patients with metastatic malignant melanoma had partial remission after three or four cycles of therapy; remission lasted 6 weeks and 10+ months, respectively. CONCLUSIONS: The dose-limiting toxicity of bryostatin 1 was myalgia. Plasma IL-6 and TNF-alpha concentrations were increased within 24 hours of therapy. Antitumor activity against malignant melanoma was observed early in the course of treatment. IMPLICATIONS: The recommended dosage of bryostatin 1 for phase II studies is 25 micrograms/m2 by intravenous infusion for 1 hour once a week for 3 weeks, with no treatment in the 4th week. IL-6 and TNF-alpha plasma concentrations may be useful in monitoring biological activity of bryostatin 1. Future studies should explore use of this drug with other conventional immune modulators and conventional cytotoxic drugs.

Adult↗

PubMed1993

In vivo administration of the anticancer agent bryostatin 1 activates platelets and neutrophils and modulates protein kinase C activity.

Bryostatin 1 is a naturally occurring macrocyclic lactone which when applied to cells in culture activates protein kinase C (PKC). In vivo bryostatin 1 functions as an anticancer agent with activity against murine lymphomas, leukemias, and melanoma. Because all organs and tissues contain PKC, normal cells would also be a likely target for this agent. Here we demonstrate that in vivo administration of bryostatin 1 activates platelets over a dose range of 0.4 to 40 micrograms/kg with half-maximal activation occurring at 3 micrograms/kg and stimulation of neutrophils over a similar dose range. This in vivo activation of neutrophils is associated with a rapid decrease in measurable cytosolic PKC, a finding consistent with translocation of the enzyme to the membrane. In contrast, no statistically significant change in PKC location was found in liver, spleen, brain, or L10A B-cell lymphoma. However, in culture the L10A lymphoma did respond to bryostatin 1 with translocation of PKC. To evaluate whether the lack of effect of bryostatin 1 on PKC in organs was secondary to rapid degradation, we developed a bioassay to measure the levels of bryostatin 1 in the blood. To measure the presence of bryostatin 1, human neutrophils were incubated with plasma from mice given injections of different concentrations of bryostatin 1. Using this assay, bryostatin 1 at levels as low as 60 nM could be measured in the plasma. A time course with this bioassay demonstrated that less than 10% of the bryostatin 1 injected was detectable after 2.5 min. These results demonstrate that bryostatin 1 is capable of activating platelets and neutrophils and modulating PKC in vivo. The lack of effect of bryostatin 1 on specific organs may be secondary to the rapid clearance/degradation of this compound from the blood.

Animals↗