Protection from arabinofuranosylcytosine and n-mustard-induced myelotoxicity using hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys monomer and dimer

Abstract

We have previously shown that the synthetic peptide pGlu-Glu-Asp-Cys- Lys (pEEDCK monomer) inhibits the cytostatic drug-induced proliferation of hematopoietic stem cells CFU-S. Keeping CFU-S quiescent by pEEDCK treatment renders them insensitive to cycle-specific cytostatic drugs and leads to reduced toxicity. Here we show that pEEDCK application during repeated (twice) administration of clinically relevant (nonlethal) 1-beta-D-arabinofuranosylcytosine (Ara-C) doses reduced the percentage of CFU-S in S-phase from 60%-70% to 25%-30% and led to a sustained stem cell number in the bone marrow (BM), whereas unprotected mice had lost about 75% of their CFU-S population. Owing to its cysteine content, the pEEDCK monomer is easily oxidized. The resulting dimer (pEEDCK)2 is a potent stimulator of hematopoiesis. As we show, it can be used for postchemotherapy acceleration of hematologic recovery, similar to the use of recombinant hematopoietic growth factors. A single injection of 30 micrograms/kg pEEDCK monomer to mice 2 hours before the second Ara-C injection retarded onset of neutropenia (by 2 to 3 days) and improved recovery after depression. The quantitative degree of neutropenia was not changed. Postchemotherapy (Ara-C administered twice, followed by N-mustard) infusion of the stimulatory (pEEDCK)2 dimer (1.4 micrograms/kg/d) produced a 4.6-fold increase of progenitor levels (6.7 CFU-GM/1,000 BM cells v 1.45 CFU-GM/1,000 in normal mice) 2 days after the end of the cytostatic treatment when CFU- GM were not detectable in unprotected mice. This increase was followed after several days by strongly elevated granulocyte counts, which remained high for approximately 1 week. Up to 75% of the peripheral leukocytes were mature polymorphonuclear leukocytes (PMN) during this phase. Ara-C (twice) and monomer treatment as above followed by dimer infusion resulted in the complete protection of hematopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression noted in unprotected animals. The inhibitory monomer appears to keep the stem cell population numerically and qualitatively intact, thus providing optimum target cell conditions for the subsequent stimulator (dimer) treatment. Our results show that the hemoregulatory peptide monomer and dimer can be used for improving the hematologic status of mice treated with clinically relevant doses of cytostatic drugs (antimetabolite and alkylating, alone and in combination). Combining both peptides can prevent occurrence of neutropenia completely. Both peptides can be obtained easily by chemical synthesis and are also active on human cells. They are thus highly promising candidates for application as multilevel hemoprotectors in cancer chemotherapy.

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5‐fluorouracil

“…However, the restricted effect of some of these factors on a specific cell lineage, and the decrease in their therapeutic effect after multiple courses of chemotherapy, suggest the necessity of alternative approaches. In mice, inhibitory factors able to maintain the stem cell and early progenitor compartments in a quiescent state have been shown to exert a protective effect against myelotoxic agents (Carde et al, 1993;Dunlop et al, 1992;Grzegorzewski et al, 1995;Paukovits et al, 1990Paukovits et al, , 1991. In particular, the tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), now termed Goralatide, a known inhibitor of stem cells (Lenfant et al, 1989;Frindel & Monpezat, 1989), has been shown to be able to protect mice against the toxicity of high doses of chemotherapy (Bogden et al, 1991;Guigon et al, 1982).…”

Long term hematopoeitic damage after chemotherapy and cytokine

“…PEEDCK has the capability of inhibiting myelopoiesis and bears a striking similarity to a 5-amino acid sequence in the effector domain of the alphasubunit of inhibitory G-proteins (73). While the dimerized peptide indirectly stimulates hematopoiesis in vitro (74), Paukovits and others have used the monomeric peptide to protect hematopoietic progenitors from damaging chemotherapeutic effect (75). Noting that the peptide appeared to be cytostatic towards CFU-S, perhaps having a physiologic role in the maintenance of low levels of CFU-S proliferation and the ability of pEEDCK to prevent ARA-C-induced cycling of CFU-S, he and co-investigators administered peptide to mice which then were given ARA-C and nitrogen-mustard (75).…”

“…While the dimerized peptide indirectly stimulates hematopoiesis in vitro (74), Paukovits and others have used the monomeric peptide to protect hematopoietic progenitors from damaging chemotherapeutic effect (75). Noting that the peptide appeared to be cytostatic towards CFU-S, perhaps having a physiologic role in the maintenance of low levels of CFU-S proliferation and the ability of pEEDCK to prevent ARA-C-induced cycling of CFU-S, he and co-investigators administered peptide to mice which then were given ARA-C and nitrogen-mustard (75). Mice receiving both peptide and cytotoxic agent endured far less severe neutropenia and had preservation or enhancement of progenitor cell activity at the CFU-S and CFU-GM differentiative level.…”

Haemopoietic stem cells: their heterogeneity and regulation

“…This has been purified to homogeneity revealing the sequence pEEDCK (Paukovits & Laerum 1982). This peptide appears to act through a protease sensitive membrane receptor (Paukovits & Paukovits 1975) and is active in inhibiting murine and human myelomonocyticrestricted progenitor cells, the granulocyte/macrophage colony-forming cells (GM-CFC) as well as CFU-S and Long-term culture initiating cells (Paukovits et al 1990(Paukovits et al , 1991(Paukovits et al , 1993. Inhibitory activity is observed at 10-13 M however, the dose response is biphasic and inhibitory properties are lost at higher concentrations.…”