Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.     

Myelopoietin, an engineered chimeric IL-3 and G-CSF receptor agonist, stimulates multilineage hematopoietic recovery in a nonhuman primate model of radiation-induced myelosuppression

Myelopoietins (MPOs) constitute a family of engineered, chimeric molecules that bind and activate the IL-3 and G-CSF receptors on hematopoietic cells. This study investigated the in vivo hematopoietic response of rhesus monkeys administered MPO after radiation-induced myelosuppression. Animals were total body irradiated (TBI) in 2 series, with biologically equivalent doses consisting of either a 700 cGy dose of Cobalt-60 ((60)Co) gamma-radiation or 600 cGy, 250 kVp x-irradiation. First series: On day 1 after 700 cGy irradiation, cohorts of animals were subcutaneously (SC) administered MPO at 200 microg/kg/d (n = 4), or 50 microg/kg/d (n = 2), twice daily, or human serum albumin (HSA) (n = 10). Second series: The 600 cGy x-irradiated cohorts of animals were administered either MPO at 200 microg/kg/d, in a daily schedule (n = 4) or 0.1% autologous serum (AS), daily, SC (n = 11) for 23 days. MPO regardless of administration schedule (twice a day or every day) significantly reduced the mean durations of neutropenia (absolute neutrophil count [ANC] < 500/microL) and thrombocytopenia (platelet < 20,000/microL) versus respective control-treated cohorts. Mean neutrophil and platelet nadirs were significantly improved and time to recovery for neutrophils (ANC to < 500/microL) and platelets (PLT < 20,000/microL) were significantly enhanced in the MPO-treated cohorts versus controls. Red cell recovery was further improved relative to control-treated cohorts that received whole blood transfusions. Significant increases in bone marrow-derived clonogenic activity was observed by day 14 after TBI in MPO-treated cohorts versus respective time-matched controls. Thus, MPO, administered daily was as effective as a twice daily schedule for multilineage recovery in nonhuman primates after high-dose, radiation-induced myelosuppression.     

In vivo effects of interleukin-6 on thrombopoiesis in healthy and irradiated primates.

We have studied the in vivo effects of recombinant human interleukin-6 (rhIL-6) on hematopoiesis in eight healthy and nine irradiated cynomolgus monkeys. Of the healthy animals, three received rhIL-6 alone (10 micrograms/kg/d, subcutaneously [SC]), one received rhIL-6 in combination with rhIL-3 (10 micrograms/kg/d, SC), one received rhIL-6 in combination with recombinant cynomolgus granulocyte-macrophage colony-stimulating factor (rcGM-CSF; 10 micrograms/kg/d, SC), two received rhIL-6 in combination with recombinant human granulocyte-CSF (rhG-CSF; 10 micrograms/kg/d, SC), and one received rhIL-6 in combination with recombinant human leukemia inhibitory factor (rhLIF; 10 micrograms/kg/d, SC). All animals were treated for at least 2 weeks with rhIL-6 or the above mentioned combinations. rhIL-6 alone significantly increased the peripheral blood platelet counts (2- to 3.5-fold). The platelets reached a plateau between days 10 and 15 of treatment. No synergistic effects on platelet numbers were observed when rhIL-6 was combined with rhIL-3, rcGM-CSF, rhG-CSF, or rhLIF. In addition to rhIL-6, only rhLIF increased the platelet numbers when administered alone. To test whether rhIL-6 might also protect the animal from thrombocytopenia or shorten the time of thrombocytopenia after irradiation, we treated nine animals with total body irradiation (3.8 Gy). Six of the animals were additional treated with rhIL-6 (4 with 10 micrograms/kg/d; and 2 with 100 micrograms/kg/d) from day -1 or +1 to day 28 post irradiation. In these animals, rhIL-6 at the same dose effective in healthy animals (10 micrograms/kg/d) was not capable of protecting the animals from platelet nadir. However, when pegylated rhIL-6 was used at a dosage of 100 micrograms/kg/d post irradiation, the mean of the nadirs was 71,000/microL as compared with 39,000/microL in control animals and the time of thrombocytopenia was shorter (3 v 5 days). In all animals (healthy and irradiated), rhIL-6 did not increase the number of bone marrow megakaryocytes but induced a right shift of DNA ploidy in megakaryocytes. These data suggest that IL-6 acts as "thrombopoietin"-like activity, but not as "megakaryocyte-CSF"-like activity.     

Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2- incompatible transplanted SCID mice

In a previous study, we found that total body irradiation (TBI) was essential to induce acute graft-versus-host disease (GVHD) after allogeneic H-2-incompatible splenocyte (SP) transplantation in SCID mice. SCID mice (H-2d) conditioned with cyclophosphamide and transplanted intravenously (IV) with 5 x 10(7) C57BL/6 (H-2b) SP developed chronic GVHD within 3 months posttransplant without any evidence of preceding acute GVHD. In this study, SCID mice were conditioned with 4 Gy TBI or non-TBI regimens, either BuCy2 (busulfan 4 mg/kg/d + cyclophosphamide 100 mg/kg/d for 2 days) or Cy5 (cyclophosphamide 100 mg/kg/d for 5 days), and then transplanted IV with 5 x 10(7) SP. The TBI-conditioned mice were further divided into tree transplant groups: (1) TBI and SP administered the same day (TBI + D0 SP), (2) SP administered 4 days post-TBI (TBI + D4 SP), and (3) SP administered 7 days post-TBI (TBI + D7 SP). The severity of GVHD was compared among these groups by clinical and histologic grading. Twenty- eight of 28 mice treated with TBI + D0 SP died of acute GVHD, with overwhelming diarrhea by day 15 posttransplantation. Sixteen mice treated with either TBI + D4 SP or TBI + D7 SP developed acute GVHD, but none of them died of this disorder during 30 days posttransplantation. The mice conditioned with non-TBI regimens developed chronic GVHD within 3 months without showing any detectable signs of acute GVHD. Serum and in situ colonic cytokines were determined by enzyme-linked immunosorbent assay and immunohistology respectively. TBI itself significantly increased both serum and colonic tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and IL-6 when compared with non-TBI regimens and normal controls. TNF-alpha appeared in the serum and colon 4 hours post-TBI and peaked in 24 hours, followed by increasing IL-1 alpha and then IL-6 levels. TNF-alpha and IL-1 alpha decreased rapidly within 3 to 5 days post-TBI if no allogeneic cells were transplanted. Histoincompatible transplantation augmented cytokine release, which remained elevated on day 10 in these animals. Mice treated with TBI + D0 SP developed the most severe acute GVHD and had the highest levels of TNF-alpha, IL-1 alpha, and IL-6. The BuCy2-conditioned mice had the lowest cytokine levels and developed no acute GVHD. When the mice transplanted with TBI + D0 SP were treated immediately with recombinant soluble human TNF receptor (rhuTNFR:Fc) 100 micrograms/d intraperitoneally and for the subsequent 15 days acute GVHD mortality was significantly reduced from 100% to 50% (P < .001).(ABSTRACT TRUNCATED AT 400 WORDS)     

The in vivo effects of recombinant human interleukin-3: demonstration of basophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in nonhuman primates

Recently human interleukin-3 (IL-3) produced by molecular cloning was characterized as a growth factor for basophils and eosinophils in human bone marrow cultures. Since we found a similar activity of the human factor on simian bone marrow cells, we investigated the in vivo effects of recombinant human (rh) IL-3 in healthy rhesus monkeys (n = 10). rh IL-3 was administered subcutaneously (SC) to monkeys at different doses (11, 33, and 100 micrograms/kg/d) for 14 days followed by subsequent rh GM-CSF administration (5.5 micrograms/kg/d SC) for another two weeks. During the second week of rh IL-3 administration monkeys responded with a twofold to threefold increase of WBCs caused by a dose-dependent elevation of basophils (up to 40% of WBCs) and eosinophils. rh IL-3 also induced a dose-dependent increase of histamine (up to 700-fold above normal values) in monkey blood cells. Administration of rh GM-CSF to rh IL-3 pretreated monkeys resulted in a twofold enhanced increase in WBCs (due mainly to eosinophils and neutrophils) compared with animals treated with rh GM-CSF alone. Simultaneous administration of both cytokines (100 micrograms/kg rh IL-3 + 5.5 micrograms/kg rh GM-CSF SC) to two separate monkeys for 14 days induced a WBC elevation similar to that observed in monkeys treated with rh GM-CSF alone. In conclusion, our results indicate that rh IL-3 is a differentiation factor for blood basophils and primes the hematopoietic system for subsequent rh GM-CSF actions.     

In vivo administration of interleukin 6 delays hematopoietic regeneration in sublethally irradiated mice.

The in vivo effects of recombinant human interleukin 6 (IL-6) on the hematopoietic system of sublethally (4.8 Gy) x-irradiated mice were investigated. Animals were injected twice daily s.c. with IL-6 (10 micrograms/kg body weight/day) for 7 days following irradiation, and the numbers of hematopoietic stem, progenitor, and circulating blood cells were evaluated at 4, 7, 13, and 23 days. IL-6 caused significant depression of early hematopoiesis (decreased numbers of spleen colony-forming units [CFU-S] and granulocyte-macrophage colony-forming cells [GM-CFC]) in the spleens of irradiated mice. Marrow hematopoiesis was less affected by IL-6 injection, although the number of hematopoietic cells was also significantly lower than in irradiated mice injected with carrier alone. The observed decrease in the numbers of hematopoietic cells was not reflected by any significant change in the circulating blood cell numbers, which were similar to those in control irradiated animals. In contrast, IL-6 administered in 100 times higher doses (1000 micrograms/kg/day) caused significant increases in bone marrow and spleen cellularity and GM-CFC numbers, thus accelerating postirradiation hematopoietic regeneration. Our studies show that IL-6, administered in relatively low doses, suppresses postirradiation hematopoietic recovery, decreasing the numbers of stem and progenitor cells in sublethally irradiated mice.     

In vivo radioprotective effects of recombinant human granulocyte colony-stimulating factor in lethally irradiated mice.

The purpose of this study was to investigate the in vivo radioprotective effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) in lethally irradiated BALB/c mice. We initially analyzed the effects of increasing doses of rhG-CSF on survival of mice receiving 700 cGy (LD100/30) single dose total body irradiation (TBI). While 1 microgram/kg to 100 micrograms/kg doses of rhG-CSF were not radioprotective, a dose-dependent radioprotection was observed at 200 micrograms/kg to 4,000 micrograms/kg rhG-CSF. We next compared four different rhG-CSF treatment regimens side by side for their radioprotective effects in LD100/30 irradiated mice. One hundred percent of control mice receiving phosphate buffered saline died within 21 days after TBI with a median survival of 14 days. The median survival was prolonged to 20 days and the actuarial 60-day survival rate was increased to 27% when mice received 2,000 micrograms/kg rhG-CSF 24 hours before TBI (P = .0002; Mantel-Peto-Cox). Similarly, the median survival time was prolonged to 24 days and the actuarial 60-day survival rate was increased to 33%, when mice were given 2,000 micrograms/kg rhG-CSF 30 minutes before TBI. Optimal radioprotection was achieved when 2,000 micrograms/kg rhG-CSF was administered in two divided doses of 1,000 micrograms/kg given 24 hours before and 1,000 micrograms/kg given 30 minutes before TBI. This regimen prolonged the median survival time of LD100/30 irradiated mice to more than 60 days and increased the actuarial 60-day survival rate to 62% (P = .0001; Mantel-Peto-Cox). By comparison, no survival advantage was observed when mice received rhG-CSF 24 hours post-TBI. Similar radioprotective effects were observed when mice were irradiated with 650 cGy (LD80/30). The presented findings provide conclusive evidence that rhG-CSF has significant in vivo radioprotective effects for mice receiving LD100/30 or LD80/30 TBI.     

A randomized placebo-controlled trial of recombinant human interleukin- 11 in cancer patients with severe thrombocytopenia due to chemotherapy

Thrombocytopenia is a complication of cancer treatment that can limit dose intensity. Interleukin-11 (IL-11) is a growth factor that increases platelet production. We conducted a multicenter, randomized, placebo-controlled trial of recombinant human IL-11 (rhIL-11) in 93 patients with cancer who had already been transfused platelets for severe thrombocytopenia resulting from chemotherapy. The patients had received platelet transfusions for nadir platelet counts of < or = 20,000/microL during the chemotherapy cycle immediately preceding study entry. Chemotherapy was continued during the study without dose reduction. Patients were randomized to receive placebo or rhIL-11 at 50 or 25 micrograms/kg subcutaneously once daily for 14 to 21 days beginning 1 day after chemotherapy. Eight of 27 (30%) evaluable patients treated with rhIL-11 at a dose of 50 micrograms/kg did not require platelet transfusions versus 1 of 27 (4%) patients who received placebo (P < .05). Five of 23 (18%) patients treated with rhIL-11 at 25 micrograms/kg avoided platelet transfusions (P = .23). Side effects were fatigue and cardiovascular symptoms, including a low incidence of atrial arrhythmias and syncope. There were no differences among treatment groups in the incidence of neutropenic fever, days of hospitalization, or number of red blood cell transfusions. This study shows that rhIL-11 treatment of a dose of 50 micrograms/kg significantly increases the likelihood that patients who have already been transfused platelets for severe chemotherapy-induced thrombocytopenia will not require platelet transfusions during a subsequent chemotherapy cycle.     

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.     

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once- daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3- treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.     

A phase I study of sequential versus concurrent interleukin-3 and granulocyte-macrophage colony-stimulating factor in advanced breast cancer patients treated with FLAC (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) chemotherapy

Cumulative thrombocytopenia is a dose-limiting toxicity of dose- intensive chemotherapy for advanced breast cancer. In this phase I study, we have studied the hematologic toxicity associated with sequential interleukin-3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF; molgramostim) administration after multiple cycles of FLAC (5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide) chemotherapy compared with that after concurrent cytokine administration or to each cytokine administered alone. Ninety- three patients with advanced breast cancer were treated with five cycles of FLAC chemotherapy and either IL-3 alone, GM-CSF alone, sequential IL-3 and GM-CSF administered by schedule A (5 days of IL-3 followed by 10 days of GM-CSF) or schedule B (9 days of IL-3 followed by 6 days of GM-CSF), or concurrent administration of IL-3 and GM-CSF for 15 days. Cohorts of patients were treated with one of four dose levels of IL-3 (1,2.5, 5, and 10 micrograms/kg) administered subcutaneously for each schedule of cytokine administration. The GM-CSF dose in all schedules was 5 micrograms/kg/day. Sequential IL-3 and GM- CSF (schedule B) was associated with higher platelet nadirs, shorter durations of platelet counts less than 50,000/microL, and the need for fewer platelet transfusions over five cycles of FLAC chemotherapy compared with concurrent cytokines, sequential IL-3 and GM-CSF schedule A, and GM-CSF alone. Concurrent IL-3 and GM-CSF was associated with unexpected platelet toxicity. The duration of granulocytopenia after FLAC chemotherapy was significantly worse with IL-3 alone compared with each of the GM-CSF-containing cytokine regimens. Although no cycle 1 maximum tolerated dose for IL-3 was defined in this study, 5 micrograms/kg was well tolerated over multiple cycles of therapy and is recommended for future studies. The data from this phase I study suggest that sequential IL-3 and GM-CSF with IL-3 administered for 9 days before beginning GM-CSF may be superior to shorter durations of IL- 3 administered sequentially with GM-CSF, to concurrent IL-3 and GM-CSF, and to either colony-stimulating factor alone in ameliorating the cumulative hematologic toxicity associated with multiple cycles of FLAC chemotherapy. Additional studies of sequential IL-3 and GM-CSF are warranted.     

Myelopoietin, a chimeric agonist of human interleukin 3 and granulocyte colony-stimulating factor receptors, mobilizes CD34+ cells that rapidly engraft lethally x-irradiated nonhuman primates.

Myelopoietin (MPO), a multifunctional agonist of interleukin 3 and granulocyte colony-stimulating factor (G-CSF) receptors, was evaluated for its ability to mobilize hematopoietic colony-forming cells (CFC) and CD34+ cells relative to control cytokines in normal nonhuman primates. Additionally, the engraftment potential of MPO-mobilized CD34+ cells was assessed in lethally irradiated rhesus monkeys. Normal rhesus monkeys were administered either MPO (200 microg/kg/day), daniplestim (a high-affinity interleukin 3 receptor agonist) (100 microg/kg/day), G-CSF (100 microg/kg/day), or daniplestim coadministered with G-CSF (100 microg/kg/day each), subcutaneously for 10 consecutive days. The mobilization kinetics were characterized by peripheral blood (PB) complete blood counts, hematopoietic CFC [granulocyte-macrophage CFC (GM-CFC), megakaryocyte CFC (MK-CFC)], and the immunophenotype (CD34+ cells) of PB nucleated cells prior to and on day 3 to days 7, 10, 12, and 14, and at intervals up to day 28 following initiation of cytokine administration. A single large-volume leukapheresis was conducted on day 5 in an additional cohort (n = 10) of MPO-mobilized animals. Eight of these animals were transplanted with two doses of CD34+ cells/kg. A maximum 10-fold increase in PB leukocytes (white blood cells) (from baseline 7.8-12.3 x 10(3)/microL to approximately 90 x 10(3)/microL) was observed over day 7 to day 10 in the MPO, G-CSF, or daniplestim+G-CSF cohorts, whereas daniplestim alone stimulated a less than onefold increase. A sustained, maximal rise in PB-derived GM-CFC/mL was observed over day 4 to day 10 for the MPO-treated cohort, whereas the daniplestim+G-CSF, G-CSF alone, and daniplestim alone treated cohorts were characterized by a mean peak value on days 7, 6, and 18, respectively. Mean peak values for PB-derived GM-CFC/mL were greater for MPO (5,427/mL) than for daniplestim+G-CSF (3,534/mL), G-CSF alone (3,437/mL), or daniplestim alone (155/mL) treated cohorts. Mean peak values for CD34+ cells/mL were noted within day 4 to day 5 of cytokine administration: MPO (255/microL, day 5), daniplestim+G-CSF (47/microL, day 5), G-CSF (182/microL, day 4), and daniplestim (96/microL, day 5). Analysis of the mobilization data as area under the curve indicated that for total CFCs, GM-CFC, MK-CFC, or CD34+ cells, the MPO-treated areas under the curve were greater than those for all other experimental cohorts. A single, large-volume (3.0 x blood volume) leukapheresis at day 5 of MPO administration (PB: CD34+ cell/microL = 438 +/- 140, CFC/mL = 5,170 +/- 140) resulted in collection of sufficient CD34+ cells (4.31 x 10(6)/kg +/- 1.08) and/or total CFCs (33.8 x 10(4)/kg +/- 8.34) for autologous transplantation of the lethally irradiated host. The immunoselected CD34+ cells were transfused into autologous recipients (n = 8) at cell doses of 2 x 10(6)/kg (n = 5), and 4 x 10(6)/kg (n = 3) on the day of apheresis. Successful engraftment occurred with each cell dose. The data demonstrated that MPO is an effective and efficient mobilizer of PB progenitor cells and CD34+ cells, such that a single leukapheresis procedure results in collection of sufficient stem cells for transplantation and long term engraftment of lethally irradiated hosts.     

The ligand for c-kit, stem cell factor, stimulates the circulation of cells that engraft lethally irradiated baboons.

Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.     

Predictors of CD4 count change over 8 months of follow up in HIV-1-infected patients with a CD4 count>or=300 cells/microL who were assigned to 7.5 MIU interleukin-2

BACKGROUND: ESPRIT is a randomized trial comparing the clinical impact of interleukin (IL)-2 plus antiretrovirals vs antiretrovirals alone. Identification of factors that influence the relationship between IL-2 and CD4 count recovery will enable better personalization of treatment with IL-2 in HIV-1-positive individuals. The IL-2 induction phase consists of three dosing cycles over 6-8 months (7.5 MIU twice a day, for 5 days every 8 weeks). METHODS: We included patients initiating IL-2 at the 7.5 MIU dose with an 8-month CD4 count, measured at least 30 days after their last cycle. We identified baseline predictors of CD4 count changes over 8 months using linear regression. RESULTS: Of 2090 patients assigned IL-2, 1673 (80%) were included in the analysis. The median (interquartile range) baseline CD4 count was 461 (370, 587) cells/microL with a median increase of 233 (90, 411) cells/microL at month 8. After adjustments, significant predictors of CD4 count change included CD4 nadir (29.8 cells/microL greater increase per 100 cells/microL higher; P<0.0001), last CD4 count before baseline (mean 36.0 cells/microL greater increase per 100 cells/microL higher; P<0.0001), time from antiretroviral start to baseline (8.3 cells/microL smaller increase per year longer; P=0.001), age (11.7 cells/microL smaller increase per 5 years older; P=0.005) and race (79.7 cells/microL greater increase for black patients vs white patients; P=0.003). A linear relationship existed between total IL-2 dose in the first cycle and CD4 count change (73.1 cells/microL greater increase per 15 MIU higher; P<0.0001). CONCLUSIONS: Prior nadir and current CD4 counts, age and IL-2 dose are major determinants of CD4 increases induced by with intermittent administration of IL-2 in HIV-1-positive individuals on antiretrovirals. The clinical function of these induced CD4 cells is under study.     

Differential effects of sequential, simultaneous, and single agent interleukin-3 and granulocyte-macrophage colony-stimulating factor on megakaryocyte maturation and platelet response in primates.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM-CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.     

Recombinant glycosylated human interleukin-6 accelerates peripheral blood platelet count recovery in radiation-induced bone marrow depression in baboons.

This report was aimed at confirming the potential clinical use for a genetically engineered glycosylated human interleukin-6 (rhIL-6) in hematopoiesis. Its tolerance and efficacy were assessed on hematopoietic restoration after neutron radiation-induced bone marrow injury on baboons, which represent an adequate model of parallelism for studying hematology in the human. The particular neutron radiation absorption pattern in the body allows the preservation of underexposed bone marrow areas that mimics an autotransplantation-like situation. An initial dose finding study (1 microgram up to 20 micrograms/kg/d for 8 consecutive days) in normal baboons established a dose-dependent response regarding the peripheral platelet count (range of increase, 1.5- to 4-fold). A significant elevation in white blood cell (WBC) count, as well as a substantial reversible normochromic normocytic anemia, were observed for the highest doses only (10 and 20 micrograms/kg/d). All rhIL-6 administered doses were clinically well tolerated. In myelosuppressed baboons, a selected dose of 10 micrograms/kg/d of rhIL-6 for 13 consecutive days significantly lessened the degree of induced thrombocytopenia as compared with the control group (P = .01) and shortened the time to occurrence of the nadir, showing that the onset of recovery occurs much earlier, ie, an average of 5 days (P = .003), in the treated group. Moreover, this accelerated platelet recovery is evidenced by an 8-day shorter mean time back to baseline values (P = .03) in the rhIL-6--treated animals. At this dose no effect was observed on the WBC recovery pattern. Importantly rhIL-6 did not accentuate the radiation-induced anemia and was clinically well tolerated. All tested monkeys recovered from their induced pancytopenia and no animal loss was recorded. IL-6, tumor necrosis factor, and IL-1 blood measurements are reported. In conclusion, rhIL-6 is a potent thrombopoietic factor for the treatment of induced thrombocytopenia in nonhuman primates at a clinically well-tolerated dose.     

Accelerated reconstitution of platelets and erythrocytes after syngeneic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice

The recent cloning of the ligand of the c-Mpl hematopoietin receptor has indicated a major role for this cytokine in the development of megakaryocytes. In this study we have applied c-Mpl ligand (thrombopoietin [TPO]) in the setting of syngeneic transplantation in an attempt to accelerate the reconstitution of platelets. Donor mice were treated with 20 kilounits (kU)/d TPO intraperitoneally (ip) for 5 days. This resulted in a 2.5-fold increment in platelet counts from 1,119 x 10(9)/L to 2,582 x 10(9)/L (mean, n = 7). Total numbers of hematopoietic progenitor cells in bone marrow (BM) and spleen, as assessed in a colony-forming unit-granulocyte erythroid monocyte macrophage (CFU-GEMM) colony assay (55.3 v 38.6 x 10(3) CFU/femur; 27.3 v 16.3 x 10(3) CFU/spleen, mean, n = 7) as well as total numbers of burst-forming unit-erythroid (BFU-E) (24.0 v 16.4 x 10(3)/femur; 10.2 v 1.9 x 10(3)/spleen, mean, n = 7), were significantly higher in TPO- treated donors than in saline-treated controls. Female Balb-C mice were lethally (8.5 Gy) irradiated and transplanted with 10(5) BM cells. After transplantation, groups of mice were treated with recombinant murine TPO at a dose of 20 to 30 kU/d ip or subcutaneously (SC) for 5 to 14 days. Using this dose and schedule, TPO did not stimulate the recovery of platelets in comparison with control animals transplanted with equal cell numbers but given vehicle alone. In other experiments, 10(5) BM cells were procured from TPO-treated donor mice and transplanted into lethally irradiated recipient mice. In comparison with animals transplanted with an equal number of BM cells derived from saline-treated controls, recipients of TPO-treated BM cells had significantly faster platelet recovery and higher platelet nadir counts (88 v 30 x 10(9)/L, mean, n = 20). Transplantation of TPO-treated BM cells also resulted in an accelerated recovery of erythrocytes and increased erythrocyte nadir counts (7.2 v 5.0 x 10(12)/L, mean, n = 20). At the day of platelet nadir (day 12 after transplantation) these animals had higher numbers of BFU-Es (770 v 422, mean, n = 5) in the marrow and also had higher reticulocyte counts (44 / 1000 v 8 / 1000 mean, n = 5) in the blood. Therefore, the accelerated recovery of erythrocytes may be a direct effect of TPO on erythropoiesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of recombinant human interleukin-6 in cancer patients: a phase I-II study

To define the toxicity profile of recombinant human interleukin-6 (rhIL- 6) and to study its effect on hematopoiesis, biochemical parameters and other cytokines, rhIL-6 was administered in a phase I-II study to 20 patients with breast carcinoma or nonsmall cell lung cancer. RhIL-6 doses were 0.5, 1.0, 2.5, 5.0, 10, and 20 micrograms/kg/d, with at least three patients per dose level. RhIL-6 was administered 24 hours by continuous intravenous infusion followed by subcutaneous (SC) administration for 6 days, partly on an outpatient basis. RhIL-6- related side effects were fever, headache, myalgia, and local erythema. Starting at 2.5 micrograms/kg/d, these side effects were compounded by nausea, reversible increase in liver enzymes, and anemia. Flu-like symptoms were controllable up to and including 10 micrograms rhIL- 6/kg/d with acetaminophen. RhIL-6 increased platelet counts with a decrease in mean platelet volume and increased leukocytes caused by neutrophil, monocyte, and lymphocyte increase, with an increase in T cells and natural killer cells at 1.0 and 2.5 micrograms rhIL-6/kg/d. The reversible anemia was characterized by a decrease in serum iron, and an increase in ferritin and erythropoietin without reticulocytosis. RhIL-6 reduced total cholesterol levels and a dose-related increase of C-reactive protein and serum amyloid A plasma levels was observed. Serum IL-6 levels were increased, especially at 10 and 20 micrograms/kg/d, whereas no change in IL-1 beta and tumor necrosis factor alpha levels was observed. RhIL-6 can be administered with controllable side effects in this setting, up to and including a SC dose of 10 micrograms/kg/d on an outpatient basis, and has a promising stimulating effect on leukopoiesis and thrombopoiesis.     

Effects of interleukin-3 after chemotherapy for advanced ovarian cancer.

To define the maximum tolerated dose and to study whether recombinant human interleukin-3 (rhIL-3) reduced chemotherapy-induced neutropenia and thrombocytopenia, 20 chemotherapy-naive patients with advanced ovarian cancer eligible for treatment with 6 cycles of carboplatin-cyclophosphamide every 4 weeks (day 1) were entered in a phase I/II open, single-center trial. Cohorts of five patients received during 7 days 1, 5, 10, or 15 micrograms/kg/d rhIL-3 (days 5 through 11) in cycles 1, 3, and 5 by continuous intravenous (IV) infusion or once daily subcutaneous (SC) administration. In control cycles 2, 4, and 6, no rhIL-3 was administered. rhIL-3 significantly increased the recovery of leukocyte, neutrophil, and platelet counts, especially at 5, 10, and 15 micrograms/kg rhIL-3. rhIL-3 also increased basophil, eosinophil, monocyte, and lymphocyte counts at this dose steps. Effects on reticulocytes were limited. No difference in efficacy between SC and IV rhIL-3 treatment was found. Chemotherapy postponement for insufficient bone marrow recovery was necessary in 22 of 45 control cycles versus 2 of 49 rhIL-3 cycles (P less than .001). Platelet transfusions were required in 7 of 45 control cycles versus 3 of 50 rhIL-3 cycles (P less than .5). rhIL-3 up to 10 micrograms/kg/d could be administered without severe side effects. At 15 micrograms/kg/d, rhIL-3 headache was dose-limiting. Other side effects were fever, flu-like symptoms, nausea, skin rash, flushing, facial erythema, and urticaria. Liver toxicity occurred in rhIL-3 and control cycles. rhIL-3 slightly increased tumor necrosis factor alpha, C-reactive protein, and serum amyloid A plasma levels, whereas no effect on IL-6 plasma levels was observed. rhIL-3 administered SC appears to be an interesting hematopoietic growth factor for reduction of chemotherapy-induced myelotoxicity.     

Effects of recombinant human interleukin-11 on hematopoietic reconstitution in transplant mice: acceleration of recovery of peripheral blood neutrophils and platelets

We have examined the effects of recombinant human interleukin-11 (rhIL- 11) on the recovery of peripheral blood cell counts and proliferation of progenitors and hematopoietic stem cells (day 12 colony-forming units-spleen-CFU-S12) in vivo using a mouse bone marrow (BM) and spleen cell transplantation model. Recovery of leukocytes was accelerated in animals receiving daily administration of rhIL-11 (100 micrograms/kg/d) and reached normal levels by day 14 posttransplantation. This increased total leukocyte count reflected mainly an increase in neutrophils. Neutropenia (absolute neutrophil count [ANC] < 1,500) was present in control transplant mice for 14 to 15 days, while in the rhIL-11-treated group, neutrophils recovered to normal by days 8 to 10 and continued to increase until day 19. Animals treated with rhIL-11 had only 1 day with ANC demonstrated < 500. Correspondingly, rhIL-11 treatment increased granulocyte-macrophage progenitors (CFU-GM) derived from both spleen and BM cells. Higher doses of IL-11 increased CFU-GM nearly threefold and CFU-Mix fourfold to fivefold, while increasing burst-forming units- erythroid to a lesser degree. BM and spleen cellularity were both increased in IL-11-treated mice, but no increase in CFU-S12 was noted. In addition, in vivo daily administration of IL-11 increased peripheral platelet counts by threefold over control transplant mice at day 10 posttransplantation during the post-irradiation platelet nadir. Further treatment led to platelet counts higher than normal 18 days posttransplantation when control animals had just attained normal platelet counts. IL-11 can accelerate the recovery of the peripheral blood leukocytes, mainly neutrophils, and platelets in transplant mice, effects that may be clinically useful in future applications for BM transplantation and chemotherapy-related cytopenias.