Dose Of Cy Research Articles

Low‐dose cyclophosphamide conditioning for haematopoietic cell transplantation from HLA‐matched related donors in patients with Fanconi anaemia

Allogeneic haematopoietic cell transplantation (HCT) is effective therapy for Fanconi anaemia (FA). FA patients do not tolerate conditioning with 200 mg/kg of cyclophosphamide (Cy), typically used in aplastic anaemia. We previously published results of studies in which Cy doses were gradually reduced from 200 to 100 mg/kg. Here we update results of the initial studies and report data on 30 new patients conditioned with Cy either at 80 mg/kg (n = 7) or at 60 mg/kg (n = 23), given over 4 days before HCT from human leucocyte antigen-matched related donors. Methotrexate and cyclosporine were given for graft-versus-host disease (GVHD) prophylaxis. All seven patients given Cy at 80 mg/kg and 21 of 23 given Cy at 60 mg/kg had sustained engraftment, while two patients, both with clonal cytogenetics abnormalities, experienced graft failure. Grades 2-3 acute GVHD rates were 57% and 14% for patients given the higher and lower Cy doses, respectively (P = 0.001). Four patients given Cy at 80 mg/kg and 22 given Cy at 60 mg/kg were alive at a median of 47 (44-58) months and 16 (3-52) months, respectively. Cy at 60 mg/kg has acceptable toxicities, low rates of GVHD, and is sufficient for engraftment of related grafts in most FA patients.

Nonmyeloablative stem cell transplantation using lymphoablative rather than myeloablative conditioning in the prefludarabine era by ATG and limiting doses of cyclophosphamide

The feasibility of using lymphoablative rather than myeloablative conditioning for durable engraftment of allogeneic stem cells and subsequent cell therapy with donor lymphocytes was pioneered in the prefludarabine era in patients with resistant lymphoma and metastatic solid tumors. Between July 1995 and August 1996, 15 patients, five males and 10 females, median age 50 (range 20-57) years, were enrolled in a protocol that consisted of different doses of cyclophosphamide (Cy), 50 mg/kg/day for 1, 2, 3 or 4 consecutive days in parallel with a fixed dose of rabbit antithymocyte globulin (ATG) (Fresenius) 10 mg/kg/day for 4 consecutive days. All patients, except one treated with a single dose of Cy, achieved full tri-lineage engraftment and no late graft failure was observed. Only three patients suffered from grade III-IV graft-versus-host disease (GVHD). Three patients out of the 15 survived long term (follow-up >93 to >96 months). We concluded that lymphoablative conditioning with ATG and intermediate-to-high-dose Cy is well tolerated and can result in durable engraftment with acceptable GVHD in heavily pretreated patients with advanced malignancies. Hence, induction of tolerance to donor alloantigens by lymphoablative conditioning while avoiding myeloablative chemotherapy or radiation therapy may serve as a platform for subsequent cell therapy with donor lymphocytes.

Virulence ofPseudomonas aeruginosain a Murine Model of Gastrointestinal Colonization and Dissemination in Neutropenia

Pseudomonas aeruginosa bacteremia in cancer patients develops from initial gastrointestinal (GI) colonization with translocation into the bloodstream in the setting of chemotherapy-induced neutropenia and GI mucosal damage. We established a reproducible mouse model of P. aeruginosa GI colonization and systemic spread during neutropenia. Mice received 2 mg of streptomycin/ml of drinking water and 1,500 U of penicillin G/ml for 4 days and then ingested 10(7) CFU of P. aeruginosa per ml of drinking water for 5 days. After GI colonization levels were determined, cyclophosphamide (Cy) was then injected intraperitoneally (i.p.) three times every other day or an antineutrophil monoclonal antibody, RB6-8C5, was injected i.p. once. Dissemination was defined by the presence of P. aeruginosa in spleens of moribund or dead mice. In this mouse model, P. aeruginosa colonizes the GI tract and then disseminates systemically once Cy or RB6-8C5 is administered. The duration and intensity of neutropenia, related to Cy dose, was found to be a means to compare the virulence of different P. aeruginosa strains, as exhibited by comparisons of strains lacking or producing the virulence-enhancing ExoU cytotoxin. The lipopolysaccharide outer core polysaccharide and O side chains were critical in establishing GI colonization, and P. aeruginosa mutants lacking the aroA gene (necessary for synthesizing aromatic amino acids) were able to establish GI colonization but unable to disseminate. Both the colonization and dissemination phases of P. aeruginosa pathogenesis can be studied in this model, which should prove useful for evaluating pathogenesis, therapies, and associated means to control P. aeruginosa nosocomial infections.

Antitumour effect of OM-174 and Cyclophosphamide on murine B16 melanoma in different experimental conditions

We report that OM-174, a purified water soluble diphosphorylated and triacetylated lipid A derived from E. coli, is able to reduce tumour progression and to prolong the survival of mice in the B16 melanoma experimental model. At the doses employed in our study OM-174 had an antitumour activity comparable to that of a single high dose of CY. More striking effects were achieved by means of the combination of the two agents in a protocol consisting of a single administration of CY (200 mg/kg) followed by five injections of OM-174 (1 mg/kg). Immunological studies of treated and control mice revealed that the antitumour activity of OM-174, alone or in combination with CY, is mediated by the stimulation of natural killer (NK) and cytotoxic T lymphocyte (CTL) responses as well as by a significant increase in the absolute number of NK1.1, CD4 and CD8 positive cells. OM-174 is thus candidate for association with cytostatic drugs in chemo-immunotherapy protocols.

Post-Transplantation Cyclophosphamide (Cy) Reduces Graft Rejection and Graft-Versus-Host Disease (GVHD) after Non-Myeloablative, Partially HLA-Mismatched (Haploidentical) Bone Marrow Transplantation (BMT).

In animal models of haploidentical BMT, a properly timed high dose of Cy post-BMT selectively eliminates host-versus-graft and graft-versus-host reactive T cells, thereby preventing graft rejection and reducing GVHD. To test the activity of post-BMT Cy in humans, 56 patients (median age 48 years) with advanced hematologic malignancies received Cy 50 mg/kg IV, either once (on day 3; n=20) or twice (on days 3 and 4; n=36) after non-myeloablative conditioning and haploidentical BMT (“mini-haploBMT”). Diagnoses included AML (n=18), MDS (n=8), ALL (n=3), CML (n=4), CMMoL (n=3), CLL (n=3), multiple myeloma (MM; n=3), Hodgkin's disease (HD; n=5), non-Hodgkin's lymphoma (NHL; n=8), and paroxysmal nocturnal hemoglobinuria (PNH; n=1). Most patients had advanced disease or failed a previous autologous transplant. All were conditioned as outpatients with fludarabine (30 mg/m2 IV q d, days −6 to −2), Cy (14.5 mg/kg IV on days −6, −5), and total body irradiation (200 cGy on day −1), transplanted with non-T cell-depleted marrow, and treated with G-CSF, FK-506 and mycophenolate mofetil beginning the day after the last dose of Cy. Median number of hospitalizations in the first 60 days after BMT was 1 (range, 0–3), and the median number of hospital days was 4 (range, 1–53). Neutrophil engraftment (>500/mm3) after one or two days of post-BMT Cy occurred at a median of 15 versus 17 days after BMT, respectively (p=.36), and platelet counts >20,000/mm3 without transfusion were achieved at a median of 31 and 31 days, respectively (p=.78). Graft rejection, which in all cases was followed by recovery of autologous hematopoiesis, occurred in 5/18 (28%) versus 4/35 (11%) evaluable patients receiving one versus two doses of post-transplant Cy, respectively (p=.24). Full donor hematopoietic chimerism in the peripheral blood on day 60 was seen in 10/13 versus 25/31 engrafting patients who received one versus two doses of post-BMT Cy; with two exceptions, mixed chimerism was an immediate harbinger of relapse. Compared to patients receiving a single dose of post-BMT Cy, those receiving two doses had significantly less Grade II–IV (43% vs. 78%; p=.01) and Grade III–IV acute GVHD (20% vs. 53%; p=.006) by day 200 after BMT. Death from GVHD occurred in 5/13 evaluable patients receiving one dose versus 2/28 evaluable patients receiving 2 doses of post-BMT Cy. This latter group also had a non-significantly lower incidence of non-relapse mortality at 100 days (4% vs. 15%, p=.13) and at 1 year (23% vs. 35%; p=.37). At a median follow-up of 172 days (range, 32–1440 days), 21 patients remain alive without disease progression, including all 3 patients with chronic phase CML and the patient with PNH. In summary, mini-haploBMT with 2 doses of post-transplantation Cy resulted in full donor chimerism without severe GVHD in 23/36 patients (64%). Its toxicity profile compares favorably to matched sibling BMT. Moreover, in a very high-risk patient group, the early event-free survival is encouraging.

The GnRH antagonist cetrorelix reduces cyclophosphamide-induced ovarian follicular destruction in mice.

It has remained controversial whether and in what way suppression of the pituitary-gonadal axis using GnRH analogues can reduce the destructive effects of chemotherapy on ovarian primordial follicles and thus prevent ovarian failure. GnRH antagonists suppress gonadotrophin levels immediately after administration. In this study we determined whether administration of the GnRH antagonist cetrorelix before exposure to increasing doses of cyclophosphamide (Cy) affected the number of surviving primordial follicles (PMF) in the mice ovary. Highly inbred young Balb/c mice (114 females) were injected with 0, 50 and 75 mg/kg of Cy. In each treatment group, half of the females were injected daily with cetrorelix starting 9 days before and 7 days post-administration of Cy. In serial sequential ovarian sections the total number of PMF in both ovaries was counted. Ovaries exposed to Cy at doses of 50 and 75 mg/kg had significantly fewer PMF than those in the control group (P < 0.01). In each of the Cy groups used, pretreatment with cetrorelix resulted in significantly higher numbers of PMF: in the 50 mg/kg Cy group only 14% were destroyed (cetrorelix group) versus 53% (P < 0.001), while in the 75 mg/kg Cy group only 35% of PMF were destroyed versus 54% in animals treated only with Cy (P < 0.004). The interaction between the effect of cetrorelix and the different doses of Cy did not reach statistical significance (P = 0.089, two-way ANOVA). Administration of the GnRH antagonist cetrorelix to mice significantly decreases the extent of ovarian damage induced by the chemotherapeutic agent Cy. The use of different substerilizing doses of Cy suggested that the extent of protection achieved by the antagonist is dose-dependent and decreases with increasing Cy doses. The results of this study may suggest a possible similar beneficial effect in women undergoing chemotherapy, can explain the discrepancy in results of existing clinical studies and indicate possible pathways for ovarian GnRH agonist protection. Further research and clinical studies are needed in order to confirm these results.

Once daily intravenous busulfan as part of a busulfan/cyclophosphamide conditioning regimen for allogeneic hematopoietic stem cell transplantation

Objective: Busulfan (Bu) is frequently used as part of myeloablative conditioning regimens prior to stem cell transplantation (SCT), especially to avoid total body irradiation. Hepatic venoocclusive disease (HVOD) is a well-recognized dose-limiting toxicity for oral Bu-preparative regimens. The purpose of this review was to evaluate the toxicity, efficacy, 100-day mortality, overall and disease free survival in patients treated with single daily dose intravenous (iv) Bu as part of a Bu/Cyclophosphamide (Cy) conditioning regimen for allogeneic SCT. Patients and Methods: 29 consecutive patients undergoing allogeneic SCT received iv Bu as part of their conditioning between March 2002 and August 2003. Conditioning consisted of iv Bu (3.2mg/kg once daily x 4 doses) followed by high dose Cy and allogeneic SCT. The donors were HLA-matched siblings (n = 23), 1 antigen mismatched related donors (n = 1) and matched unrelated donors (n = 5). All patients received graft versus host disease (GVHD) prophylaxis consisting of Cyclosporine A and Methotrexate. Results: 28 patients engrafted with a median time for neutrophil recovery of 21 days (12,29) and median time for platelets recovery of 18 days (10,49). 14 patients (48.3%) developed HVOD, 10 mild (34.5%), 2 moderate (6.9%) and 2 severe HVOD (6.9%). Acute GVHD grade II-IV occurred in 18 patients (64.3%). 3 patients (10.3%) died in the first 100 days after the transplant, with 1 death due to severe HVOD (3.4%), 1 due to graft failure (3.4%) and 1 to other transplant related toxicity (3.4%). After a median follow up of 8 months (1,16) chronic GVHD occurred in 12 out of 24 evaluable patients (50%), 2 patients relapsed (6.9%), 1 patient is still in a partial remission (3.4%) and a total of 7 patients died (24.1%). 22 patients (75.9%) were alive at their last follow up. Overall survival at 6 months was 84.5%, and at 1 year 69.4%. 6-month disease-free survival was 80.7% and at 1 year 67.3%. Conclusions: Conditioning regimens with single daily dose iv Bu allowed consistent engraftment from related and unrelated donors. Only one graft failure occurred in a patient undergoing a second allogeneic SCT from a second matched related donor. 14 patients met criteria for HVOD, but the majority of them were mild. Treatment related mortality was limited. Overall survival and disease-free survival rates are encouraging thus meriting further studies for defining the role of iv Bu as a preferred substitute for oral Bu. Objective: Busulfan (Bu) is frequently used as part of myeloablative conditioning regimens prior to stem cell transplantation (SCT), especially to avoid total body irradiation. Hepatic venoocclusive disease (HVOD) is a well-recognized dose-limiting toxicity for oral Bu-preparative regimens. The purpose of this review was to evaluate the toxicity, efficacy, 100-day mortality, overall and disease free survival in patients treated with single daily dose intravenous (iv) Bu as part of a Bu/Cyclophosphamide (Cy) conditioning regimen for allogeneic SCT. Patients and Methods: 29 consecutive patients undergoing allogeneic SCT received iv Bu as part of their conditioning between March 2002 and August 2003. Conditioning consisted of iv Bu (3.2mg/kg once daily x 4 doses) followed by high dose Cy and allogeneic SCT. The donors were HLA-matched siblings (n = 23), 1 antigen mismatched related donors (n = 1) and matched unrelated donors (n = 5). All patients received graft versus host disease (GVHD) prophylaxis consisting of Cyclosporine A and Methotrexate. Results: 28 patients engrafted with a median time for neutrophil recovery of 21 days (12,29) and median time for platelets recovery of 18 days (10,49). 14 patients (48.3%) developed HVOD, 10 mild (34.5%), 2 moderate (6.9%) and 2 severe HVOD (6.9%). Acute GVHD grade II-IV occurred in 18 patients (64.3%). 3 patients (10.3%) died in the first 100 days after the transplant, with 1 death due to severe HVOD (3.4%), 1 due to graft failure (3.4%) and 1 to other transplant related toxicity (3.4%). After a median follow up of 8 months (1,16) chronic GVHD occurred in 12 out of 24 evaluable patients (50%), 2 patients relapsed (6.9%), 1 patient is still in a partial remission (3.4%) and a total of 7 patients died (24.1%). 22 patients (75.9%) were alive at their last follow up. Overall survival at 6 months was 84.5%, and at 1 year 69.4%. 6-month disease-free survival was 80.7% and at 1 year 67.3%. Conclusions: Conditioning regimens with single daily dose iv Bu allowed consistent engraftment from related and unrelated donors. Only one graft failure occurred in a patient undergoing a second allogeneic SCT from a second matched related donor. 14 patients met criteria for HVOD, but the majority of them were mild. Treatment related mortality was limited. Overall survival and disease-free survival rates are encouraging thus meriting further studies for defining the role of iv Bu as a preferred substitute for oral Bu.

Effect of different doses of cyanamide on striatal salsolinol formation after ethanol treatment

To assess the dose-dependent effect of cyanamide (CY, a potent aldehyde dehydrogenase inhibitor) on salsolinol release in the striatum, rats were treated with CY (25, 50 and 100 mg/kg) plus ethanol (EtOH,1 g/kg) intraperitoneally. Striatal salsolinol was detected using in vivo microdialysis coupled with high-performance liquid chromatography with an electrochemical detector in free-moving rats, and blood acetaldehyde (AcH) and EtOH were detected using the head-space gas chromatographic method. With the increase in the doses of CY following EtOH, the peak concentrations of striatal salsolinol and blood AcH were increased significantly. Our study indicated that the magnitude of striatal salsolinol levels may depend on the concentration of blood AcH, and that there is a correlation between the blood AcH and striatal salsolinol.

CD40 ligand-specific antibodies synergize with cyclophosphamide to promote long-term transplantation tolerance across MHC barriers but inhibit graft-vs-leukemia effects of transplanted cells.

We previously demonstrated that allogeneic bone marrow transplantation (BMT) after low-dose total lymphoid irradiation (200 cGy) and depletion of donor-reactive cells with cyclophosphamide (Cy) converted recipients to graft-vs-host disease (GVHD)-free chimeras tolerant to donor skin grafts. BMT also generated strong graft-vs-leukemia (GVL) response. However, clinical application of the protocol was hampered by the requirement for a relatively high dose of Cy (200 mg/kg). In this study we have tried to minimize the Cy dose by a concomitant blockade of CD40-CD40L interaction. Mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (BM(1)) and skin graft on day 0, injected with Cy (200 mg/kg or less) on day 1, and transplanted with a second C57BL/6 BM cell inoculum (BM(2)) on day 2. CD40L-specific antibody (MR1) was given with BM(1), BM(2), and 2 days later. Treated animals were monitored for survival, chimerism, and skin allograft rejection. The GVL potential of transplanted cells was examined in mice inoculated with BCL1 leukemia cells before irradiation. Blocking CD40-CD40L interaction with MR1 mAb allowed the reduction of a tolerance-generating Cy dose by 50%. Unfortunately, adding MR1 to the protocol reduced the GVL potential of the transplanted cells. Neither low-dose Cy nor antibodies alone could downregulate donor or recipient immune response. CD40L-specific antibodies synergize with Cy to induce bilateral transplantation tolerance. Therefore, their use may be beneficial for safer allogeneic BMT for nonmalignant indications. However, due to MR1-associated reduction of GVL effects, MR1 should be considered with caution as conditioning for BMT for leukemia-bearing recipients.

Th2/Th1 switch induced by a single low dose of cyclophosphamide in a rat metastatic lymphoma model.

Cyclophosphamide (Cy) is an alkylating agent widely used in cancer chemotherapy. It has a bimodal effect on the immune system, depending on the dose and schedule of administration. We have previously demonstrated that a single low dose of Cy has an antimetastatic effect, achieved through immunomodulation, in lymphoma bearing rats. Such a treatment reduced the splenic production of IL-10, TGF-beta, and NO, restoring the lymphoproliferative capacity. A shift from immunosuppression to immunopotentiation induced by low-dose Cy treatment was mainly mediated by a decrease in IL-10 production. The present study focused on the analysis of the modulation of type-1 cytokine levels by treatment with a single low dose of Cy and the effect these cytokines (IL-2 and IFN-gamma) and IL-10 have on primary tumor and metastatic cell growth. Our results suggest that a single low dose of Cy induces a Th2/Th1 shift in the cytokine profile of lymphoma-bearing rats, which may be responsible for its antimetastatic effect. A direct action of IL-10 as a growth factor and IFN-gamma as a cytotoxic factor on metastatic cells is also shown.

Immunomodulating anticancer alkylating drugs: targets and mechanisms of activity.

CY and L-PAM potentiated specific anti-tumor response in addition to their killing effect. The immunomodulating effect of a low dose of either CY or L-PAM was expressed in mice bearing large s.c. MOPC-315 plasmacytoma tumors. Cured mice were resistant to a challenge dose of the syngeneic tumor and their spleens contained specific cytotoxic T cells. Induction of specific anti-tumor response by a low dose of alkylating drugs was due to expression of "latent anti-tumor" capability. This fitted with the conception that "suppressed concomitant immunity" occurring in tumor-bearing animals can be activated. The immunomodulating activity of alkylating drugs was related to enhancement of T-cell functions:impairment of suppressor T-cell activity,enhancement of effector T-cell activity and increase in production of cytokines at the tumor site. The target tumor killing activity of a low dose alkylating drug was dissociated from its immunomodulating activity by treating mice bearing a tumor resistant to an alkylating drug. A low dose of CY had an immunomodulating effect in human cancer such as reduction of ConA-induced suppressor cell activity in melanoma, some improvement in addition to use of melanoma vaccine, and potentiation of DTH in cancer patients. The immunomodulating effect of alkylating drugs suggest that their use might be beneficial not only for killing tumor cells but also for promoting specific anti-tumor immune response.

High doses of cyclophosphamide in the treatment of severe systemic lupus erythematosus

TREATMENT of patients with systemic lupus erythematosus (SLE) has not changed substantially in the last 10 years. In general, low to moderate doses of corticosteroids and antimalarics are sufficient for mild forms of the disease, whereas high doses of corticosteroids, combined with other immunosuppressive drugs, are required for cases with affected kidneys or central nervous system (CNS). As an immunosuppressive drug, cyclophosphamide (Cy) may be applied according to different protocols, but the protocol from the National Institutes of Health (NIH), is used most frequently. 4 Side effects of therapy with Cy are numerous, some of which are very serious. The most common adverse effects include alopecia, amenorrhea, bone marrow suppression, hemorrhagic cystitis, risk of infection, and malignancy. If the dose of Cy and route of administration are carefully selected, even severe forms of SLE could be successfully treated with minimal side effects. The aim of this work was to report on our 10-year experience with high “pulse” doses of Cy in the treatment of most severe forms of SLE. For the period 1988 to 1999, a total of 178 patients (162 women and 16 men) who met the ARA criteria for diagnosis of SLE were submitted to treatment at the Clinic of Rheumatology and Clinical Immunology, MMA, Belgrade. Among these patients, 78% (139 of 178) had arthritis, 64% (114 of 178) facial butterfly erythema, 42% (75 of 178) glomerulonephritis, 19% (34 of 178) CNS manifestations according to ARA criteria, 10% (18 of 178) a severe form of skin vasculitis, and 7% (12 of 178) carditis. Of the patients with glomerulonephritis, type IIb was diagnosed in 21, type III in 21, type IV in 30, and type V in 9, according to WHO classification. In the group of patients with affected CNS, 26 had epilepsy and 8 had psychosis. High “pulse” doses of Cy were applied in 68 patients with the most severe disease: 38 patients (56%) had glomerulonephritis, about half of whom had kidney insufficiency; 24 patients (35%) had glomerulonephritis and CNS involvement; and 6 patients (9%) had CNS manifestations only (Table 1). For 22% (15 of 68) of patients, therapy with Cy came first. Another 78% (53 of 68) patients were treated previously with other therapeutic modalities, but unsuccessfully. Cyclophosphamide was administered at a dose of 10 to 15 mg/kg body weight, depending on creatinine clearance in infusion of physiologic solution, together with 40 mg methylprednisolone. Antiemetics were given before and after each infusion. In addition, extensive hydration with at least 2 L of liquid was performed. The above procedure was repeated monthly during 6 months and, thereafter, a maximum of six more times in intervals of 3 months, depending on the effect achieved after the first 6 months. Between infusions, patients were treated with corticosteroids at a dose of 15 to 30 mg/d, and also with antimalarics. Treatment efficacy was evaluated as positive if clinical, biochemical, and immunoserologic signs of disease activity were absent (complete remission), or biochemical and immunoserologic signs persisted (incomplete remission). Complete remission of disease was achieved in 75% (51 of 68) and incomplete remission in 10% (7 of 68) of patients (Table 2). A positive effect was usually obtained after a third “pulse” of Cy (range 1 to 8). These patients were treated further with low doses of corticosteroids or antimalarics, or in eight cases with azathioprine. Therapy with Cy was ineffective in 15% (10 of 68) of patients. Four patients from this group developed terminal kidney insufficiency. They did not reach remission after 6-month treatment. In such cases, cyclosporine or high doses of human intravenous immunoglobulins were applied. At present, treatment lasting 6 to 24 months

Synergistic anti-tumor activity of a novel immunomodulator, BCH-1393, in combination with cyclophosphamide

N, N-dimethylaminopurine pentoxycarbonyl D-arginine (BCH-1393) is a novel low molecular weight synthetic immunomodulator that has been shown to significantly stimulate cytotoxic T-lymphocyte responses both in vitro and in vivo (Zacharie B, Gagnon L, Attardo G, Connolly TP, St-Denis Y, Penney CL. Synthesis and activity of 6-substituted purine linker amine immunostimulants. J. Med. Chem. 1997;40:2883–94). Prompted by this evidence, we extended evaluation of BCH-1393 for anticancer activity in syngeneic mouse experimental tumor models. Consistent with previous findings, in vitro assessment of BCH-1393 activity demonstrated a significant increase in the CTL responses in the range of 10 −9–10 −5 M. Treatment of mice with four consecutive daily intraperitoneal injections at 25 and 50 mg/kg resulted in a significant increase of the relative percentage of blood CD4+, CD8+, NK and monocyte subsets without any evidence of toxicity. In vivo anti-tumor activity of BCH-1393 was evaluated, either alone or in combination with subtherapeutic doses of cyclophosphamide (Cy), against weakly immunogenic mouse breast carcinoma DA-3 and strongly immunogenic colon adenocarcinoma MC38. Daily intraperitoneal injection of BCH-1393 at 50 mg/kg alone was well tolerated but produced a relatively weak anti-tumor effect in both tumor models. However, a significant inhibition of tumor outgrowth and suppression of established tumor growth was observed when BCH-1393 was administered in combination with subtherapeutic doses of Cy. Combination treatment of 50 mg/kg BCH-1393 with 100 mg/kg Cy (given as single intravenous bolus injection) starting 2 days prior to DA-3 tumor cell inoculation prevented tumor outgrowth in 70–80% of treated mice. In the remaining 20–30% of mice that had developed tumors, a nearly complete (90%) tumor growth inhibition was observed at days 22–24 post tumor implant. In the MC38 tumor model, combination treatment of established tumors with BCH-1393 and Cy (CTX) at 50 mg/kg resulted in a significant delay in tumor growth compared to CTX treatment alone. The observed concomitant anti-tumor activity of BCH-1393 with cyclophosphamide warrants further investigation of this immunomodulator as an adjunctive treatment of cancer.

A within-subjects microdialysis/behavioural study of the role of striatal acetylcholine in D 1-dependent turning

In rats lesioned with 6-hydroxydopamine (6-OHDA) the effect of the noncompetitive N-methyl d-aspartate (NMDA) receptor antagonist, MK-801, the dopamine (DA) D 2 receptor agonist quinpirole and the A 2A adenosine antagonist SCH 58261 was studied on acetylcholine (ACh) release in the lesioned striatum and contralateral turning behaviour stimulated by the administration of the DA D 1 receptor agonist CY 208-243. Administration of CY 208-243 (75, 100 and 200 μg/kg) to 6-OHDA-lesioned rats dose-dependently stimulated ACh release and induced contralateral turning. MK-801 (50 and 100 μg/kg) reduced basal ACh release (max 22%) and did not elicit any turning. MK-801 (50 and 100 μg/kg) potentiated the contralateral turning, but failed to modify the stimulation of ACh release elicited by 100 and 200 μg/kg of CY 208-243. MK-801 (100 μg/kg) prevented the increase in striatal ACh release evoked by the lower dose of CY 208-243 (75 μg/kg) but contralateral turning was not observed. The D 2 receptor agonist quinpirole (30 and 60 μg/kg) elicited low-intensity contralateral turning and decreased basal ACh release. Quinpirole potentiated the D 1-mediated contralateral turning behaviour elicited by CY 208-243 (100 μg/kg), but failed to affect the increase in ACh release elicited by the D 1 agonist. The adenosine A 2A receptor antagonist SCH 58261 (1 mg/kg i.v.) failed per se to elicit contralateral turning behaviour. SCH 58261 potentiated the contraversive turning induced by CY 208-243 but failed to affect the increase of ACh release. The results of the present study indicate that blockade of NMDA receptors by MK-801, stimulation of DA D 2 receptors by quinpirole and blockade of adenosine A 2A receptors by SCH 58261 potentiate the D 1-mediated contralateral turning behaviour in DA denervated rats without affecting the action of the D 1 agonist on ACh release. These observations do not support the hypothesis that the potentiation of D 1-dependent contralateral turning by MK-801, quinpirole or SCH 58261 is mediated by changes in D 1-stimulated release of ACh in the striatum.

Cyclophosphamide metabolism in children with Fanconi's anaemia.

Although patients with Fanconi's anaemia (FA) exhibit a heightened sensitivity to DNA cross-linking agents, modified doses of CY continue to be used in their conditioning prior to BMT. We measured the pharmacokinetics and metabolism of CY in six children with FA using an established high performance thin layer chromatography technique. CY doses ranged between 5 and 20 mg/kg (median 10 mg/kg). The median CY clearance was 0.6 l/h/m2 (range 0.4-1.1 l/h/m2), t1/2 was 8.1 h (range 6.7-9.5 h) and volume of distribution was 0.19 l/kg (range 0.16-0.34 l/kg), respectively. These results contrast with those previously reported from a comparable group of non-FA children in whom the median CY clearance was 3.2 l/h/m2 (range 2-5 l/h/m2) (P = 0.035), t1/2 was 2.4 h (range 2-3.8 h) (P = 0.035) and volume of distribution 0.5 l/kg (range 0.26-0.95 l/kg) (NS). Unlike the control group in whom the presence of inactive metabolites of CY was common, metabolites could not be found in any FA patient. The enhanced sensitivity of children with FA to CY may in part result from altered drug metabolism.

Efficient activation of antitumor immunity by IL-6 gene-modified leukemia vaccine in combination with low dose cyclophosphamide and low dose IL-2

Objective: To investigate the antitumor effect of the IL-6 gene-modified erythroleukemia cells combined with low dose cyclophosphamide (Cy) and low dose IL-2. Methods: Mice inoculated with FBL-3-IL-6 in combination with low dose IL-2 and low dose cyclophosphamide (Cy). Results: Mice received combined therapy of FBL-3-IL-6, IL-2 and Cy developed tumors most slowly and survived much longer when compared with mice in control groups, with 5 out of 8 leukemia-bearing mice being tumor free 100 days after the combined treatment. To further explain the mechanism of the antitumor effects by the combined therapy. It was found that combined therapy with low dose Cy, low dose IL-2 and FBL-3-IL-6 achieved maximal cytotoxic effects of nature killer cells and specific cytotoxic T lymphocytes, increased production IL-2, TNF and GM-CSF from spleen lymphocytes in tumor-bearing mice. Vaccination with the FBL3-IL-6 also enhanced the cytotoxic activity of the peritoneal macrophages. The results demonstrated that administration of low dose Cy and low dose IL-2 in combination with IL-6 genemodified leukemia vaccine could elicit potent antileukemia effects, and the mechanisms involved in the antitumor process may include the induction of specific and nonspecific antitumor immunity, reversal of T suppressor cells that mediated local immuno-suppression in tumor bearing mice. Conclusion: The combined therapy with cytokine gene-modified tumor vaccine, low dose of Cy and IL-2 might be a promising approach for the treatment of leukemia.

MAINTENANCE IMMUNOSUPPRESSION WITH CYCLOSPORINE MONOTHERAPY IN LIVER TRANSPLANT ADULT RECIPIENTS

181 Introduction: Cyclosporine (Cy) monotherapy as maintenance immunosuppression is rarely attempted after liver transplantation (LT). We reduce immunosuppression after LT and attempt Cy monotherapy in all patients. Methods: We reviewed our long term results in a cohort of LT patients on CsA monotherapy for at least 1 or more years. All patients were induced with rabbit-ATG, Azathioprine (AZA), Prednisone (P), and Cy. AZA was withdrawn at a median of 1.6mos (range 1 week - 18mos) post LT. Prednisone was withdrawn at a median of 15.5 mos (range 2 - 37mos). The median follow-up on Cy monotherapy is 30 mos (range 7 - 58 mos). Results: Presently 51 of 59 patients (86.4%) with greater than 2yrs follow-up after LT and not on other immunosuppressive protocols are on Cy monotherapy. Three of 51 pts (6%) experienced a total of 4 rejection episodes of which three were steroid sensitive. Two of these three patients are back on daily prednisone. Three patients died, one each from recurrent hepatocellular carcinoma, hepatitis C (HCV), and reactivation of HBV. Median serum glucose and cholesterol improved on Cy monotherapy from 6.2 to 5.1 and 6.6 to 4.9 mmol/L respectively (P< 0.05). In 9 of 12 pts diabetes control improved on Cy monotherapy. Liver enzymes and bilirubin (TB) improved on Cy monotherapy and this was most pronounced in HCV positive pts (ALT from 119 to 44 IU, p < 0.01; TB from 21 to 14 mmol/L, p <0.05). Cy dose after start of Cy monotherapy has actually been reduced in most patients from a median daily dose of 275 mg/d to 175mg/d. Conclusion: Cy monotherapy is achievable and highly desirable in most LT adult recipients with minimal risk of rejection, significant improvements in serum cholesterol glucose and especially liver biochemistry most notably in HCV patients.

Modulation of the Antimetastatic Effect of a Single Low Dose of Cyclophosphamide on Rat Lymphoma

The aim of this paper was to identify the mechanism/s responsible of the antimetastatic effect of a single low dose of cyclophosphamide (Cy), previously demonstrated by us in the rat lymphoma L-TACB. No direct cytotoxic antimetastatic activity of Cy could be proved. In vitro treatment of L-TACB cells with mafosfamide did not alter their invasiveness or their motility. The adoptive transfer of splenocytes from Cy-treated tumor-bearing rats, together with L-TACB cells inhibited their metastatic growth. The single low dose Cy treatment of T-immunodeficient nude mice did not show the antimetastatic effect on L-TACB observed in immunocompetent mice. An inhibition of the metastatic ability due to immunomodulation by Cy is proposed.

Conditioning with cyclophosphamide/antithymocyte globulin for allogeneic bone marrow transplantation from HLA-matched siblings in children with severe aplastic anemia.

Graft rejection has been a problem after bone marrow transplantation for patients with severe aplastic anemia (SAA). Ten children with SAA were conditioned for bone marrow transplantation from HLA-identical siblings, using cyclophosphamide (CY, 50 mg/kg) plus antithymocyte globulin (ATG, 15 mg/kg) for 4 successive days. Marrow was infused 36 h after the last dose of CY. Cyclosporin A and methotrexate were administered as graft-versus-host disease (GVHD) prophylaxis. All patients achieved durable engraftment at follow-up of 7-41+ months (mean, 25) without significant GVHD. Since investigators have used different sources, doses, and time schedules of ATG, we compared our results with other published reports. We conclude that CY/ATG conditioning is well tolerated and effective in children with SAA.

Adoptive Transfer of Anti-CD3–Activated CD4+ T Cells Plus Cyclophosphamide and Liposome-Encapsulated Interleukin-2 Cure Murine MC-38 and 3LL Tumors and Establish Tumor-Specific Immunity

AbstractThe infusion of anti-CD3–activated murine T cells plus interleukin-2 (IL-2) exerts antitumor effects against several tumors in murine immunotherapy models. This study compares the therapeutic efficacy of anti-CD3–activated CD4+ or CD8+ T-cell subsets, when given with cyclophosphamide (Cy) and liposome-encapsulated IL-2 (L-IL2) in a murine model. C57BL/6 mice bearing subcutaneous (SC) MC-38 colon adenocarcinoma, 3LL Lewis lung carcinoma, or 38C13 lymphoma for 7 to 14 days were pretreated with low-dose intraperitoneal (IP) Cy before intravenous (IV) injection of anti-CD3–activated T cells or T-cell subsets. Cell administration was followed by IP administration of L-IL2 for 5 days. Mice receiving activated CD4+ T cells showed significantly reduced tumor growth or complete remissions with prolonged disease-free survival in MC-38, 3LL, and 38C13. The timing of Cy doses in relation to adoptive transfer was critical in obtaining the optimal antitumor effect by CD4+ cells. Injecting Cy 4 days before the infusion of CD4+ cells greatly enhanced the antitumor effect of the CD4+ cells and improved survival of the mice compared with other Cy regimens. C57BL/6 mice cured of MC-38 after treatment with CD4+ T cells developed tumor-type immunologic memory as demonstrated by their ability to reject rechallenges with MC-38, but not 3LL. Similarly, mice cured of 3LL tumors rejected rechallenges of 3LL, but not MC-38. The immunologic memory could be transferred with an IV injection of splenocytes from mice cured of MC-38 or 3LL. No cytotoxic T-lymphocyte activity was detected in T cells or T-cell subsets from mice cured of MC-38 or 3LL. Increased IL-2 and interferon-γ (IFN-γ) production was observed from CD4+ subsets in cured animals when stimulated in vitro with the original tumor, but not with an unrelated syngeneic tumor. These results suggest that tumor-specific immunity can be achieved in vivo with anti-CD3–stimulated CD4+ T cells in this cellular therapy model.