Clinical use of recombinant human hematopoietic growth factors (GM-CSF, IL-3, EPO) in patients with myelodysplastic syndrome.

We have conducted several phase I/II clinical studies in a total of 65 MDS patients utilizing recombinant human hematopoietic growth factors including GM-CSF, IL-3, and EPO. Twenty-seven patients with MDS were treated with either continuous i.v. infusion or single daily s.c. injection of rhGM-CSF at dosages from 15 micrograms/m2 to 1000 micrograms/m2. All of them exhibited white cell responses during the treatment cycles, but no sustained rise in reticulocytes or platelets was recorded. In four of the patients, all with > or = 15% blast cells in the bone marrow, the percentage of circulating blast cells increased during treatment with rhGM-CSF (at dosages of 500 micrograms/m2 and 1000 micrograms/m2, respectively), although no leukemic conversion occurred. Of 9 patients treated so far with rhIL-3 at single daily s.c. dosages of 60 micrograms/m2, all exhibited white cell responses; 8 exhibited significant improved platelet and reticulocyte counts. Nineteen further patients received rhEPO for a period of 14 weeks by s.c. (10,000 U five times weekly) or i.v. bolus administration (150-450 U/kg). None of these patients experienced an increase in white cell and platelet counts. A significant increase of the reticulocyte count was recorded in 3 patients only. Another strategy involves the recruitment of leukemic cells into the cell cycle by hematopoietic growth factors followed by treatment with cycle-specific cytostatic agents. Therefore in 10 patients administration of rhGM-CSF (250 g/m2/day x 14, s.c.) was combined with Ara-C treatment (20 mg/m2/day x 14; s.c.). Initial results of this pilot study available in 5 patients indicated that this approach may control leukemic cell proliferation and may increase number of mature myeloid cells in both bone marrow and peripheral blood. A similar approach utilizing rhIL-3 in conjunction with Ara-C is on-going.     

Immunologic effects of AS101 in the treatment of cancer patients

AS101 [ammonium trichloro(O,O'-dioxyethylene)tellurate] is a new immunomodulator previously shown to stimulate the production of different cytokines in vitro and in vivo. We report here our results of a phase I clinical trial conducted on 47 cancer patients with advanced malignancies. AS101 was administered intravenously at escalating doses from 1 to 10 mg/m2, twice or thrice a week. The maximal tolerated dose has not yet been determined. However, significant immunologic responses were noted at dose levels of 1-3 mg/m2 administered three times a week. At these doses statistically significant rises in gamma-interferon, natural killer cell activity, tumor necrosis factor and interleukin-2 (IL-2) levels as well as the expression of IL-2 receptors were noted. In most of the immunologic parameters the maximal response was seen at 3 mg/m2. Throughout the study toxicity was minimal. In view of these results phase II studies are currently being initiated.     

A new paradigm for the treatment of high-risk prostate cancer: radiosensitization with docetaxel

Survival and biochemical outcome of patients with localized, high-risk prostate cancer treated with definitive three-dimensional conformal radiation therapy (3-D CRT) with or without hormonal therapy are poor. Other therapeutic strategies are needed to improve outcome in these poor-prognostic-group patients. One such strategy involves the use of chemotherapeutic agents to radiosensitize the effects of local 3-D CRT. Very few investigators have tested this novel concept of chemotherapeutic radiosensitization. Two studies evaluated the combination of estramustine phosphate and vinblastine (EV) with radiation therapy (RT). In both studies, the combination of EV and RT resulted in moderate to severe acute and late toxicity. A recently completed, phase I trial evaluated the maximally tolerated dose (MTD) of weekly docetaxel that could be concurrently delivered with 3-D CRT (70.2 Gy) in men with high-risk prostate cancer. The MTD of concurrent weekly docetaxel with 3-D CRT was determined to be 20 mg/m(2), and this combination was shown to be safe and well tolerated. This was the first trial to evaluate taxane radiosensitization in prostate cancer. Other phase I/II studies are needed to further assess chemotherapeutic radiosensitization in localized, high-risk prostate cancer.     

In vitro and in vivo action of recombinant human GM-CSF (rhGM-CSF) in patients with myelodysplastic syndromes

The availability of rhGM-CSF has allowed the in vivo treatment of patients with cytopenia. Therefore, a phase I-II trial was initiated to study the effect of rhGM-CSF in patients with myelodysplastic syndromes who were not eligible for other kinds of therapy. rhGM-CSF has been tested in 10 patients in doses from 15 micrograms/m2 to 150 micrograms/m2 given intravenously over 8 hours for a cycle of 7 days followed by an interval of 14 days and a second 7-day treatment course. A dose-dependent increase in leukocyte count was observed in 9 of 10 patients. No change in reticulocyte numbers was seen and only one patient experienced an increase in platelet count. Toxicity mainly consisted of mild phlebitis at the site of infusion and sternal pain after bolus injection. An increase in blast cell counts in some patients necessitated the start of low-dose Ara-C therapy.     

Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in human plasma. An Oasis((R)) HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 microm, 120A, [Formula: see text] mm) column. The initial composition of the mobile phase was 2% methanol/98% 5mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3min, followed by a linear gradient over 4 min to 30% methanol/70% 5mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5'-deoxy-5-fluorouridine (5'-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC-H](-), m/z 263 for [dFdU-H](-), and m/z 245 for [5'-DFUR-H](-). The average recoveries for dFdC, dFdU, and 5'-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5-1000 ng/ml for dFdC, and 5-5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were 相似文献   

Interaction of recombinant IL-1 and recombinant tumor necrosis factor in the induction of mouse acute phase proteins

Recombinant mouse and human IL-1 (alpha and beta forms), as well as rTNF-alpha when administered in vivo, induced the production of the mouse acute phase reactants: serum amyloid P-component (SAP), C3, and fibrinogen. The SAP response to all three rIL-1 proteins reached a maximum at a dose of 10(4) U/mouse, which corresponds to 1 to 10 micrograms of protein. The maximum in vivo response consisted of a 10-fold increase in SAP levels, a 2-fold increase in C3 levels, and a 3-fold increase in fibrinogen concentration. By contrast, rTNF-alpha induced a much smaller acute phase (AP) protein response (4-fold increase in SAP) when administered in vivo. Administration of a combination if rIL-1 and rTNF resulted in an AP response that was additive for SAP, synergistic for fibrinogen, but resulted in only the same amount of C3 induced by IL-1 alone. Both recombinant monokines induced new SAP synthesis by isolated hepatocytes in vitro with an optimal response occurring with either 1 U of rIL-1/ml per 2 x 10(5) hepatocytes or 10(-3) U/ml of rTNF. The hepatocyte response to IL-1 was of the same magnitude as the response of intact mice; however, the response to TNF was approximately 10(4) times more efficient in vitro. A mixture of the monokines induced an in vitro SAP response that was additive when suboptimal doses of rIL-1 were combined with optimal amounts of rTNF-alpha. Overall, the findings indicate that both monokines directly trigger hepatocyte synthesis of SAP and that their combined effect probably accounts for a substantial portion of the synthesis of these AP proteins in mice.     

Administration of recombinant bovine interferon-alpha I at the time of maternal recognition of pregnancy inhibits prostaglandin F2 alpha secretion and causes luteal maintenance in cyclic ewes.

The antiluteolytic protein, ovine trophoblast protein-1, which is secreted by sheep embryos at about the time of the maternal recognition of pregnancy, exhibits significant structural homology with alpha interferons. Experiments were conducted to examine the effects of intra-uterine and systemic administration of a recombinant bovine interferon-alpha I (rboIFN-alpha I) upon the interoestrus interval, endometrial oxytocin receptor concentrations and secretion of prostaglandin (PG) F2 alpha in cyclic ewes. In Expt 1, each ewe had a cannula placed in the tip of a uterine horn ipsilateral to a corpus luteum, 7 days after an induced oestrus. From day 9 after oestrus until day 19, ewes received either 200 (n = 4), 667 (n = 5) or 2000 (n = 9) micrograms/24 h of rboIFN-alpha I, meclofenamic acid (n = 4) or vehicle (n = 11). Other ewes received 2000 micrograms rboIFN-alpha I/24 h (n = 5) between days 12 and 15 only. All ewes were killed on day 19. Mean luteal phase, as determined by daily plasma progesterone measurements, was significantly longer (P less than 0.01) and mean concentrations of 13,14-dihydro-15-keto PGF 2 alpha (PGFM) in plasma were lower (P less than 0.05) in ewes receiving 667 or 2000 micrograms rboIFN-alpha I between days 9 and 19, or 2000 micrograms between days 12 and 15, than in animals from other treatment or control groups. A similar protocol was used in Expt 2, in which further ewes received either 2000 micrograms rboIFN-alpha I/24 h (n = 5) or vehicle (n = 5) by bolus infusions twice a day into one uterine horn. Mean luteal phase was significantly (P less than 0.05) longer in treated than in control animals, but differences in PGFM concentrations were not significant. In Expt 3, after a synchronized oestrus, ewes received either 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 12 and 15 (n = 10), 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 9 and 15 (n = 11), i.m. injection of vehicle alone twice a day (n = 20), or continual intra-uterine infusion of 2 mg rboIFN-alpha I/day between days 12 and 15 (n = 7). The mean luteal phase of ewes receiving rboIFN-alpha I by intrauterine infusion or i.m. injection between days 9 and 15 was significantly longer than for animals from the other two groups (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

Unique histological changes in lung metastases of osteosarcoma patients following therapy with liposomal muramyl tripeptide (CGP 19835A lipid)

Summary We have recently begun a phase II trial in patients with osteosarcoma who developed pulmonary metastases during adjuvant chemotherapy or who presented with pulmonary metastases that persisted despite chemotherapy. Eligible patients were rendered free of visible disease by surgery. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (MTP-PE, CGP 19835A lipid) (2 mg/m²) was infused twice weekly for 3 months. In five patients, a single tumor nodule recurred within 6 weeks after completion of therapy. These lesions were resected and submitted for pathological examination. Tissue specimens obtained after therapy were compared to those obtained before therapy. All the patients showed a histological change in the characteristics of the pulmonary tumors. In three patients, peripheral fibrosis surrounded the tumor and inflammatory cell infiltration and neovascularization were present. This is in contrast to central necrosis, with viable peripheral tumor cells and no inflammatory response observed in lesions resected following chemotherapy. In a fourth case, evidence of early fibrotic changes was found. This and the fifth case showed a change in malignant characteristics, from high grade before liposomal therapy to low grade after therapy. The present study provides evidence for a biological effect of liposomal MTP-PE.     

Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.     

Experiences with continuous intraarterial administration of recombinant interferon alpha-2C (rIFN-alpha 2) for treatment of patients with advanced transitional cell bladder cancer

The response of locally-advanced transitional cell carcinoma of the urinary bladder to intraarterial regional treatment with recombinant interferon alpha-2C (rIFN-alpha 2) was investigated. Patients received 4-weekly courses of i.a. continuous infusion of rIFN-alpha 2 (5 megaunits/24 hours administered into the a. iliaca interna via a subcutaneously implanted port-a-cath) alternating with 4 weeks i.m. treatment cycles (5 megaunits/day x 3 weekly). None of the 5 patients entered in the study showed evidence of objective tumor regression after a median of 4 i.a./i.m. treatment cycles. Constitutional, organ and local toxic effects were considerable even with that low-dosage schedule employed. The lack of efficacy and poor tolerance of the regimen suggest that i.a. administration of IFN is unlikely to yield an acceptable therapeutic index.     

Phase I Trial of Radiotherapy Concurrent with Twice-Weekly Gemcitabine for Head and Neck Cancer: Translation From Preclinical Investigations Aiming to Improve the Therapeutic Ratio

BACKGROUND: Once-weekly gemcitabine concurrent with radiotherapy was highly effective in the treatment of head and neck cancer (HNC) but limited by high mucosal toxicity. Pre-clinical investigations suggested that delivering gemcitabine at substantially lower doses twice weekly during radiotherapy improved the therapeutic ratio. We sought to translated these preclinical findings to a phase I trial. METHODS: Twenty-five patients with non-resectable HNC were scheduled to receive gemcitabine twice weekly during the last 2 weeks (total 5 infusions) of hyperfractionated radiotherapy delivering 1.2 Gy twice daily to total 76.8 Gy. Tumor biopsies to measure active intracellular (phosphorylated) gemcitabine were planned after the first drug delivery. Patients were assigned to escalating dose cohorts using the Continuous Reassessment Method. RESULTS: Twenty-one patients evaluable for toxicity were divided into cohorts receiving twice weekly treatment with 10, 20, 33, or 50 mg/m² gemcitabine. Dose-limiting toxicity was grade 3-4 confluent mucositis/pharyngitis, and the maximally tolerated dose (MTD) was 20 mg/m². Median survival was 20 months, with no difference between cohorts receiving lower (10, 20 mg/m²) or higher (33, 50 mg/m²) gemcitabine doses. Tumor biopsies after the first drug delivery showed only a minority of tumor cells in the specimens. CONCLUSION: These findings validate preclinical models that show that gemcitabine is radiation sensitizer at doses far below those used for systemic chemotherapy. However, the improvement in the therapeutic ratio predicted from the preclinical study did not translate into a substantial relative increase in the MTD of the drug in the clinical phase I trial.     

Elaboration of the combination of antitumor preparations bleomycetin + 5-fluorouracil + cisplatin

Clinical trials on efficacy and toxicity of combined use of bleomycetin, 5-fluorouracil and cisplatin in patients with disseminated tumor processes were conducted. Two regimens were applied. Regimen I included intravenous administration of cisplatin in a dose of 100-150 mg/m2 on day 1, intramuscular administration of bleomycetin in a dose of 10 mg on days 2-4 and intravenous jet injection of 5-fluorouracil in a dose of 400 mg/m2 on days 2-4. Regimen II consisted of intramuscular administration of bleomycetin in a dose of 10 mg on days 1-3, intravenous jet injection of 5-fluorouracil in a dose of 400 mg/m2 on the same days and intravenous administration of cisplatin in a dose of 100-150 mg/m2 on day 4. The intervals between the courses amounted to 4 weeks. Complete regression of cervical carcinoma relapsing was observed in 1 patient. In 5 patients i.e. 1 with small-cell lung cancer, 3 with squamous cell lung cancer and 1 with metastases of low-differentiated cancer from an undetected focus to supraclavicular lymph nodes the effect was partial. Long-term stabilization of the disease at the background of the treatment for 6-7 months was stated in 3 patients. On the whole the objective response was in 6 out of 22 patients or in 27 per cent. 7 of them were treated with cisplatin in a dose of 150 mg/m2. The regimens of the combined use of 5-fluorouracil, bleomycetin and cisplatin were low toxic. The therapeutic effect showed that the combination was of practical value.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.

Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.     

Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial

Dendritic cells (DC) derived from peripheral blood monocytes are currently being investigated in clinical trials for their role in stimulating the immune system. We performed a phase I/II clinical trial using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. After liposomal transfection, flow cytometry testing showed that 2% to 53% of the DC expressed mucin epitopes. Patients were immunized two or three times with 1 million transfected DC injected subcutaneously (s.c.). A vaccine-specific delayed-type hypersensitivity (DTH) reaction was observed in 3 out of 10 patients. After vaccination, 4 patients showed a 2- to 10-fold increase in the frequency of mucin-specific interferon-gamma (IFN-gamma)-secreting CD8+ T cells. We demonstrated the feasibility and safety of a vaccine consisting of autologous gene-transfected DC, and that immunologic responses could be induced even in patients with pretreated and advanced disease.     

A phase I/II trial of oxidized autologous tumor vaccines during the “watch and wait” phase of chronic lymphocytic leukemia

Based on their activity in patients with advanced stage chronic lymphocytic leukemia (CLL), a phase I/II study was designed to evaluate the feasibility, safety, and efficacy of autologous vaccines made from oxidized tumor cells in patients with earlier stage CLL, and to determine an optimal schedule of injections. Eighteen patients (at risk for disease progression and with white blood cell counts between 15 and 100×10⁶ cells/ml) were injected intramuscularly with 10 ml of oxidized autologous blood (composed mainly of CLL cells) either 12 times over 6 weeks (group 1), 12 times over 16 days (group 2), or 4 times over 6 weeks (group 3). Fourteen out of eighteen patients had Rai stage 0–II disease, while 4/18 had stage III–IV disease but did not require conventional treatment. Partial clinical responses, associated with enhanced anti-tumor T cell activity in vitro, were observed in 5/18 patients of whom three were in group 2. Stable disease was observed in six patients while disease progression appeared not to be affected in the remaining patients. Toxicity was minimal. Vaccination with oxidized autologous tumor cells appears worthy of further investigation and may be a potential alternative to a watch and wait strategy for selected CLL patients.     

A phase I/II study of recombinant tumor necrosis factor and recombinant interferon gamma in patients with AIDS-related complex

A clinical trial was conducted to determine the tolerance and toxicity of recombinant tumor necrosis factor (rTNF) and recombinant interferon gamma (rIFN-gamma) when administered concurrently by continuous intravenous infusion to 11 patients with the AIDS-related complex (ARC). In addition, HIV culture, p24 antigen levels, and CD4 positive lymphocytes were monitored to obtain preliminary evidence of antiviral and immunologic effects. Two 5-day treatment cycles were separated by a 9-day washout period. Two patients were entered at each dosage level and each patient received the two 5-day treatment cycles at two sequential dose levels ranging from 1 to 25 micrograms/m2. Two patients did not complete their second treatment cycle--one due to the development of a rash, the second due to central venous catheter discomfort. The occurrence of phlebitis with peripheral vein administration of these agents necessitated administration via central venous catheter. With the exception of a single patient who developed severe headache at the 25 micrograms/m2 dose, severe clinical toxicities were not observed. Fever, chills, headache, and myalgias were the most significant clinical toxicities observed and all were dose dependent. The percentage fall in total granulocytes was dose dependent and ranged from 17% at the 1 microgram/mm2 dose to 48% at both the 15 and 25 micrograms/mm2 dose levels. The mean nadir granulocyte count was 1694/mm3. No significant renal or hepatic toxicity was observed. Of 22 treatment cycles the CD4 cell number was increased in 11, unchanged in 7, and decreased in 4. The mean CD4 cell number did not change significantly (176 +/- 143/mm3 pretherapy versus 279 +/- 305/mm3 posttherapy).(ABSTRACT TRUNCATED AT 250 WORDS)     

A glycoconjugate from corms of saffron plant (Crocus sativus L.) inhibits root growth and affects in vitro cell viability

A glycoconjugate has been characterized from saffron corms (Crocus sativus L.) that inhibits the growth of roots of Nicotiana tabacum and Arabidopsis thaliana, at concentrations ranging from 1-100 micrograms m-3. Roots of seedlings grown in the presence 0.1 microgram m-3 glycoconjugate showed bulging of epidermal cells, whereas at 10 micrograms m-3, roots were completely devoid of hairs. At 100 micrograms m-3 glycoconjugate the cell walls of the root vascular tissues were thicker and, overall, the vascular tissue was enlarged. In addition, this glycan is cytotoxic to isolated tobacco cells and protoplasts, with 50% cell death induced by 0.5 and 2 micrograms m-3 glycoconjugate, respectively. Morphological and biochemical changes induced by the exposure to the glycoconjugate included cell size decrease, loss of regular cell shape, cytoplasm collapse, and release of intracellular proteins. This molecule at low concentrations (0.1 microgram m-3) mimics the effects of Yariv phenylglycosides and of mutant Arabidopsis which present defective or missing arabinogalactan-proteins (AGPs) in roots, indicating the glycoconjugate might interact with cell surface AGPs.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.