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Purpose: The targeted cytotoxic somatostatin analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier octapeptide RC-121, is scheduled for clinical trials. To extend previous findings we tested AN-238 on human androgen sensitive MDA-PCa-2b prostate cancers grown subcutaneously and androgen independent LNCaP derived C4-2 prostate cancers xenografted into the tibiae of nude mice., Materials and Methods: Changes in serum prostate specific antigen (PSA) levels were monitored by radioimmunoassay. Somatostatin receptors in tumor samples were characterized., Results: Three intravenous injections of AN-238 at 150 nmol/kg doses inhibited the growth of subcutaneous MDA-PCa-2b tumors by 62% vs controls (p <0.05) and were more effective than equimolar doses of the radical AN-201 (p <0.05). AN-238 also decreased serum PSA levels by 62% vs controls (p <0.01). In nude mice bearing intra-osseous implanted C4-2 prostate cancers AN-238 decreased serum PSA levels by 65% compared with controls after 5 weeks of therapy (p <0.05), while AN-201 was ineffective. All AN-238 treated mice were alive at the termination of the experiment, while only 50% of controls and 60% of animals treated with AN-201 survived (p <0.01). Histological evaluation of intraosseous C4-2 tumors showed that AN-238 induced a significant increase in apoptosis (p <0.05). MDA-PCa-2b and C4-2 tumors showed high affinity binding for somatostatin and the expression of mRNA for somatostatin receptor subtypes 1, 2A and 5., Conclusions: The current study demonstrates the efficacy of the somatostatin analogue AN-238 for subcutaneous MDA-PCa-2b as well as for intraosseous C4-2 prostate cancers xenografted into nude mice. This targeted cytotoxic analogue could represent a new therapy for patients with advanced metastatic prostate carcinoma.

A restricted receptor for interleukin 13 (IL-13R alpha2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R alpha2, we established an IL-13R alpha2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R alpha2. G-26-IL-13R alpha2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R alpha2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R alpha2 in vivo. These characteristics of the G-26-IL-13R alpha2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R alpha2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R alpha2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R alpha2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R alpha2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

There is no consensus on treatment of locally advanced or metastatic breast cancer after failure of first-line cytotoxic chemotherapy. Common options are continuous infusion of a taxane (docetaxel or paclitaxel), vinorelbine or fluorouracil. Capecitabine is now licensed for use in breast cancer, both in combination with IV docetaxel after anthracycline failure, and as single-agent therapy after failure of anthracyclines and taxanes. The clinical evaluation dossier on capecitabine fails to answer the most important questions about comparative efficacy and safety. In second-line treatment, after anthracycline failure, the only available comparative trial showed that the capecitabine + docetaxel combination increased median survival time by about three months relative to placebo + docetaxel, but caused more adverse events. There are no trials comparing capecitabine with other options. There is no evidence that capecitabine increases the length or quality of survival, relative to intravenous vinorelbine, in women with breast cancer that is resistant to both anthracyclines and taxanes. The classical adverse effects of capecitabine are also observed in women with breast cancer, namely palmoplantar erythrodysesthesia, diarrhoea, nausea and vomiting, and major hyperbilirubinemia. Capecitabine can be taken by mouth and this may be an advantage. However, current evidence is too limited to justify using capecitabine outside of clinical trials.

This audit was designed to assess whether existing UK vaccination programmes mean that patients receiving chemotherapy are being vaccinated against influenza. One hundred and ten adult patients receiving chemotherapy at a south London tertiary referral centre were interviewed when they attended for their chemotherapy. Thirty-six of the 110 (33%) patients had received their influenza vaccination at the time of the study. Vaccination rates were significantly higher in those patients older than 65 years (53% vs 17%, p<0.001), and in those with co-morbidities (49% vs 25%, p<0.05). The vaccination rate in this at-risk population is lower than the overall national uptake in those aged 65 and over. Those patients most likely not to receive their influenza vaccination are those who have no indication for vaccination other than the fact they are receiving chemotherapy. Increased awareness of the benefits of influenza vaccine and its safety is needed among general practitioners, patients and oncologists.

The accumulation of radioactive somatostatin analog [111In]pentetreotide in non-small cell lung cancer (non-SCLC) during scintigraphy of patients provides a rationale for investigating the efficacy of somatostatin receptor-based chemotherapy in non-SCLC. Consequently, in this study, we evaluated the antitumor effects of cytotoxic somatostatin analog AN-238 on H838 human non-SCLC xenografted into nude mice in comparison with its cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201). The expression of messenger RNA (mRNA) for human somatostatin receptor subtypes 2 (hsst2) and 5 (hsst5) in H838 cells, and tumors was also investigated using reverse-transcription polymerase chain reaction (RT-PCR). Somatostatin receptors on H838 tumors were characterized by ligand competition assay using radiolabeled somatostatin analog, RC-160. Three i.v. injections of AN-238 at 150 nmol/kg, given on days 1, 7 and 21, resulted in a significant (p<0.05) tumor growth inhibition, the final tumor volume being 60% smaller than in the controls. The tumor doubling time was also extended significantly (p<0.05) from 9.65+/-0.56 days in the controls to 17.52+/-3.3 days. Only one of 8 mice died due to toxicity. In contrast, cytotoxic radical AN-201 was ineffective and more toxic, killing 2 of 7 animals. mRNA for hsst2 was found in H838 xenografts, but not in H838 cells from which the xenografts originated. Interestingly, H838 cells grown in a special, serum-free medium did express mRNA for hsst2. mRNA for hsst5 was not found in any samples tested. Binding studies demonstrated the presence of high affinity (K(d) = 7.3+/-1.2 nM) binding sites for RC-160 with a mean maximal binding capacity (B(max)) of 953.3+/-45.3 fmol/mg protein. AN-238 at 3.14+/-0.93 nM concentration displaced 50% of radiolabeled RC-160 binding to somatostatin receptors in H838 tumors. Our results indicate that patients with inoperable non-SCLC may benefit from chemotherapy targeted to somatostatin receptors based on AN-238.

The development of targeted cytotoxic analogs of hypothalamic peptides for the therapy of various cancers is reviewed and various oncological studies on experimental tumors are summarized. Novel therapeutic modalities for breast, prostate and ovarian cancer consist of the use of targeted cytotoxic analogs of LH-RH containing doxorubicin (DOX) or 2-pyrrolino-DOX. The same radicals have been incorporated into cytotoxic analogs of somatostatin which can be also targeted to receptors for this peptide in prostatic, mammary, ovarian, renal and lung cancers, brain tumors and their metastases. A targeted cytotoxic analog of bombesin containing 2-pyrrolino-DOX has also been synthesized and successfully tried in experimental models of prostate cancer, small cell lung carcinoma and brain tumors. The development of these new classes of peptide analogs should lead to a more effective treatment for various cancers.

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.

Advances in immunoconjugate technology have revitalized the "magic bullet" concept of immunotherapeutics for the treatment of cancer. The growing availability of "human" antibodies, the increased epitope repertoire due to genomics and proteomics efforts, and advances in the means of identification and production of tumor-specific antibodies have greatly increased the potential for cancer therapeutic opportunities. Furthermore, the realization that effector molecule potency must be sufficiently high to be effective at concentrations that might realistically be delivered to the tumor site on an antibody carrier has greatly spurred the fields of medicinal chemistry and radionuclide chelate chemistry to produce such molecules.

We evaluated the effectiveness of targeted cytotoxic analog of somatostatin (SST) AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked covalently to SST octapeptide carrier RC-121 in DU-145 human androgen-independent prostate cancers xenografted into nude mice. We also investigated the expression of mRNAs for SST receptor subtypes 2A and 5 (sst2A and sst5) in DU-145 tumors. After 8 weeks of treatment, AN-238 practically arrested the proliferation of DU-145 cancers. The tumor volume in nude mice that received 4 injections of AN-238 at the dose of 150 nmol/kg was 63.4+/-6.7 mm3, nearly 4 times smaller than that in controls which measured 249.1+/-36.3 mm3 (p<0.001). Treatment with AN-238 lowered tumor weight by 68% (p<0.01) compared with the control group and extended the tumor volume doubling time to 184.1+/-69.4 days, versus 32.1+/-6.6 days in controls (p<0.05). No toxicity-related deaths occurred during treatment with AN-238. Cytotoxic radical AN-201 administered alone or in an unconjugated mixture with carrier RC-121 inhibited the growth of DU-145 tumors only after the third and fourth injection and was toxic. The expression of mRNA for sst2A and sst5 was detected in all specimens of control DU-145 tumors and in tumors treated with AN-238. The present study demonstrates the high efficacy of SST-receptor-targeted chemotherapy in a model of human androgen-independent prostatic carcinoma.

Receptors for interleukin 4 (IL-4R) are overexpressed on the surface of various human solid tumors including renal cell carcinoma, glioblastoma, Kaposi's sarcoma, and head and neck squamous cell carcinoma (SCCHN). On the basis of this preferential receptor overexpression, a novel IL-4R-targeted cytotoxin, IL-4 (38-37)-PE38KDEL, was developed in which circularly permuted IL-4 [IL-4 (38-37)] was fused to mutated form of Pseudomonas exotoxin (PE38KDEL). Despite the recognized expression of the IL-4R on SSCHN, the utility of a receptor-specific fusion protein for the treatment of this disease remains unknown. The purpose of this study was to establish the utility of IL-4 (38-37)-PE38KDEL for the treatment of established SSCHN in animal models of human disease. Expression of IL-4R in SCCHN was confirmed by immunohistochemistry with eight of eight tissue sections expressing IL-4R. Protein synthesis inhibition assays demonstrated growth inhibition of two cell lines in IL-4 (38-37)-PE38KDEL in a dose-dependent fashion. In two SCCHN s.c. xenografted nude mouse models, i.p. and intratumoral injection of IL-4 (38-37)-PE38KDEL mediated tumor regression with no visual toxicity observed in any of the animals. Subcultured tumor cells after intratumoral treatment with IL-4 toxin did not develop resistance to the drug. These data demonstrate that IL-4 (38-37)-PE38KDEL is effective in mediating significant antitumor effects in SCCHN and may represent an attractive therapeutic option for patients with advanced cancers of the upper aerodigestive tract.

Thrombotic thrombocytopenic purpura (TTP) is a rare and occasionally fatal haematologic disorder that can coexist with systemic lupus erythematosus (SLE) and other autoimmune diseases. We identified all cases of TTP seen in our institution over a 3 year period using a computerized database. We found that SLE activity (measured by the SLE Disease Activity Index) and TTP activity ran a parallel course in three patients with coexistent SLE and TTP. TTP in these three patients, although refractory to plasmapheresis, responded to cytotoxic therapy. These observations further support an autoimmune contribution to the pathogenesis of some cases of TTP. A literature review revealed that mortality in SLE patients with more severe, refractory TTP treated with plasmapheresis and cytotoxics, may not be higher than in patients responding to plasmapheresis alone (who are likely to have milder disease). These data suggest that cytotoxics may have a role in treatment of patients with active SLE and TTP refractory to plasmapheresis.

In the past two decades, cytogenetic and molecular genetic investigations have revealed that sarcomas are frequently characterized by specific chromosomal translocations, often involving genes encoding transcription factors. These translocations result in the expression of chimeric oncoproteins that contain functional domains contributed by each of the parental genes. Functioning as deregulated transcription and signaling factors, these novel proteins contribute to the malignant phenotype of the tumor cell by disrupting the tightly regulated process of target gene expression. Several therapeutic strategies that exploit the tumor-specific nature of these oncogenes are currently being investigated. These targeted approaches seek to manipulate the specific biology of these gene fusions, and along with more traditional therapeutic modalities, could augment current approaches to cancer management.

Combined radioimmunotherapy (RAIT) and hypoxic cytotoxin therapy (SR4233 or NLCQ-1) have been evaluated with both modalities administered on the same day with only moderate improvement compared with the effects of RAIT alone. In a series of studies using oxygen electrodes, immunohistochemistry and radiotracers, we have demonstrated that RAIT induces a prolonged state of hypoxia in most tumors, without affecting the pO(2) levels in normal tissues. Using serial microelectrode measurements through subcutaneous (s.c.) GW-39 human colonic xenografts, we established that the median pO(2) was unrelated to the initial size of the tumor, over a range of sizes from 1.0 to 4.0 cm. Fourteen days after mice were given a 240-microCi dose of (131)I-MN-14 anti-carcinoembryonic antigen immunoglobulin G, their median pO(2) declined from 26.1 +/- 9.6 mmHg to 9.8 +/- 3.9 mmHg (p < 0.001). Using the radiotracer (3)H-MISO that accumulates in hypoxic regions, uptake in GW-39, LoVo and LS174T s.c. human colonic tumors increased 3.0- to 4.2-fold from day 14 through day 28 post-RAIT, but uptake of (3)H-MISO in CALU-3 tumors remained unchanged after RAIT. Normal tissue (liver, kidney, lung) uptake of (3)H-MISO did not exhibit significant changes. The increase in tumor hypoxia was also demonstrated visually using anti-PIMO staining of tumor sections. We postulated that sequential delivery of the 2 therapeutic agents, with the hypoxic cytotoxin given 2 weeks after RAIT when tumor pO(2) levels were at their nadir, would improve the therapeutic response above either modality alone or above the 2 agents delivered on the same day. Tumor growth was compared in mice given either RAIT or cytotoxin alone, the combined treatment on the same day or with the cytotoxin delivered 14 days after RAIT. Tumor size on day 35 for RAIT-treated and SR4233-treated GW-39 were 3.56 +/- 0.40 and 7.98 +/- 2.50 cm(3). When RAIT + SR4233 were delivered on the same day, tumor size dropped to 2.78 +/- 0.80 cm(3). If RAIT was given on day 0 and SR4233 on day 14, size further declined further to 1.74 +/- 0.32 cm(3) (p < 0.05 compared with same day delivery). For LS174T, tumor size on day 28 for RAIT-treated and SR4233-treated tumors were 1.14 +/- 0.36 cm(3) and 3.65 +/- 0.78 cm(3), respectively. When RAIT + SR4233 were delivered on the same day, size was 0.51 +/- 0.174 cm(3). If RAIT was dosed on day 0 and SR4233 was given on day 14, tumor size was 0.13 +/- 0.07 cm(3) (p < 0.05). Similar results were obtained for LoVo, but not for CALU-3 tumors. Another hypoxic cytotoxin, NLCQ-1, was also more efficacious 2 weeks after RAIT, compared with same-day dosing. Thus, information on tumor hypoxia after radioantibody therapy could be important for ascertaining a window of opportunity when the surviving tumor regions are most responsive to hypoxic cytotoxins., (Copyright 2001 Wiley-Liss, Inc.)

The protective efficacy of toxoids prepared from crude cytotoxin (polymyxin-B extract, PBE) and purified Klebsiella cytotoxins (KCTs) was studied in rabbits and mice. The toxoids of KCT-I and PBE were found to be protective against homologous as well as heterologous Klebsiella challenges, while toxoids of KCT-II and KCT-III afforded protection in mice against homologous Klebsiella infections. KCT-I and PBE toxoids also induced good humoral anti-Klebsiella response in rabbits with ELISA titres ranging from 20480 to 81480. Immunized female rabbits passed protective anti-Klebsiella immunoglobulins to their offsprings through colostra. Baby rabbits fed on colostrum of immunized rabbits withstood lethal Klebsiella infection up to 1 month of age, but not on the 50th day. Baby rabbits having an anti-Klebsiella IgG titre > or =1280 were fully protected against lethal dose of Klebsiella. The study revealed a significant protective efficacy of KCT-I and PBE toxoids against klebsiellosis in mice and rabbits.

A vast majority of high-grade gliomas over-express a receptor for interleukin 13 (IL13). This glioma-associated receptor for IL13 is interleukin 4 (IL4)-independent. This is in contrast to the physiological and IL4-shared receptor for the IL13, IL13/4 receptor, which is found on many normal organs. IL13-based Pseudomonas exotoxin (PE)-containing cytotoxic fusion proteins have been shown to be very potent anti-glioma agents. However, native IL13-based cytotoxins interact with both forms of the IL13 receptor. Therefore, mutations in IL13 were made in order to diminish/eliminate IL13's interaction with the shared IL13/4 receptor of normal tissue. These mutations encompassed amino acids located on alpha-helix A and C of IL13. We have engineered double or triple mutants of IL13 linked to various forms of PE. We found that these mutations could be successfully incorporated into IL13 without the loss of the protein's ability to selectively deliver the toxin to glioma cells while reducing their toxicity.

Objective: To analyze predictive factors for the development of osteonecrosis (ON) in a large cohort of patients with systemic lupus erythematosus (SLE) followed in a single center., Methods: A nested matched case control design was used. Patients with SLE who developed ON during followup were identified from the University of Toronto Lupus Clinic database. The diagnosis of ON was confirmed by either radiographs, bone scans, tomograms, or magnetic resonance imaging. A comparison group of patients with SLE without ON was selected from the same database, matched by year of birth. sex, and year of entry to the clinic to the patients with ON. Clinical, laboratory, and therapeutic factors thought to be relevant to the development of ON were compared between the 2 groups., Results: Seventy patients with SLE developed ON in the course of followup at the clinic. In univariate analysis, arthritis was the only clinical feature predictive of the development of ON. Use of glucocorticosteroid therapy, dose and duration, as well as Cushingoid appearance and cytotoxic therapy were also predictive for the development of ON. Multivariate analysis revealed that glucocorticosteroid use, the presence of arthritis, and the use of cytotoxic medications remained significant., Conclusion: Glucocorticosteroid therapy, the presence of arthritis, and use of cytotoxic medication are independent risk factors for development of ON in patients with SLE.

Objective: To evaluate the use of disease modifying antirheumatic drugs (DMARDs), cytotoxic agents, and corticosteroid therapy in patients diagnosed with rheumatoid arthritis (RA) in two periods, 1979 to 1987, and 1988 to 1996., Materials and Methods: Review of the records of 788 patients with RA diagnosed at the Department of Rheumatology, the University Hospital of Tromsø., Results: We found a significant increase in the proportion of patients who started with auranofin, sulfasalazine, methotrexate, and corticosteroids in 1988 1996 compared to 1979 1987. The initiation of use of gold salts, antimalarials, and D-penicillamine declined significantly from the first to the second period., Conclusion: Patients diagnosed with RA between 1988-1996 were treated more actively than patients diagnosed in the period 1979-1987. During 1988 to 1996 auranofin, sulphasalazine, methotrexate, and corticosteroids replaced gold salts, antimalarials, and D-penicillamine.

Human JC virus is ubiquitous in human populations and is reactivated frequently in immunosuppressed patients. Fifty-one patients with autoimmune disease receiving immunomodulating therapy were evaluated to study the possible relationship between immunosuppression and JCV viruria. Patients were divided into cytotoxic and noncytotoxic treatment groups based on their prescription. The incidence of JCV viruria in the cytotoxic treatment group was significantly higher than that in the noncytotoxic group (67% vs. 28%; P < 0.05). Most patients with JCV viruria were receiving corticosteroid (P = 0.03 for any dose and P < 0.001 for higher-dose treatments) and cytotoxic agents (P = 0.02). Age, disease duration, and medication duration appeared not to be the precipitating factors of JCV viruria in this study. The results of clinical evaluation indicate that cytotoxic immunosuppression may play an important role in JC virus reactivation., (Copyright 2000 Wiley-Liss, Inc.)

Targeted toxins, consisting of tumor-selective ligands coupled to polypeptide toxins, represent a new class of cancer therapeutics that kills malignant cells by inactivating cytosolic protein synthesis and inducing apoptosis. A number of these molecules have been produced under good manufacturing practice conditions and given systemically to patients with a variety of neoplasms. The promising results to date and the remaining pharmacological hurdles are discussed.

Background/purpose: Ischemia-reperfusion injury is encountered frequently in conditions that diminish intestinal blood flow. Caffeic acid phenethyl ester (CAPE), which is a specific component of the honeybee hive product propolis, exhibits potential antioxidant properties. This experimental study was designed to determine the effect of CAPE on ischemia-reperfusion injury in rat intestine., Methods: Fifty rats were divided into 5 groups; sham (SH), saline ischemia (SI), saline reperfusion (SR), CAPE ischemia (CI), and CAPE reperfusion (CR). Either CAPE, 10 micromol/kg, or saline was administered intraperitoneally 30 minutes before ischemia. Intestinal ischemia for 30 minutes and reperfusion for 60 minutes were applied. Ileum specimens were obtained to determine the tissue levels of malondialdehyde, superoxide dismutase, catalase, and histological changes., Results: Malondialdehyde levels in the CR group did not increase after reperfusion when compared with the CI group. However, statistically significant differences were observed between the SR and SI groups. Additional mucosal injury in the CR group when compared with the CI group was not observed. Whereas, there was a statistically significant increase in mucosal injury in the SR group. Reperfusion did not cause further injuries through both biochemical and histological parameters in the CR group., Conclusions: Results of this study showed that prophylactic administration of CAPE in ischemic condition prevents reperfusion injuries by eliminating oxygen radicals and inhibiting polymorphonuclear leukocyte infiltration. CAPE may be useful in combating the diseases of oxidative stress.

We retrospectively analyzed factors influencing PBPC mobilization during steady-state hematopoiesis in 52 patients with malignant lymphoma (n=35) or multiple myeloma (n=17) who received 77 cycles of G-CSF (12.5-50 microg G-CSF/kg/day). For 15 of these patients, the first mobilization cycle (12.5 microg G-CSF/kg/day) was followed by a second course with an increased dose of G-CSF (25 or 50 microg/kg/day). Leukapheresis was started on day 4, about 2 h after s.c. G-CSF administration, and repeated on 2-5 consecutive days. CD34+ cells were determined by flow cytometry in each apheresis product and in the peripheral blood prior to G-CSF administration, beginning on day 4. Colony assays were performed on cryopreserved samples prior to autografting. In the 15 patients receiving two mobilization cycles the higher G-CSF dose was associated with higher levels of CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful (>2x10(6) CD34+ cells/kg) collections (p=0.058). Patients with limited previous cytotoxic therapy (n=19, up to six cycles of a standard regimen such as CHOP and/or less than 20% marrow irradiation) who received a daily dose of 12.5 microg G-CSF/kg had higher levels of circulating CD34+ cells, a higher mean yield of CD34+ cells per apheresis (p<0.05), and a higher percentage of successful collections (p<0.05) compared with patients previously treated with more intensive radiochemotherapy (n=15). Ten of 20 patients (50%) who failed during the first cycle were successful during subsequent cycles with escalated doses of G-CSF. Trough levels of circulating CD34+ cells on day 4 were predictive for success or failure to achieve >2x10(6) CD34+ cells/kg, especially in heavily pretreated patients. In conclusion, a daily dose of 12.5 microg G-CSF/kg seems sufficient to mobilize PBPC during steady-state hematopoiesis in the majority of patients who have received limited previous radiochemotherapy. Higher doses of G-CSF, up to 50 microg/kg/day, mobilize more PBPC and should be considered for patients previously treated with intensive radiochemotherapy or those failing to mobilize sufficient numbers of CD34+ cells with lower doses of G-CSF.

Background: Basic fibroblast growth factor receptors on DU145 human prostatic carcinoma xenografts serve as targets for the delivery of a growth factor-toxin chimera, basic fibroblast growth factor-saporin (bFGF-SAP), which produces significant antitumor activity in a nude mouse model. However, DU145 tumors often become resistant to prolonged treatment., Methods: Nude mice bearing DU145 xenografts were intravenously administered bFGF-SAP (0.05 microg/kg weekly for 4 weeks), and a panel of eight tumors was isolated from the treated animals and established in monolayer culture., Results: In cell-survival assays, sensitivity of the treated tumor-derived cell lines to bFGF-SAP (IC50 = 12-100 nM) varied widely from cells derived from a vehicle-treated control tumor (IC50 = 10 nM). A significant inverse correlation was observed between increased IC50 values in vitro and increased tumor growth delay in vivo. Pretreatment of tumor cells with suramin or neutralizing antibodies to bFGF or keratinocyte growth factor (KGF) circumvented resistance in one of the tumor lines, confirming autocrine-mediated resistance. In another tumor subline, a 3-fold decrease in bFGF high-affinity receptor sites, which concurred with a 4-fold decrease in ability to internalize the bFGF ligand, was consistent with a decrease in total cellular expression of the FGF2 receptor (Bek). Resistance was circumvented by alternatively targeting FGF1 receptor (Flg) on these cells with a saporin immunotoxin., Conclusions: These studies identify alterations in the ligand-targeted receptor as a frequent contributor to resistance arising in DU145 tumors to in vivo treatment with a bFGF receptor-directed-toxin chimera, and provide the basis for designing methods to circumvent resistance for the purpose of enhancing efficacy of receptor-directed therapies in the treatment of prostate cancer., (Copyright 1999 Wiley-Liss, Inc.)

Elucidation of naturally occurring toxins is very important from various points of view. Of these toxin studies, search for cytotoxins which show selective toxicity against tumor cells is a challenging subject of study. In search of new bioactive substances from marine organisms, we have been investigating cytotoxic constituents in marine sponges. Through bioassay-guided separation of marine spongean extracts by use of L1210 and KB cell lines, we isolated several potent cytotoxins. This article reviews our recent investigations of three spongean cytotoxins: 1) altohyrtin A and its allied compounds (macrolides, IC50 0.01-0.4 ng/ml (KB cells)) from Hyrtios altum, 2) arenastatin A (a depsipeptide, IC50 5 pg/ml (KB)) from Dysidea arenaria, and 3) callystatin A (a polyketide, IC50 10 pg/ml (KB)) from Callyspongia truncata.

Verotoxin 1 (VT1) is an E. coli elaborated subunit toxin active only against (tumor) cell lines that express the VT1 receptor, globotriaosyl ceramide-Gb3. Astrocytomas can be highly malignant brain tumors that remain refractory to clinical treatment. Some human astrocytoma cell lines are particularly sensitive to VT1 in vitro. To address whether this represents a feasible approach to the elimination of these tumors in man, human astrocytoma tumor xenografts in nude mice were treated with verotoxin. Following a single low-dose intratumoral injection of VT1, complete regression of a 1-cm-diameter tumor within 10 days was observed in all treated animals, without reoccurrence (up to 60 days). Apoptosis was demonstrated in both tumor and vascular cells within the treated xenograft. Verotoxin binding to tumor cells and blood vessels in sections of primary glioblastoma multiforme was found.

The majority of adenocarcinoma of the pancreas are non-resectable at diagnosis due to locally advanced or metastatic disease. There will be an estimated 28,900 new cases of pancreatic cancer diagnosed in the United States in 1998. In data collected from 1986-1993, the five year survival of all stages combined was 4%. Realizing that most patients present with advanced disease, and there are no acceptable screening methods to detect early stage disease, efforts to develop active anti-cancer agents with minimal toxicity are essential in order to improve the quality of life and survival. Several 5-fluorouracil based regimens have been tried without a significant impact on palliation or survival. Recently, the anti-metabolite gemcitabine has been approved for use in individuals with locally advanced and metastatic disease, primarily on the basis of improved functional status. Many cytotoxic agents have proven ineffective in the treatment of this disease. There are several ongoing studies investigating the role of new cytotoxic and biologic agents.

TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate.

The vast majority of brain cancers (gliomas) express a receptor (R) for interleukin 13 (IL13). In order to achieve specific targeting of the IL13R in gliomas, we have mutagenized human (h) IL13. The mutation was made to alter IL13 interaction with the shared functional IL13/4 normal tissue receptor, but not with the glioma-associated receptor. We have thus produced hIL13.E13K (glutamic acid at position 13 changed to lysine) and fused it to derivatives of Pseudomonas exotoxin A. The hIL13.E13K-based cytotoxins are less active on normal cells and thus less toxic, and are better antitumor agents compared with the cytotoxins containing nonmutagenized hIL13.

The use of cytotoxic chemotherapy for cancer therapy has been very successful in the treatment and often cure of patients with particular neoplasms, such as testicular carcinomas and some lymphomas. In addition, the use of adjuvant chemotherapy in patients whose primary tumor has been surgically removed contributes significantly to cure rates in some of the more common malignancies such as breast carcinoma and colon cancer. Nonetheless, for most patients with metastatic malignancies, current antineoplastic drugs provide only brief remissions with few or no long term cures. In addition, the side effects of therapy lead to substantial morbidity in nearly all patients. Insights derived from model system studies on two glutathione based lead compounds, TER286 and TER199, suggest new clinical strategies and raise interesting basic research questions regarding the cell biology foundations of cancer chemotherapy.

Several novel nonthermal ablative modalities for the palliation of malignant esophageal stenoses have been developed over the past decade. In this article, the authors review techniques and clinical experience with photodynamic therapy as well as the intratumoral injection of alcohol, cytotoxins, and immunomodulators.

Immunocytochemical analyses of human plaques and experimental arterial lesions have implicated activated lymphocytes and monocytes in the pathogenesis of atherosclerosis, as demonstrated by the expression of interleukin-2 (IL-2) membrane receptors and major histocompatibility complex class II epitopes. The objective is to determine if targeting these cells with an IL-2 receptor-specific chimeric toxin, DAB486-IL-2, can inhibit experimental post-angioplasty vascular neointimal thickening. Twenty-two atherogenically modeled rabbits were treated in vivo with DAB486-IL-2 (0.1 mg/kg per day i.v.; n = 11) or placebo (n = 11) for 10 days following aortic balloon angioplasty (4 atm x 30 s each x 2 dilatations). In vitro 3H-leucine incorporation studies of mononuclear leukocyte and vascular smooth muscle cell protein synthesis inhibition by DAB486-IL-2 were also performed. Angioplasty sites were examined for evidence of hyperproliferative atherosclerotic narrowing by quantitative angiography and histomorphometry of neointimal cross-sectional area at baseline and 6 weeks after injury. In vitro Concanavalin-A stimulated rabbit mononuclear leukocyte protein synthesis was 50% inhibited by DAB486-IL-2 at a concentration (IC50) of 6 x 10(-11) M. Rabbit vascular smooth muscle cells were approximately 150-fold less sensitive to DAB486-IL-2 (IC50 = 10(-8) M). In vivo studies showed no change in angioplasty site angiographic minimum luminal diameter at 6 weeks in DAB486-IL-2 treated animals (from 2.96 +/- 0.52 to 2.96 +/- 0.48 mm; percent cross-sectional area reduction = 1 +/- 10%; P = N.S.). In control animals, luminal diameter decreased from 2.79 +/- 0.4 to 2.32 +/- 0.52 mm at 6 weeks, and percent cross-sectional area was reduced by 34 +/- 14% (P < 0.01 vs. placebo). Quantitative histomorphometric angioplasty segmental intimal cross-sectional area reduction of treated and placebo vessels also differed significantly (19 +/- 16% vs. 31 +/- 21%; P < 0.05). DAB486-IL-2 caused no adverse effects on animal survival, weight or hepatic transaminase levels. We conclude that post-angioplasty administration of the chimeric toxin DAB486-IL-2 inhibits angiographic narrowing and neointimal thickening in the atherogenic rabbit model. Although this IL-2 receptor-specific molecule was cytotoxic in vitro for activated mononuclear leukocytes and vascular smooth muscle cells, systemic toxicity did not occur in vivo at a dose comparable to that evaluated in clinical trials of this agent. Potential anti-proliferative effects of this chimeric toxin may be mediated by direct local inhibition of leukocyte-mediated inflammation, or through the indirect modification of vascular cell mitogenesis and cytokine release.

Purpose: The antitumor activity of the mitotoxin basic fibroblast growth factor-saporin (bFGF-SAP) against human prostatic carcinoma DU 145 was examined in athymic nude mice. Therapeutic efficacy was evaluated on the basis of dose, route of administration and treatment schedule., Materials and Methods: Chemical conjugate or recombinant bFGF-SAP (0.02 to 50 micrograms/kg.) was administered by intravenous tail injection, intraperitoneal injection, or local or distal subcutaneous injection beginning 5 days (or 60 to 121 days for large tumor studies) after subcutaneous implantation of DU 145 cells. Tumor growth was monitored as long as 140 days by external caliper measurements., Results: Recombinant bFGF-SAP, though less cytotoxic than its chemical conjugate form, effectively targeted DU 145 tumors growing as xenografts in nude mice in a dose-dependent manner. Antitumor response to treatment by intravenous, intraperitoneal, or distal subcutaneous injection suggested similar bioavailability of the mitotoxin administered by each route; local subcutaneous injection to the tumor site resulted in statistically better antitumor response. Schedules that included at least 1 bFGF-SAP treatment beyond day 23 increased long-term antitumor efficacy independent of total dose. Moreover, recombinant bFGF-SAP induced dramatic reduction of large, established tumors., Conclusions: These studies suggest a therapeutic potential for bFGF receptor-directed toxins in targeting prostate cancer; further, these data suggest that treatment of established tumors (> 3 weeks) results in qualitatively and quantitatively improved tumor responses.

The principal therapeutic procedures and when they are clinically indicated in the management of essential mixed cryoglobulinemia (EMC) have been the subject of much debate. This paper reviews current knowledge and our experience in the treatment of this complex disease. It is generally agreed that patients with purpura, the primary symptom of EMC, should avoid long periods of sitting or standing in the same position. Non-steroidal antiinflammatory drugs can be used for the management of arthralgias and/ or arthritis. Low dose steroids (0.5-0.3 mg/kg/die) are usually sufficient to control the purpura, arthralgias, arthritis and weakness, while larger doses (0.5-1.5 mg/kg/die) are needed to treat the renal involvement, peripheral neuropathy and serositis. Since the discovery of the association between EMC and viral infections, the appropriateness of cytotoxic drugs has been re-evaluated and they are no longer used. With the low antigen content diet, a regimen designed to restore a saturated mononuclear phagocytic system, good results have been obtained in the treatment of purpura, arthralgias, weakness and peripheral neuropathy. Furthermore, this dietary regimen may play a steroid sparing role. Plasma exchange is widely used in the management of severe renal involvement, hyperviscosity syndrome, sensory motor neuropathy and liver involvement in EMC.

We studied the efficacy of captopril, an angiotensin-converting enzyme inhibitor in treating persistent moderate or severe proteinuria in children with various glomerular diseases other than minimal-change nephrotic syndrome. Captopril was administered for 3 months to 15 normotensive and nonazotemic or mildly azotemic patients (12 boys, 3 girls) in whom corticosteroid and cytotoxic treatment had failed to induce remission. Urinary protein excretion decreased from 2873.14 +/- 1937.50 (mean +/- s.e.m.) to 1684.71 +/- 1463.13 mg/day (P < 0.05). The reduction in proteinuria was not related to a significant fall in systemic blood pressure or a change in renal function. Serum albumin did not rise and side effects due to captopril were not observed. We concluded that, in the short term, captopril can be used safely and effectively for decreasing the proteinuria of nephrotic children unresponsive to conventional therapy.

Anti-neutrophil cytoplasmic antibody (ANCA) determinations appear to be useful in the diagnostic evaluation and therapeutic monitoring of patients with necrotizing vasculitis. The purpose of this study was to determine whether semiquantitative ANCA measurements are useful in distinguishing between increases in primary disease activity and complications of cytotoxic or immunosuppressive therapy. The authors reviewed clinical data from 27 consecutive ANCA-positive patients who had at least three ANCA determinations performed at the University of Michigan Medical Center. Eleven patients had Wegener's granulomatosis (WG), 3 had "limited" WG; 9 had "ANCA-positive" vasculitis; and 4 had "pauciimmune" rapidly progressive glomerulonephritis. During a period of 3 months to 3.8 years, 159 ANCA determinations were performed, primarily for the purposes of diagnosis and monitoring disease activity. Eleven episodes were identified in which a single ANCA assay was used to distinguish between increased primary disease activity and a suspected therapeutic complication. The suspected therapeutic complications included pneumonia (8 of 11), bacterial sinusitis (1 of 11), cyclophosphamide-induced pneumonitis (1 of 11), and cyclophosphamide-induced vestibular toxicity (1 of 11). In 10 of 11 instances in which an ANCA assay was used to distinguish between an exacerbation in vasculitic disease activity and a therapeutic complication, interpretation of the ANCA titer was pivotal in arriving at a therapeutically appropriate decision either to increase or discontinue cytotoxic therapy. This study suggests that ANCA determinations can be used reliably to distinguish between increased primary disease activity and complications of therapy that mimic an increase in disease activity.

Existing hypoxia-selective cytotoxins (HSCs) are designed to kill only the hypoxic subpopulation in tumours, and to be used in conjunction with other therapies (e.g., radiation). A new class of drugs, hypoxia-activated prodrugs of diffusible cytotoxins (HPDCs) are proposed. These are designed to exploit, rather than merely deal with, tumour hypoxia, by releasing diffusible cytotoxins on bioreduction in hypoxic regions. Such diffusible cytotoxins are required to be much more cytotoxic than the parent prodrug, to be sufficiently stable (half lives from 0.1 to 10 min) to allow them to diffuse up to 200 microns from the hypoxic regions, and to be equally effective against all major tumour cell subpopulations, including non-cycling cells. Nitrogen mustards, which show little cell cycle specificity, which kill cells by a well-understood mechanism (DNA cross-links), and which have stabilities and reactivities able to be predictably controlled by structural variations, are proposed as suitable candidates fur such diffusible cytotoxins. Design parameters for two classes of potential HPDCs are discussed; nitro-deactivated aromatic mustards, and cobalt (III) complex-deactivated aliphatic mustards. Examples of both classes show greater cell-killing activity against intact compared with dissociated multi-cellular spheroids. This suggests they may indeed function as HPDCs, by penetrating to the hypoxic core of the spheroid and there releasing potent cytotoxins which diffuse out to kill surrounding cells at lower oxygen tensions.

The synthesis of B-ring and C-ring trifluoroacetamide-substituted colchicinoids and fluoro-substituted colchicineethylamides is presented. The B-ring trifluoroacetamido-substituted analogues exhibit moderate enhancement of potency compared to the nonfluorinated analogues for tubulin assembly inhibition and cytotoxicity toward two wild type cell lines. The C-ring substituted fluoroethylamides have reduced relative potencies in the same systems due to the strong electron-withdrawing effect of the fluoro derivatives. The fluoro colchicinoids are much more cytotoxic toward drug-resistant cell lines than to the wild type cell lines. Their enhanced potency is probably due to an effect of the fluoro moiety on functions specific to resistant cells and/or their higher hydrophobicity that may result in higher intracellular drug content. This finding may suggest the application of designed fluorinated anticancer drugs to overcome acquired resistance which may develop after several regiments of treatment with a nonfluorinated chemotherapeutic agent.

A series of 5-nitrofuran-2- and 3-carboxamides bearing alkylating side-chains has been synthesized and tested for their ability to radiosensitize selectively hypoxic Chinese hamster cells (V79) to the lethal effects of ionizing radiation and also for their ability to act directly and selectively as cytotoxic drugs on hypoxic V79 cells. The compounds were extremely efficient radiosensitizers of such cells in vitro and were more efficient than known nitroimidazoles of similar type. Their efficiencies as radiosensitizers correlated with their high electron affinity (E7(1] as measured by pulse-radiolysis. However the compounds showed little radiosensitizing activity towards KHT sarcomas in C3H mice. The compounds in this series of nitrofurans were generally more toxic towards hypoxic cells than towards oxic cells in vitro but were less effective upon the basis of a differential effect than were similar nitroimidazoles reported previously.

The medical treatment of patients with uveitis often necessitates the use of systemically administered drugs, including corticosteroids, cytotoxic agents, and cyclosporine. Because of the potential side effects of these medications, physicians unfamiliar with inflammatory ocular disease may be asked to participate in the care of these patients. The most important criteria on which to base the decisions of when and how to treat patients with uveitis are the site and severity of the inflammation, the degree of visual acuity, and the potential for restoration of vision with treatment. Slit-lamp examination, indirect ophthalmoscopy, and fluorescein angiography are useful for evaluating the loss of vision in patients with endogenous uveitis. The main goal of treatment is to prevent permanent scarring and deterioration of vision as a result of the disease or the drug side effects. Frequent reassessment will help ensure adequate treatment and will minimize adverse drug effects.

The isolation of potent antibacterial proteins and peptides from human and animal phagocytes has raised the possibility that these 'antibiotics from within' can be used as therapeutic agents. Problems in delivery to and toxicity towards the host must be faced, but rapid progress in defining the structural determinants of the cytotoxic action of these naturally occurring cytotoxins provides a basis for biotechnological development of new classes of antibiotics.