Gerhard Hamilton

DOTA-Lanreotide: A Novel Somatostatin Analog for Tumor Diagnosis and Therapy*

Long acting somatostatin-14 (SST) analogs are used clinically to inhibit tumor growth and proliferation of various tumor types via binding to specific receptors (R). We have developed a 111In-/90Y-labeled SST analog, DOTA-(d)βNal1-lanreotide (DOTALAN), for tumor diagnosis and therapy. 111In-/90Y-DOTALAN bound with high affinity (dissociation constant, Kd, 1–12 nm) to a number of primary human tumors (n = 31) such as intestinal adenocarcinoma (n = 17; 150-4000 fmol/mg protein) or breast cancer (n = 4; 250-9000 fmol/mg protein). 111In-/90Y-DOTALAN exhibited a similar high binding affinity (Kd, 1–15 nm) for the human breast cancer cell lines T47D and ZR75–1, the prostate cancer cell lines PC3 and DU145, the colonic adenocarcinoma cell line HT29, the pancreatic adenocarcinoma cell line PANC1, and the melanoma cell line 518A2. When expressed in COS7 cells, 111In-DOTALAN bound with high affinity to hsst2 (Kd, 4.3 nm), hsst3 (Kd, 5.1 nm), hsst4 (Kd, 3.8 nm), and hsst5 (Kd, 10 nm) and with lower affinity to hsst1...

Role of the MDR-1-Encoded Multiple Drug Resistance Phenotype in Prostate Cancer Cell Lines

The treatment of advanced metastatic prostate cancer by hormone manipulation or orchiectomy is frequently followed by the appearance of hormone-insensitive and highly chemoresistant tumor cells. In this study we have investigated the contribution of the P-glycoprotein-mediated drug efflux (multidrug-resistance; MDR) to the cellular resistance of prostate carcinoma-derived cell lines to diverse cytotoxic drugs by detection of P-glycoprotein (P-gp) measurement of P-gp-mediated drug transport and reversal of MDR by chemosensitizers. The in vitro chemosensitivity of three prostate cancer cell lines (PC-3, DU-145 and LNCaP) to doxorubicin was measured in a thymidine incorporation proliferation assay. Growth of the partially hormone-sensitive cell line LNCaP is inhibited by low doses of doxorubicin (IC50:27 ng./ml.), but PC-3 and DU-145 are highly resistant to the drug, with IC50 values of 10 micrograms./ml. and 7.5 micrograms./ml., respectively. The chemosensitivity of the PC-3 and DU-145 cells is increased in response to 1 microM. verapamil, 1 micrograms./ml. cyclosporine A and 2 microM. tamoxifen, which are known to partially reverse the MDR phenotype in other resistant tumors. A verapamil-sensitive drug efflux has been demonstrated for the PC-3 and Du-145, but not for the LNCaP, cell lines, using flow cytometric measurements of the P-gp substrate rhodamine 123 efflux from preloaded cells. In agreement with the functional measurements, the expression of the P-glycoprotein was detected in the PC-3 and Du-145 cell lines in Western blots using the monoclonal C 219 antibody. In conclusion, the chemoresistant and hormone-insensitive PC-3 and Du-145 cell lines express P-gp and exhibit verapamil-sensitive drug efflux, indicative of MDR. However, the low MDR-reversal rates observed in these cell lines in response to chemosensitizers in clinically achievable concentrations (approximately 2- to 3-fold reversal), point to non-MDR-associated cellular mechanisms as dominant factors of chemoresistance in prostate cancer.

Endotoxin, TNF-alpha, interleukin-6 and parameters of the cellular immune system in patients with intraabdominal sepsis

The correlation of endotoxin (ET), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and cellular immune parameters with multiple organ failure and lethal outcome in intraabdominal infections was studied in a group of 18 patients with peritonitis, abscess or pancreatitis. Of these patients, 7 developed respiratory failure and 5 died due to multiple septic organ failure. The peak levels of ET (2.7 +/- 1.3 ng/ml) in the course of the disease were followed by moderate increases of TNF-alpha (mean 147 +/- 41 pg/ml) and IL-6 (170 +/- 61 pg/ml) within 2 days. Analysis of the parameters for the last 12 days prior to death or discharge showed, that the patient group with lethal outcome was characterized by significant lower mean plasma levels of TNF-alpha (less than 75 pg/ml versus greater than 160 pg/ml) and IL-6 (less than 130 pg/ml versus greater than 270 pg/ml), as well as high rates of unstimulated thymidine uptake into peripheral mononuclear blood cells (greater than 44000 cpm/8 x 10(6) PMBC/18 h versus less than 24000 cmp), T-lymphocyte depression (CD3; approximately greater than 40% reduction) with lower T-helper/inducer subset cell numbers (mean CD:CD8 ratio 1.0 +/- 0.55 versus 1.8 +/- 0.2) and lower lectin (PHA) stimulation values (1.9 +/- 1.4 versus 4.1 +/- 1.0). These data demonstrate an anergic immune status with low mediator levels and depressed T-lymphocyte function in patients with poor prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-mediated bioadhesion: Proteolytic stability and binding-characteristics of wheat germ agglutinin and Solanum tuberosum lectin on Caco-2, HT-29 and human colonocytes

Abstract For the development of lectin-mediated drug delivery systems, the proteolytic stability of the non-toxic lectins from Arachis hypogea, Lens culinaris, Dolichus biflorus, Solanum tuberosum (STL) and Triticum vulgare was investigated by in vitro exposure to gastrointestine-located enzymes. No degradation products were observed within 24 h of incubation on sodium dodecyl sulfate polyacrylamide gels. Binding to human colon carcinoma cell lines was investigated by flow cytometry. The fluorescein-labelled derivatives of N-acetylglucosamine-specific wheat germ agglutinin (WGA) and STL exhibited the highest cell-associated fluorescence intensity. As determined by dilution experiments, the number of WGA-binding sites on Caco-2, HT-29 and human colonocytes exceeded those for STL by 5-, 1.7- and 1.4-fold, respectively. By a competitive flow cytometric assay using N,N′, N″-triacetylchitotriose for inhibition, WGA-affinity exceeded STL-affinity by ten-fold. The affinity of each lectin to Caco-2, HT-29 and human colonocytes was about the same, indicating that similar lectin receptors were involved. Preventing N-glycosylation of the carcinoma cells by pretreatment with 0.001% tunicamycin for 40 h resulted in 30% inhibition of WGA- and STL-binding. When WGA was covalently attached to Sepharose beads (250–350 μm), the interaction with HT-29 and Caco-2 cells showed stable and tight binding. Therefore, especially considering the comparable affinity of human colonocytes and monolayer-forming Caco-2 and HT-29 cells, this system is proposed as a model for the development of lectin-mediated particulate pharmaceutical devices.

Current status of intermittent androgen suppression in the treatment of prostate cancer

Treatment of advanced prostate cancer by continuous androgen suppression results in excellent short-term response but poor long-term survival. Intermittent androgen suppression (IAS) aims to maintain androgen responsiveness of tumor cells by regular cycles of treatment cessations and tumor regrowth to specific prostate-specific antigen limits. First clinical trials demonstrate consistent responses and improved quality of life in most patients on androgen suppression retreatment for up to five cycles, with mean off-treatment periods of approximately 5 to 16 months. Most patients with metastatic disease exhibit early disease progression or androgen independency under IAS, but a subgroup including patients with metastatic disease respond to a single androgen suppression cycle with off-treatment times for up to 48 months. In conclusion, IAS improves the quality of life in patients with primarily hormone-dependent tumors without adverse effects and seems to be most effective in patients with prostate cancer with asymptomatic biochemical progression and low tumor burden. Patients should be treated within the framework of randomized trials and characterized for survival and prognostic factors associated with response to IAS treatment.

Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R>1,400 Ω cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitisers inhibited drug flux (137 pmol h−1 cm−2; range, 133–142 pmol h−1 cm−2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 μg/ml (0.9 μM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 μM; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhdamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of r-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation. Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly. As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.

Insulin-like growth factor-I-dependent growth and in vitro chemosensitivity of Ewing's sarcoma and peripheral primitive neuroectodermal tumour cell lines

Serum-free growth of Ewings sarcoma (ES) and primitive peripheral neuroectodermal tumour (pPNET) cell lines was achieved by supplementing a basal medium with insulin-like growth factor-I (IGF-I). These cultures were used to investigate the sensitivity of 3 ES (EW-2, RD-ES, SK-ES-1) and 3 pPNET (SIM-1, KAL, SAL) cell lines to a panel of anti-tumour agents in short-term (48-h) proliferation assays. Of the four cytostatic drugs included in the currently used multi-drug regimens, cyclophosphamide, doxorubicin and actinomycin-D inhibit the proliferation of the cell lines with high efficacy, whereas the vinca alkaloids were less effective. Cisplatin, etoposide, mitomycin-C and mitoxanthrone were also found to have a high inhibitory activity in this in vitro ES/pPNET system. The most remarkable effect was observed for cytosine arabinoside (ARA-C), which gave a half-maximal inhibition at drug concentrations approximately 5000 times below the clinical peak plasma concentrations (250 micrograms/ml). The ARA-C sensitivity of ES and pPNET cell lines is comparable with the established ARA-C sensitivities of leukaemia-derived cells. The different ES and pPNET cell lines showed a rather uniform response to the different cytostatic drugs with decreased sensitivity of individual pPNET cell lines to vinblastin, ARA-C and mitoxanthrone. Modulation of the IGF-I/IGF-I receptor/IGF-I binding protein system, which seems to constitute an important stimulator of cell growth in neuroectoderm-derived or -related tumours, can be used to enhance the drug sensitivity of the tumour cells in vivo or in vitro therapeutic procedures. According to our results, serum-free conditions for autologous bone marrow purification are expected to result in significantly increased chemosensitivity of ES and pPNET cells in response to anthracyclines and cisplatin.

Cytokine Patterns in Patients Who Undergo Hemofiltration for Treatment of Multiple Organ Failure

AbstractThe excessive uncontrolled activation of inflammatory cells and mediators after trauma or major surgery plays a key role in the development of adult respiratory distress syndrome and multiple organ system failure (MOSF). In the past elevated cytokine levels were shown to influence the outcome of these patients adversely. There are diverging results regarding the removal of circulating cytokines by various methods of hemopurification for clinical improvement of MOSF. Seven patients after trauma or major surgery underwent continuous venovenous hemofiltration (CVVH) for the treatment of severe organ failure of the heart and lungs (Murray score 2.74) but not for renal or liver failure. The cytokine levels were measured at the beginning and 15, 60, 120, and 240 minutes after initiation of CVVH (measure points MP1–5). Clinical improvement during the treatment was monitored, and correlation with cytokine levels was evaluated. Arterially measured tumor necrosis factor α rose from 11.14 ng/ml to 17.86 ng/mln(p < 0.05). Arterial interleukin-6 (IL-6) levels significantly decreased during CVVH from 1284.7 ng/ml to 557.9 ng/ml; IL-8 levels simultaneously decreased from an initial peak of up to 154.4 ng/ml at MP3 to 97.3 ng/ml at MP5. The drop in serum IL-6 and IL-8 levels closely correlated with clinical improvement. After 2 hours of CVVH the hemodynamic situation improved significantly, as revealed by a decrease in catecholamine expenditure, an increase in arterial pressure, and a decrease in pulmonary artery pressure. Moreover, 2 hours after the initiation of CVVH the oxygenation index rose significantly and correlated well with the drop in shunt fraction. The Murray score significantly fell to 1.86. The removal of IL-6 and IL-8 by CVVH after initial stimulation correlates with clinical improvement, which was demonstrated by significantly improved oxygenation and hemodynamics from 2 hours after the initiation of CVVH onward. The elimination of cytokines and several mediators by CVVH may contribute to the cardiopulmonary improvement of critically ill patients. In comparison with the clinical control group (n= 7), which was comparable in terms of MOSF, no intervention led to a similar improvement in cardiorespiratory failure, and overall two of these patients died. Moreover, patients of the control group experienced a significant longer stay at in the intensive care unit.

Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line

Mechanisms of intracellular pH (pHi) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pHi regulation in colonic tumors. pHi changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [3H]thymidine uptake. Resting pHi in Hepes-buffered solution was 7.53 +/- 0.01 (n = 36). Both 1 mM amiloride and Na(+)-free solution inhibited pHi recovery from acidification and decreased pHi in resting cells. In HCO3-/CO2-buffered media resting pH1 was 7.42 +/- 0.01 (n = 36). Recovery from acidification was Na(+)-dependent, CI(-)-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 microM H2DIDS pHi recovery was completely inhibited. In Na(+)-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 (n = 6) and no alkalinization was observed in CI(-)-free medium. Addition of 5 microM tributyltin bromide (an anion/OH-exchange ionophore) caused pHi to decrease from 7.43 +/- 0.05 to 7.17 +/- 0.08 (n = 5). The effects of pH0 on steady-state pHi, pHi recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [CO2] and [HCO3-]. In general, increases in pH0 between 6.7 and 7.4 increased pHi recovery, steady-state pHi and growth rates. In summary, SW-620 cells have a resting pHi > 7.4 at 25 degrees C, which is higher than other intestinal cells. Acid extrusion in physiological bicarbonate media is accomplished by a pHi-sensitive Na+/H+ exchanger and a pHi-insensitive Na(+)-HCO3-cotransporter, both of which are operational in control cells at the resting pHi. No evidence for activity of a CI-/HCO3- exchanger was found in these cells, which could account for the high pHi observed and may explain why the cells continue to grow in acidic tumor environments.

Intraoperative Course and Prognostic Significance of Endotoxin, Tumor Necrosis Factor-alpha and Interleukin-6 in Liver Transplant Recipients

Early events in reaction of the host immune system to an allograft were studied by intraoperative measurements of endotoxin (ET), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in human liver transplantation. Blood samples were collected prior to operation, before clamping of the liver vessels, at the beginning and end of the anhepatic phase, and 5 and 30 min after recirculation. Diagnosis of rejection and infection in the graft recipients was established by assessment of spontaneous blastogenesis and T-lymphocyte subpopulations in addition to clinical symptoms and results from biopsies and blood chemistry. Of the 27 unmatched liver transplantations included in this study, 8 patients had infections in the first two postoperative weeks and 6 patients showed rejection of their grafts under antithymocyte globulin immunoprophylaxis. Endotoxin was transiently elevated in plasma in the anhepatic phase (2-fold in comparison to normal values) as expected for an accumulation of intestine-derived endotoxin during clamping of liver vessels, but no correlation was found with TNF-alpha levels and specific post-transplant complications. All patients with rejections had high plasma levels of TNF-alpha immediately after recirculation (mean value 240 pg TNF-alpha/ml), in contrast to low TNF-alpha levels in graft recipients without complications or infections. These results indicate that the initiation of rejection in liver transplantation is associated with increased plasma concentrations of TNF-alpha. The measured TNF-alpha concentrations are adequate to promote the binding of lymphocytes to allograft endothelial tissue and/or to induce expression of MHC antigens in the graft. Subsequent viral or bacterial infections were preceded by high intraoperative plasma concentrations of interleukin-6 (mean value 1400 pg IL-6/ml). The correlations of rejection with high intraoperative TNF-alpha levels and of infection with those of IL-6 are statistically significant in Wilcoxon tests for the direct measurements and in Fishers exact tests for positive test values, with limits of 90 pg/ml for TNF-alpha and 800 pg/ml for IL-6.