Klaus Nebendahl

Therapeutic advantages of Auger electron- over β-emitting radiometals or radioiodine when conjugated to internalizing antibodies

Abstract.Recent studies suggest a higher anti-tumour efficacy of internalizing monoclonal antibodies (MAbs) when labelled with Auger electron emitters, as compared with β-emitters. The aim of this study was to compare the anti-tumour efficacy and toxicity of the internalizing MAb, CO17-1A, labelled with Auger electron emitters (125I, 111In) versus conventional β–-emitters (131I, 90Y) in a colon cancer model, and to assess whether the residualizing radiometals may have therapeutic advantages over the conventionally iodinated conjugates. Biodistribution studies of 125I-, 111In- or 88Y-labelled CO17-1A were performed in nude mice bearing subcutaneous human colon cancer xenografts. For therapy, the mice were injected with either unlabelled or 125I-, 131I-, 111In- or 90Y-labelled CO17-1A IgG2a, whereas control groups were left untreated or were given a radiolabelled isotype-matched irrelevant antibody. The influence of internalization was assessed by comparing the results with those obtained with an anti-carcinoembryonic antigen (CEA) antibody which does not internalize to a relevant extent. The maximum tolerated activities (MTA) and doses (MTD) of each agent were determined. Myelotoxicity and potential second-organ toxicities, as well as tumour growth, were monitored. Bone marrow transplantation (BMT) was performed in order to enable dose intensification. Radiometals showed significantly better tumour-to-blood ratios than the respective iodinated conjugates. The MTAs of 131I- and 125I-CO17-1A without artificial support were 11.1 MBq (300 µCi) and 111 MBq (3 mCi), respectively; the MTA of the metals was reached at 4 MBq (100 µCi) for 90Y-, and at 85 MBq (2.3 mCi) for 111In-CO17-1A. Myelotoxicity was dose limiting in all cases. BMT enabled an increase in the MTA to 15 MBq (400 µCi) of 131I-labelled CO17-1A, to 4.4 MBq (120 µCi) of 90Y-labelled CO17-1A, and to 118 MBq (3.2 mCi) of 111In-labelled CO17-1A, while the MTA of 125I-CO17-1A had not been reached at 185 MBq (5 mCi) with BMT. Whereas no significant therapeutic effects were seen with unlabelled CO17-1A, tumour growth was retarded significantly with its radiolabelled forms. The therapeutic results were significantly (P<0.01) better with both Auger electron emitters (125I and 111In) than with the β-emitters, and, in accordance with the biodistribution data, a trend towards better therapeutic results was found with radiometals (more complete remissions) as compared with radioiodine. In contrast, at equitoxic doses, no significant difference was observed in the therapeutic efficacy of 131I- versus 125I-labelled non-internalizing anti-CEA antibody, F023C5. These data suggest that, at equitoxic doses, the therapeutic efficacy of internalizing MAbs labelled with Auger electron emitters, such as 125I or 111In, is superior to that of internalizing MAbs labelled with conventional β-emitters. The lower toxicity of Auger electron emitters may be due to the short path length of their low-energy electrons, which can reach the nuclear DNA only if the antibody is internalized (as is the case in antigen-expressing tumour tissue, but not in the stem cells of the red marrow).

Combined treatment with vitamins E and C in experimental myocardial infarction in pigs

The effect of two different combined treatments with vitamin E acetate and vitamin C on infarct size and recovery of regional myocardial function was investigated in ischemic, reperfused porcine hearts. The left anterior descending coronary artery was distally ligated in 30 thoracotomized pigs for 45 minutes followed by 3 days of reperfusion. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic (dye technique) myocardium. Regional myocardial function was assessed by sonomicrometry. Ten pigs received vitamin E acetate (12 gm intravenously three times for 1 week) before ischemic and vitamin C (4.4 gm intravenously) before reperfusion (therapy A). Another 10 pigs were treated with vitamin E acetate (12 gm intraarterially) and vitamin C (4.4 gm intravenously) during ischemia (therapy B). An additional 10 pigs served as a control group. Global hemodynamics did not differ significantly among the groups before and during ischemia. Mean plasma concentrations of vitamin E amounted to 107 micrograms/ml in group A, 16 micrograms/ml in group B, and 0.9 micrograms/ml in the control group at the onset of reperfusion. Therapy A reduced the size of the infarct from 73 +/- 12% to 47 +/- 16% of the region at risk (p less than 0.005) and improved regional systolic shortening from 0 +/- 7% to 11 +/- 6% at 3 days after reperfusion (p less than 0.01). Therapy B decreased the size of the infarct to 64 +/- 9% of the region at risk (p = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/H+ Exchange Inhibitor Cariporide Attenuates Cell Injury Predominantly During Ischemia and Not at Onset of Reperfusion in Porcine Hearts With Low Residual Blood Flow

BackgroundThis study investigated whether myocardial protection by inhibition of Na+/H+ exchange (NHE) occurs during ischemia and/or during reperfusion. Methods and ResultsThe left anterior descending coronary artery was occluded in 32 pigs for 60 minutes and then reperfused for 24 hours. Infarct sizes (nitroblue tetrazolium [NBT] stain, histology) were determined at the end of the experiments. An extracorporeal bypass was used to achieve a constant residual blood flow of 3 mL/min in the myocardium at risk during ischemia. The NHE-1 inhibitor cariporide or distilled water was infused into the extracorporeal bypass system. In group 1, active treatment was administered from the onset of ischemia until 10 minutes of reperfusion (n=8). In group 2, active treatment was infused during the first 30 minutes of ischemia only (n=8). The group 3 animals (n=8) received intracoronary cariporide after 45 minutes of ischemia until 10 minutes of reperfusion. The control animals (group 4, n=7) were treated similarly to group 1 animals, with the cariporide solution being replaced by distilled water. Infarct sizes of group 1 (NBT stain, 41.5±20%; histology, 44.6±12%) and group 2 (NBT stain, 33.5±14%; histology 34.9±15%) differed significantly (at least P =0.012) from infarct sizes of group 3 (NBT stain, 71.6±15%; histology, 69.2±12%) and the control group (NBT stain, 76±9%; histology 72.4±12%). Cariporide treatment in group 1 and group 2 significantly improved functional recovery after 24 hours of reperfusion. ConclusionsMyocardial protection by cariporide is predominantly achieved by NHE inhibition during ischemia and not during early reperfusion.

Low-molecular-weight heparin reduces neointimal proliferation after coronary stent implantation in hypercholesterolemic minipigs.

BackgroundIntracoronary stents have been suggested as a method of reducing the restenosis rate after balloon angioplasty. Proliferation of vascular smooth muscle cells is a major contributing factor to the restenosis process. Heparin and some of its derivatives have been shown to inhibit smooth muscle cell proliferation. We investigated the effect of low-molecular-weight heparin on the proliferative response after implantation of a balloon-expandable tantalum stent in previously deendothelialized coronary artery segments of hypercholesterolemic minipigs. Methods and ResultsMinipigs were fed a diet containing 2% cholesterol, starting 1 month before balloon denudation of the endothelium in a coronary artery. One month later, a stent was implanted at this site. Animals were killed after 4 weeks (group 1, n = 6) or 3 months (group 2, n = 6). Animals in group 3 (n = 6), also followed for 4 weeks after stenting, received subcutaneous low-molecular-weight heparin at a dose of 200–300 units/kg anti-factor Xa activity per day in addition to the chronic acetylsalicylic acid (100 mg/day) also administered to groups 1 and 2. Eighteen of 22 animals survived to the end of the study. Angiography revealed patent stents in all surviving animals. In group 1, histological analysis showed extensive neointimal proliferation around stent struts. Maximal neointimal thickness seen in group 1 averaged 0.93±0.11 mm, was lower after 3 months (0.8±0.14 mm) in group 2, but was significantly reduced (0.44±0.18 mm, p < 0.01) in group 3. ConclusionsThese data show a significant reduction of the neointimal proliferative response to coronary stent implantation by low-molecular-weight heparin.

The effect of two different diltiazem treatments on infarct size in ischemic, reperfused porcine hearts.

The effect of diltiazem on the development of infarcts was investigated in porcine hearts. The left anterior descending coronary artery was occluded in each of 32 anesthetized pigs for 75 min and was reperfused for 4 hr. Diltiazem (15 micrograms/kg X min) was infused in 11 pigs for 30 min before occlusion (therapy A) and in another eight pigs before reperfusion (therapy B). Eleven pigs served as controls. Tissue concentrations of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) were determined in transmural needle biopsy samples taken from the ischemic apex after 70 min of ischemia. The infarct size, expressed as the ratio of the infarcted tissue over the area at risk of necrosis multiplied by 100, amounted to 79 +/- 20% in the control group. Although there was no significant difference between hemodynamics in the control and the treated groups, pretreatment with diltiazem significantly reduced infarct size (53 +/- 26%; p = .025). Reduction of infarct size by therapy B did not reach the required level of significance (66 +/- 33%). The ischemic loss of ATP and NAD was significantly lower in the pretreated group, which further indicates that the beneficial effect of diltiazem was exerted primarily during ischemia and not during reperfusion.

Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy

Whereas bivalent fragments have been widely used for radio‐immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of 131I‐labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose‐limiting organ toxicity and anti‐tumor efficacy. The maximum tolerated doses (MTDs) and dose‐limiting organ toxicities of the 131I‐labeled anti‐CEA antibody MN‐14 [IgG, F(ab′)2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D‐lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 131I‐labeled immunoconjugates [IgG, F(ab′)2 and Fab], the red marrow was the only dose‐limiting organ; MTDs were 260 μCi for IgG, 1,200 μCi for F(ab′)2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab′)2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti‐tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments. Int. J. Cancer 77:787–795, 1998.

Reduced acute thrombus formation results in decreased neointimal proliferation after coronary angioplasty

OBJECTIVES We tested the hypothesis that reduced acute platelet deposition after angioplasty results in reduced late neointimal proliferation. BACKGROUND Platelet-mediated mechanisms contribute to smooth muscle cell proliferation and migration. METHODS Indium-111-labeled platelets were injected 16 h before coronary stent angioplasty in 10 Göttinger minipigs: group 1 (n = 5) = heparin (100-U/kg bolus) before angioplasty; group 2 (n = 5) = recombinant hirudin (CGP 39393, 1.0-mg/kg body weight bolus intravenously), followed by subcutaneous doses of 6 to 10 mg/kg every 8 h. Furthermore, stent angioplasty was performed in coronary arteries of 16 minipigs: group 3 (n = 5, nine stents) = 100 U/kg heparin only; group 4 (n = 5, 10 stents) = 1-mg/kg bolus hirudin before and 45 min after angioplasty; group 5 (n = 6, 11 stents) = hirudin (1-mg/kg intravenous bolus) before and 45 min after angioplasty, followed by 6 to 10 mg/kg subcutaneously every 8 h. RESULTS In segments with deep arterial injury, the number of platelets/angioplasty segment in group 2 after 72 h (mean 21, range 9.7 to 39.7 x 10(6)) was significantly less than that in group 1 (mean 375, range 72 to 787 x 10(6)). Morphometric analysis after 4 weeks showed no difference between groups in degree of vessel wall injury. Mean (+/- SD) neointimal thickness was 0.70 +/- 0.06 mm in group 3 and was significantly reduced in both group 4 (0.46 +/- 0.11 mm) and group 5 (0.48 +/- 0.21 mm). CONCLUSIONS The direct thrombin inhibitor hirudin significantly reduces platelet deposition up to 72 h after coronary stent angioplasty. A hirudin bolus alone as well as continued subcutaneous administration for 14 days substantially reduced neointimal proliferation compared with heparin 4 weeks after coronary stent angioplasty in minipigs.

Time delay of cell death by Na+/H+-exchange inhibition in regionally ischemic, reperfused porcine hearts.

Studies in different preparations have suggested that Na+/H+ exchange is one mechanism causally involved in cell death in myocardial ischemia and reperfusion. The time delay of cell death by pretreatment with the Na+/H+-exchange inhibitor HOE642, cariporide (4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate), was investigated in regionally ischemic, reperfused porcine hearts. HOE642 (1 mg/kg) was injected intravenously in 14 thoracotomized pigs 10 min before occlusion of the left anterior descending coronary artery (45 min of ischemia, six pigs; 70 min of ischemia, six pigs; 90 min of ischemia, two pigs). Ischemia was followed by 24 h of reperfusion. Six animals (45 min of ischemia) served as controls. Infarct size was determined as a ratio of infarcted (tetrazolium stain, histology) to ischemic myocardium (dye technique), and regional myocardial function was assessed by sonomicrometry. HOE642 did not affect global hemodynamic parameters. In the pretreated group with 45 min of ischemia, HOE642 significantly decreased histochemical infarct size from 51.2 +/- 12.6% (control group) to 13.2 +/- 12% (p < 0.005) and histologic infarct size from 44.5 +/- 9% to 17.1 +/- 7% (p < 0.005). Recovery of regional systolic shortening after 24 h of reperfusion was improved from 2 +/- 6% (control group) to 12 +/- 7% (p = 0.02). In addition, myocardial contracture and increase in heart rate during early reperfusion were attenuated. When ischemia was prolonged to 70 min after pretreatment with HOE642, infarct size, recovery of systolic shortening, myocardial contracture, and increase in heart rate did not differ from those of the control group. Pretreatment with HOE642 increased the tolerance to ischemia/reperfusion by approximately 20-25 min. Inhibition of Na+/H+ exchange appears to be very promising in the clinical treatment of acute myocardial ischemia and reperfusion.

Routes of administration

Publisher Summary Substances such as chemical elements, compounds, drugs, antibodies, cells, or other agents may be administered by different routes. This chapter discusses the administration routes, techniques and guidelines for safe injection volumes, and sites of administration. Rats can be trained to accept handling and restraining and can become familiar with their handlers. Though time-consuming, it is essential to minimize distress. All procedures should therefore be carried out only by persons well known to the animal. The simplest method for administering substances is to mix them with food or drinking water. It is possible to administer substances directly into the stomach with accurate dosages and reliable timing. When a liquid volume is administered by gavage a substantial amount of the dose passes rapidly through the stomach to the small intestine. Intragastric administration is carried out using a special blunt-ended, curved, or straight needle with a ball tip. The ball tip helps to prevent the needle from damaging the oesophagus and from passing through the glottal opening into the trachea. Further, parenteral administration involves application of a substance to the body in a manner that passes the gastrointestinal tract. Giving small amounts of solutions is called an injection, while administration of larger quantities of solutions is named an infusion. In both cases the skin must be penetrated by a needle. Other methods of parenteral administration include subcutaneous or intraperitoneal implantation of an osmotic pump and, without penetrating the skin, inhalation or topical application.

An orthotopic model of ductal adenocarcinoma of the pancreas in severe combined immunodeficient mice representing all steps of the metastatic cascade.

Introduction Clinically relevant animal models are needed to evaluate new therapeutic strategies against pancreatic adenocarcinoma, which is almost incurable by established treatment. Aims To establish and characterize a metastatic orthotopic transplant model for pancreatic ductal adenocarcinoma in severe combined immunodeficient (SCID) mice. Methodology Human pancreatic ductal carcinoma cells, PancTu 1, were implanted either subcutaneously or orthotopically into the pancreas. Results After 4 weeks, orthotopic transplantation resulted in an extensive local tumor growth of an undifferentiated ductal adenocarcinoma with slight to moderate desmoplastic reaction. The tumor growth and spread resembled the situation in humans, including invasion into adjacent organs causing biliary and stomach obstruction. In addition, tumor metastases to regional lymph nodes of the pancreas, lung, liver, mesentery, and diaphragm, and attached to the kidneys, spleen, and reproductive organs were observed. In contrast, no invasion or metastases could be demonstrated by subcutaneous implanted PancTu 1 cells. Using immunohistochemical analysis, even single human tumor cells could be detected in blood vessels and metastatic organs, providing evidence that the orthotopic transplant model appropriately reflects the entire process of the metastatic cascade. Conclusion This cancer model in SCID mice appears to be a powerful tool to investigate the identity of metastasis-associated genes and to evaluate preclinically the potency of novel antimetastatic agents in ductal adenocarcinoma of the pancreas.