Preclinical assessment of hypoxic marker specificity and sensitivity.

PURPOSE: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. MATERIALS AND METHODS: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. RESULTS: The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. CONCLUSIONS: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.

Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays.

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

Prediction of tumour hypoxia and radioresistance with nuclear medicine markers.

Second-generation nuclear medicine markers of tumour hypoxia have been synthesised and screened for hypoxic marking activity in cell cultures and in mouse tumours (EMT-6). Markers of the iodinated azomycin nucleoside class with greater water solubility and faster plasma clearance rates relative to iodoazomycin arabinoside (IAZA) were of particular interest. The test systems used to characterise hypoxic marking activity of compounds included (1) covalent linkage of radiolabelled markers to cells in suspension culture equilibrated with specific O2 concentrations; (2) biodistribution of radiolabelled markers in EMT-6 tumour-bearing mice; and (3) biodistribution in R3327-AT tumour-bearing rats by nuclear medicine procedures. Of the iodinated azomycin nucleosides produced to date, beta-D-iodoazomycin galactoside (beta-D-IAZG) and beta-D-iodoazomycin xylopyranoside (beta-D-IAZXP) exhibited high metabolism-dependent hypoxic cell uptake, rapid clearance kinetics from the blood and excellent tumour marking activity in vivo. Tumour-blood (T/B) ratio (a measure of tumour hypoxic fraction) was dependent upon EMT-6 tumour size and implantation site. The radioresistance of individual tumours was measured by in vivo/in vitro assay and correlated well with the T/B ratio of hypoxic marker. These studies have identified beta-D-IAZG and beta-D-IAZXP as effective hypoxic markers for planar and single photon emission computerised tomography (SPECT) imaging studies of tumour oxygenation.

Uptake by cells and photosensitizing effectiveness of novel pheophorbide derivatives in vitro.

Pheophorbide a prepared from the algae Spirulina was derivatized at the C(7)-carboxylic group by linking amino alkyls of various lengths and terminal functional groups. The compounds were purified by thin-layer chromatography (TLC) and by high-pressure liquid chromatography (HPLC). Solubilization of compounds by serum lipoproteins, the kinetics of compound uptake into mammalian cells, and photosensitizing effectiveness when activated by 673 nm laser light have been studied. Optimal photosensitizer uptake into cells and the greatest photosensitizing activity were observed with compounds having side-chain lengths of 4-6 carbon atoms which terminated in -OH and -CH3 groups. The most effective compounds were 3 orders of magnitude more potent than Photofrin in the degree of photoinactivation of cultured EMT-6 tumor cells. HDL and LDL significantly promoted the efflux of these photosensitizing drugs from cells, suggesting that their long-term retention in normal tissues in vivo would be minimal and produce little phototoxicity.

Synthesis and evaluation of some nitrobenzenesulfonamides containing nitroisopropyl and (ureidooxy)methyl groups as novel hypoxic cell selective cytotoxic agents.

Basic nitrobenzenesulfonamides containing nitroisopropyl and (ureidooxy)methyl groups were prepared and evaluated as novel hypoxic cell selective cytotoxic agents. In vitro, N-(2-aminoethyl)-N-methyl-3-nitro-4-(1-methyl-1-nitroethyl)benzene sulfonamide hydrochloride (11) proved to be preferentially toxic to hypoxic EMT6 mammary carcinoma cells. At 1 mM concentration in vitro, 11 reduced the surviving fraction of these hypoxic cells to 3 x 10(-3) with no effect on aerobic cells. In radiation experiments, 11 appeared to function as a hypoxic cell radiosensitizer as well as a selective cytotoxic agent. However, administration of 11 at 200 mg/kg ip or 100 mg/kg iv to BALB/c mice implanted with solid EMT6 tumors produced no evidence of significant in vivo cytotoxic or radiosensitizing activity. N-Methyl-N-[2-(methylamino)ethyl]-3-nitro-4- [(ureidooxy)methyl]benzenesulfonamide hydrochloride (20) showed slight differential toxicity toward EMT6 cells at 3 mM concentration and radiosensitizing activity comparable to misonidazole at 1 mM concentration.

Effect of cholesterol deprivation on piglet small intestine and serum lipids.

Using the neonatal piglet, the effects of dietary cholesterol deprivation on growth, intestinal enzyme activity, intestinal and hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), and serum lipid were studied. Six litters of piglets were randomly assigned to one of two feeding regimens: restricted (800 ml of formula/24 h) versus unrestricted (1,200 ml of formula/24 h). Within litters, piglets were separated by sex, then randomly assigned to a formula containing low cholesterol (less than 2 mg/dl) or high cholesterol (145 mg/dl). Piglets were fed for 2 weeks. Male piglets in the restricted low cholesterol group gained significantly less weight per milliliter of formula than the restricted high cholesterol males. No effect was observed in the females. Microvillus membrane lactase activity was greater in males fed a high versus low cholesterol diet. Intestinal and hepatic HMG-CoA reductase activities and serum lipid profiles showed a trend toward compensation for dietary cholesterol deprivation but did not differ statistically between the cholesterol-fed versus -deprived groups. It is concluded that dietary cholesterol deprivation in the male neonatal pig causes alterations in growth, but no other statistically significant responses were detectable in this study.

Cyclization-activated prodrugs. Basic esters of 5-bromo-2'-deoxyuridine.

Some 3'- and 5'-[[(alkylamino)ethyl]glycyl] esters of 5-bromo-2'-deoxyuridine were prepared and evaluated in vitro as progenitors of the parent alcohol. The esters proved to be relatively stable at low pH but released 5-bromo-2'-deoxyuridine cleanly at rates which were pH and structure dependent. These basic esters are examples of cyclization-activated prodrugs in which generation of active drug is not linked to enzymatic cleavage but rather results from an intramolecular cyclization-elimination reaction.

Cyclization-activated prodrugs. Basic carbamates of 4-hydroxyanisole.

A series of basic carbamates of 4-hydroxyanisole was prepared and evaluated as progenitors of this melanocytotoxic phenol. All of the carbamates were relatively stable at low pH but released 4-hydroxyanisole cleanly at pH 7.4 at rates that were structure dependent. A detailed study of the N-methyl-N-[2-(methylamino)ethyl]carbamate showed that generation of the parent phenol followed first-order kinetics with t1/2 = 36.3 min at pH 7.4, 37 degrees C, and was accompanied by formation of N,N'-dimethylimidazolidinone. These basic carbamates are examples of cyclization-activated prodrugs in which generation of the active drug is not linked to enzymatic cleavage but rather depends solely upon a predictable, intramolecular cyclization-elimination reaction.

Changes in phospholipid and cholesterol concentrations of the rat microvillus membrane during maturation.

We studied the cholesterol and phospholipid content and the cholesterol-to-phospholipid molar ratio of the small intestinal microvillus membrane in fetal, weanling, and adult rats. We also investigated the effect of glucocorticoid administration on these lipids and the ontogeny of 1,2-diglycerol-CDP choline phosphocholine transferase (PCT). Cholesterol and phospholipid concentrations decreased with maturation, phospholipids declining more than cholesterol. Thus, the cholesterol-to-phospholipid molar ratio rose with maturation. A similar decline was seen with PCT activity. Glucocorticoid treatment had no effect on cholesterol, phospholipids, or PCT. However, the maturational increase in the cholesterol-to-phospholipid molar ratio was blunted by glucocorticoid administration in the weanling and adult rats.

Alterations in piglet small intestine after cholesterol deprivation.

Mammalian cells require cholesterol for normal cell function. This requirement can be fulfilled by endogenous biosynthesis or by extracellular supplementation. Infants fed with human milk receive greater quantities of cholesterol than those fed commercial formulas. Whether this lack of cholesterol in commercial formulas poses a threat to normal neonatal cell function is not known. We compared small intestinal microvillus membrane fluidity, hydrolase activities, protein concentration, permeability to nonabsorbable markers, and weight gain in neonatal piglets receiving restricted intake of isocaloric formulas containing either normal amounts of cholesterol (145 mg/dl) or very low levels of cholesterol (less than 2 mg/dl). Using the fluorescent probe, diphenylhexatriene, and fluorescence polarization, microvillus membranes from cholesterol deprived piglets demonstrated higher fluidities than did microvillus membranes from animals fed normal concentrations of cholesterol. Cholesterol-deprived animals, even though their caloric intake was similar to cholesterol-fed animals, demonstrated a net weight loss per animal whereas the cholesterol-fed animals demonstrated a weight gain. These results demonstrate that in a pig model on a restricted intake, cholesterol deprivation alters the biophysical properties of the microvillus membrane.

Synthesis and activity of novel nitropyrazines for use as hypoxic cell radiosensitizers.

A series of eight novel nitropyrazines has been prepared by oxidation of sulfoximine intermediates. The partition coefficient, one-electron reduction potential, sensitizer enhancement ratio, and chronic and acute aerobic cytotoxicity have been measured for each. Two representatives of this series were tested in the Ames test and were not found to be mutagenic.

Preparation and analgesic properties of amino acid derivatives of (-)-5,9 alpha-diethyl-2'-hydroxybenzomorphan.

The N-arginyl derivative of methionine-enkephalin (fragment 60-65 of beta-lipotropin) has been shown to be equiactive with the parent pentapeptide, despite the fact that the tyrosine amino group in this compound has been neutralized by the formation of an amide linkage. A series of N-(amino acid) derivatives of (-)-5,9 alpha-diethyl-2'-hydroxybenzomorphan was prepared and evaluated for analgesic activity. In vitro activities were found to vary greatly, depending on the nature of the amino acid used. The N-arginyl derivative was found to be equipotent to (-)-5,9 alpha-diethyl-2'hydroxybenzomorphan and also to methionine-enkephaline in the naloxone binding assay.

Approaches to vasodilating/beta-adrenergic blocking agents: examples of the dihydrolutidine type.

The aminohydroxypropoxy moiety has been incorporated into the dihydrolutidine class of vasodilators. In the spontaneously hypertensive rat, one of these, (S)-4-[2-methyl-4-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl]-3,5-dicarboethoxy-1,4-dihydrolutidine (4c), exhibited antihypertensive activity on the order of the standard 4-[2-(trifluoromethyl)phenyl]-3,5-dicarboethoxy-1,4-dihydrolutidine (2a). This antihypertensive activity could not be explained in terms of a vasodilating effect, as determined in the dog. In this latter model, 2a decreased both mean arterial and hindlimb perfusion pressures.

Piperazinylquinoxalines with central serotoninmimetic activity.

Regioselective syntheses of substituted 2-chloroquinoxalines and derived 2-(1-piperazinyl)quinoxalines are described. Selectivity in regards to serotonin reuptake blocking and serotoninmimetic activities of the piperazinylquinoxalines is reported. In general, introduction of a 6-substituent into the piperazinylquinoxaline enhanced serotonin reuptake blocking activity and diminished serotoninmimetic activity. Unsubstituted and 3-hydroxypiperazinylquinoxalines had primarily serotoninmimetic activity.

Heterocyclic analogues of the antihypertensive beta-adrenergic blocking agent (S)-2-[3-(ter-butylamino)-2-hydroxypropoxy]-3-cyanopyridine.

The synthesis of a series of isoelectronic analogues of (S)-2-[3-(tert-butylamino)-2-hydroxypropoxy]-3-cyanopyridine (1) are described; included in this group are examples of thiazole, isothiazole, thiadiazole, pyrazine, and the structurally related naphthyridines. All of the compounds are similar to 1 in that they contain a cyano group ortho to the aminohydroxypropoxy side chain and meta to the nitrogen heteroatom. In addition, several related examples, having additional nuclear substituents and/or groups other than CN in the position adjacent to the aminohydroxypropoxy group, were prepared, and beta-adrenoceptor antagonist activity and vasodilating potency were determined. Three compounds, thiazole 2 and isothiazoles 3 and 27, effectively lowered mean arterial pressure in the SH rat at 5 mg/kg. Compounds 2, 3, and 27 increased iliac blood flow and exhibited beta-adrenergic blocking properties in the dog.

beta-Adrenergic blocking agents with acute antihypertensive activity.

Modification of the pharmacological profile of the vasodilating/beta-adrenergic blocking agent 2-[4-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl]-4-(trifluoromethyl)imidazole (1) has been investigated. Introduction of selected substitutents onto the imidazole ring, in place of the trifluoromethyl group, has yielded highly cardioselective beta-adrenergic blocking agents such as 7, 17, and 18. The placement of alkyl or chloro groups onto the aryl ring of 1, as illustrated by 33, has produced a class of compounds characterized as antihypertensive beta-adrenergic blocking agents. In these examples, the acute antihypertensive activity does not appear to be due to either vasodilating or beta 2-agonist properties.

Trichloroacetamidines, a new class of positive inotropic agents.

A series of trichloroacetamidine derivatives, obtained by addition of amines to trichloroacetonitrile, was evaluated for positive inotropic activity on isolated cat heart papillary muscles. Increased contractility, not antagonized by beta-adrenergic blockade with sotalol or reserpine pretreatment, was observed in this assay with a variety of N-substituted trichloroacetamidine derivatives. More extensive pharmacological studies with the 3-indolylmethyl analogue 2 showed that this amidine in dogs, 5 mg/kg iv, produced a positive inotropic effect more pronounced than that of ouabain, 50 microgram/kg iv. Several of the trichloroacetamidines were found to be inhibitors of guinea pig kidney and calf heart Na-K-dependent ATPase and to have specificity for these enzymes different from that of ouabain. Bacterial mutagenic activity was observed with three members, 2,3, and 12, of the series.

Synthesis and antihypertensive activity of some ester progenitors of methyldopa.

A variety of esters of methyldopa was synthesized with the objective of obtaining derivatives that would be more efficiently absorbed from the gastrointestinal tract than the free amino acid and would undergo conversion to methyldopa readily in the blood or target tissues. Two of the esters, alpha-pivaloyloxyethyl (4u) and alpha-succinimidoethyl (4w), were found to be more potent antihypertensive agents than methyldopa in animal models and were selected for further study in man. The amino esters were prepared by three different methods, including direct esterification of methyldopa without the use of N- or O-protecting groups.