Antitumour evaluation of dolastatins 10 and 15 and their measurement in plasma by radioimmunoassay

Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia that have been shown to interact with tubulin. Their growth-inhibitory properties were compared using panels of human ovarian and colon-carcinoma cell lines. Both agents were very potent inhibitors of cell growth, with dolastatin 10 being an average of 9.1-fold more potent than dolastatin 15 [mean 50% inhibitory concentrations (IC₅₀ values) 2.3×10^–10 and 2.1×10^–9 M, respectively; P <0.05] and more potent than paclitaxel or vinblastine. While neither dolastatin exhibited marked cross-resistance in cisplatin- or etoposide-resistant cell lines, contrasting effects were observed using an acquired doxorubicin-resistant (CH1doxR, 100-fold resistant, P-glycoprotein overexpressing) cell line. Resistance was significantly higher to dolastatin 15 (12.7-fold) than to dolastatin 10 (only 3.2-fold; P <0.05) and was reversible in both cases by verapamil. In vivo, using a s.?c. advanced-stage human ovarian carcinoma xenograft and equitoxic doses, greater activity was observed with dolastatin 10 (6.1-day growth delay) versus 0.4 days for dolastatin 15. A radioimmunoassay for dolastatin 10 (limit of detection in mouse plasma 5 ng/ml) was developed. The rabbit antiserum also cross-reacted by 65% with dolastatin 15. Comparative mouse pharmacokinetics following i.?v. administration of 1 mg/kg showed that both compounds are rapidly eliminated, but with a shorter second-phase half-life (t ¹ _{/2 β}) being observed for dolastatin 15 (being detectable for only up to 4 h post-administration), the t ¹ _{/2 β} being 3 times longer for dolastatin 10. In addition, areas under the plasma concentration-time curve (AUC values) were 1.6-fold higher for dolastatin 10 (333 versus 208 ng ml^–1 h). Plasma binding of dolastatin 10 exceeded 90%. The highly sensitive RIA will be useful for pharmacokinetic studies in conjunction with the planned phase I clinical trials of these novel, extremely potent, tubulin-binding agents, of which dolastatin 10 appears to possess the more promising preclinical features.     

Assessment of dynorphin-A depletion in the anorexia of Walker-256 tumor bearing rats

Rats bearing the Walker-256 (W-256) tumor display an anorexic profile which resembles that of normal animals forced to drink 2% NaCl [2,24], a regimen which depletes neurohypophyseal dynorphin-A (DYN) [3,9]. As expected, the naloxone reversible feeding induced by 2-deoxy-D-glucose (2-DG) was attenuated (36%) in the W-256 tumor bearing rats (TBR). Interestingly, immunoreactive (ir) levels of dynorphin-A 1–17 (DYN-17) and its postulated breakdown product, dynorphin-A 1–18 (DYN-8), were also reduced in the neurohypophysis of W-256 TBR by 42 and 50%, respectively. However, ir-DYN levels were not reduced in TBR in those brain regions which are probably involved in the regulation of appetite (e.g., hypothalamus). 2-DG itself did not consistently affect ir-DYN levels in any tissue for either controls or TBR. The ratio of DYN-8 to DYN-17 did not change in response to any treatment, including the depletion of both peptides from the NIL of TBR. In summary, the present data do not support DYN depletion as being a factor which contributes to the anorexia of the W-256 TBR.     

Magnetic field mapping

Homogeneous radiofrequency magnetic fields are necessary for production of high-quality magnetic images and for most forms of magnetic resonance spectroscopy. It is often convenient to map the radiofrequency homogeneity associated with a resonant device by measuring the magnetic image intensity of an aqueous phantom placed within the resonator. The rf field intensity is not related trivially to the magnetic image intensity, and the relationship is different for different image acquisition methods. In this report relationships between rf field intensity and magnetic image intensity are derived and radio-frequency field maps presented for comparison using (1) an rf probe moved about within the resonant volume, (2) spin-echo images, and (3) small tip angle gradient refocused echo images.     

Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers

A quantitative method has been developed for the analysis of 4-aminobiphenyl (4-ABP) covalently bound as the sulfinic acid amide to the 93 beta cysteine of human hemoglobin. The method uses mild basic hydrolysis of hemoglobin to release the parent amine, derivatization to form the pentafluoropropionamide, and capillary gas chromatography with detection by negative-ion chemical ionization mass spectrometry. The method is precise and gives reproducible results on multiple blood samples taken from individuals over 48 h. Application of this method to blood samples from cigarette smokers and nonsmokers revealed consistently higher adduct levels in smokers. The mean value for smokers was 154 pg 4-ABP per g Hb compared to 28 pg/g Hb for nonsmokers, with no overlap of adduct levels between the two groups. Studies on quitting smokers revealed that adduct levels declined over a period of 6-8 weeks to nonsmoker levels. The finding of 4-ABP adducts in all nonsmokers was not anticipated but is consistent with low-level ubiquitous contamination of air, food, or water. In other animals sampled, rats and dogs had measurable adduct levels, but monkeys and fish did not. The hemoglobin adduct of 4-ABP is the product of a series of reactions between the hemoprotein and N-hydroxy-4-ABP. The formation of hydroxylamines from carcinogenic aromatic amines and their subsequent reactions with DNA are generally thought to be critical events in the initiation of bladder tumors. We suggest that the observed hemoglobin adduct levels formed by this proximate carcinogen will reflect the extent to which these steps have occurred. This is the first report of 4-ABP adducts in human blood.     

Acute Alcohol Effects on Opiomelanocortinergic Regulation

To assess acute effects of alcohol on forebrain and pituitary opiomelanocortinergic regulation, a model was developed in which “experienced” (previously introduced to ethanol administration, so the subjective response was not a novel stimulus) male Sprague-Dawley rats received pulsatile intragastric ethanol infusions during the dark (active) photophase to produce and sustain (for 3 hr) behaviorally relevant (0, 40 to 70, 80 to 110, or 120 to 150 mg/dl) plasma ethanol levels. The effects of alcohol on hypothalamo-pituitary-adrenal (H-P-A) axis function were biphasic with respect to dosage (inhibition with low dosage and stimulation with higher dosages) and time (initial stimulation with higher dosages was followed by rapid return to control levels even though elevated plasma ethanol levels were maintained). The effects of alcohol on H-P-A activation were also inconsistent; some of the animals did not appear to respond even though elevated (i.e., > 100 mg/dl) plasma ethanol levels were produced. Induction of moderate (80 to 110 mg/dl) plasma ethanol levels acutely (within 30 min) increased immunoreactive (i) β-endorphin concentrations in the ventral tegmental area of the brain; higher (120 to 150 mg/dl) plasma ethanol levels increased iβ-endorphin concentrations in both the ventral tegmental area and the nucleus accumbens, whereas iβ-endorphin concentrations were not significantly altered in other brain areas. High (120 to 150 mg/dl) plasma ethanol levels also increased mediobasohypothalamic pro-opiomelanocortin (biosynthetic precursor of forebrain β-endorphin) mRNA concentrations at 3 and 6 hr after initiation of ethanol infusions. Results demonstrate that atraumatic induction of physiologically meaningful plasma alcohol levels by gastric ethanol infusion activates the forebrain opiomelanocortinergic opioid system and exerts complex effects on the interrelated H-P-A system, consistent with evidence that these systems may interact to mediate or modulate some responses to alcohol ingestion.     

Immune responsiveness of monkeys exposed chronically to cigarette smoke

Eleven adult male stumptailed monkeys (Macaca arctoides) were chronically exposed to either a low dose (human equivalent of 1 pack/day) or a high dose (human equivalent of 3 packs/day) of high-tar, high-nicotine University of Kentucky reference cigarette smoke for 4-8 years. Several parameters of their immunological response were compared to six nonsmoked control animals. The results from these experiments suggest that cigarette smoking does not significantly affect the response of spleen cells to the mitogens phytohemagglutinin or lipopolysaccharide. However, spleen cells from animals subjected to the heavy dose of cigarette smoke demonstrated a significant reduction in their natural killer cell-mediated lytic activity and a decreased response to concanavalin A. These results suggest that cigarette smoking may have a differential effect on lymphocyte subpopulations, and that the effects on the immune response are related to the dose of cigarette smoke.     

Pleomorphic lipoma of the bulbar conjunctiva

We describe a patient who had most of the histologic features of the pleomorphic lipoma. Floret giant cells and nuclear pyknosis were well demonstrated in this case, and characteristic bubbly nuclear vacuolations were also seen. There was an absence of mitotic activity. The variable size of the adipocytes and nuclear pyknosis and the presence of collagen indicate an atrophying degenerative process in the formation of pleomorphic lipoma.     

Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: Effects of raloxifene HCl, tamoxifen, estrogen, and alendronate

Bone loss associated with postmenopausal osteoporosis can be reduced by treatment with antiresorptive agents such as estrogen or bisphosphonates. Whereas bisphosphonates primarily affect bone loss, estrogens have an advantage of also lowering serum cholesterol levels, although they have a detrimental effect in the uterus. Recently, raloxifene HCl, a selective estrogen receptor modulator (SERM), has been shown to decrease both bone loss and cholesterol levels without the negative uterine effects. These antiresorptive agents reduce bone turnover, which can be evaluated by measuring bone turnover markers. To compare the effects of estrogen, two SERMs (raloxifene HCl and tamoxifen), and alendronate, a bisphosphonate that inhibits bone loss by an estrogen-independent pathway, on metabolic bone markers and cholesterol levels, rats were ovariectomized 2 weeks prior to 3 weeks of daily oral treatment with raloxifene HCl (3 mg/kg), ethynyl estradiol (0.1 mg/kg), tamoxifen (3 mg/kg), or alendronate (3 mg/kg). Raloxifene HCl, tamoxifen, and ethynyl estradiol reduced serum cholesterol to levels below control values within 4 days after initiation of treatment, whereas alendronate had no effect. After 3 weeks of treatment, serum cholesterol values in ethynyl estradiol treated animals, although still below the control value, had risen 6.4-fold; raloxifene HCl and tamoxifen values rose by only 1.4–1.5-fold. Therefore, compared with estrogen, SERMs may have a longer-term suppressive effect on serum cholesterol. At 4 days of treatment, ovariectomized rats had a 1.4-fold increase in serum osteocalcin level compared with controls. Ethynyl estradiol lowered this level within 1 week of treatment by 18%, with a more pronounced reduction of 34% at 3 weeks. In contrast, raloxifene HCl, tamoxifen, or alendronate had very little effect after the first week (6% to 13% reduction), although there was an 18% to 25% reduction by 3 weeks. Urinary pyridinoline levels, elevated 1.4-fold in the ovariectomized rat compared with controls 2 weeks after surgery, were reduced to control values after 2 weeks of treatment with raloxifene HCl, ethynyl estradiol, tamoxifen, or alendronate. These data support the concept that estrogen, raloxifene HCI, tamoxifen, and alendronate inhibit bone loss in the ovariectomized animal by reducing bone resorption. The results also indicate that for treatment of postmenopausal osteoporosis, raloxifene HCl may have an advantage over the other antiresorptives studied in having both non-uterotrophic and hypocholesterolemic effects in addition to its ability to inhibit bone resorption.