In the ewe, estradiol and progesterone inhibit luteinizing hormone (LH) secretion during the breeding season. Endogenous opioid peptides (EOP) are also inhibitory to LH secretion, and both estrogen and progesterone have been reported to enhance EOP inhibition of LH release. Which EOP are involved in this inhibition is unclear. In this study, we concentrated on β-endorphin because evidence for its ability to inhibit LH secretion exists in ewes. We first studied the distribution of β-endorphin-immunoreactive neurons in 4 cycling ewes using immunocytochemistry. Cell bodies were found only within the medial basal hypothalamus (MBH) and were concentrated in arcuate nucleus and mammillary recess of the third ventricle, with a few in the median eminence. Extensive fiber tracts were seen in preoptic area (POA) and median eminence. We next tested the hypothesis that gonadal steroids increase the synthesis of EOP by measuring levels of mRNA for proopiomelanocortin (POMC), the precursor to β-endorphin. Ovariectomized ewes were treated with no steroids (n = 7) or given subcutaneous Silastic implants containing either estradiol (n = 6) or progesterone (n = 6). After 4 days of treatment, EOP inhibition of LH secretion was measured by determining the LH response to WIN 44,441-3 (WIN), an EOP antagonist. LH pulse frequency and pulse amplitude were determined in blood samples collected at 12-min intervals for 3 h before and after intravenous administration of 12.5 mg WIN. WIN injection increased (p < 0.01) the LH pulse frequency only in progesterone-treated and pulse amplitude only in estradiol-treated ewes. After blood sampling, the ewes were killed, and POA, MBH, and pituitary gland were removed. Total RNA was extracted from these tissues and dot blotted onto nitrocellulose membranes for hybridization with a DNA probe complementary to the POMC mRNA. The resulting autoradiographs were quantified densitometrically. Levels of POMC mRNA in the MBH were increased (p < 0.01) by both estradiol and progesterone as compared with the no steroid group. There was no detectable POMC mRNA in the POA. These results suggest that estrogen and progesterone enhance EOP inhibition of LH secretion by increasing POMC mRNA levels and thus synthesis of β-endorphin.

Background: Chest wall recurrence (CWR) causes much morbidity when not controlled by local measures. Lyso-thermosensitive liposomal doxorubicin (LTLD) provides accelerated release of doxorubicin (Dox) at ≥ 39° C, and is being studied for CWR based on the pioneering thermochemotherapy work of Dewhirst and colleagues at Duke. We now report on its tolerance, pharmacology, and preliminary antitumor effects in a dose-escalation study combined with local hyperthermia (LH) for radiation-refractory CWR. LH selectively increases liposomal permeability in tumor microvasculature, and promotes release of Dox from LTLD, and Dox tumor uptake, in addition to LH anti-tumor effects. Methods: This phase I study entered patients (pts) with CWRs &lt; 3 cm deep failing all standard Tx including surgery, radiation, and chemotherapy: pts received up to 6 LTLD/LH treatments every 21 days at a starting dose of 40 mg/m2 (cohort 1) and escalated to 50 mg/m2 (cohort 2). LTLD was infused IV over 30 minutes (min); followed within 30 min by microwave or ultrasound LH. The thermal dose goal was 40°C-42°C for 60 min. Pharmacokinetic samples for total plasma Dox and doxorubicinol (Doxol) were taken at 0.5, 5, 10 and 24 hours after starting infusion. Left ventricular ejection fraction was monitored every other cycle. Results: Eleven pts with a median of 4 prior chemotherapy regimens (range 2 — 12) were enrolled; all but one had prior anthracycline (AC). All pts received ≥ 2 cycles. The within subject variability in Dox and Doxol exposure was small with mean Cycle 2 vs Cycle 1 ratios ranging from 0.99 to 1.06. Cmax/dose (ng/ml)/(mg/m2) Cycle 1 Cycle 2 Dox 499.82 512.00 Doxol 0.46 0.45 AUClast/dose ((ng*hr/ml)/(mg/m2) Dox 1338.12 1381.82 Doxol 7.96 8.04 Grade 3 and 4 toxicities included reversible neutropenia in 17 (40.5%) and one case (each) of mucositis (grade 1), chest wall thermal burn, and chest wall cellulitis (both grade 4); these occurred in only ≥ 5% of 42 cycles given. No cardiomyopathy or hand-foot toxicity occurred. The rate of clinically-significant (≥ 6 point) QoL improvement on the FACT-B after 2 cycles was 54.5% (95% CI: 25.1%–83.9%), including 1 lasting &gt; 3 months. The local objective response rate was encouraging: 45.5% (95% CI: 16.1%–74.9%), with 1 complete and 4 partial local responses. Conclusion: LTLD + LH is safe in CWR after prior radiation and doxorubicin. A phase II study is planned for pharmacologic evaluations and to add pts with less or no anthracycline treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-13-01.

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