Effect of Dose, Timing and Delivery of Tumor Necrosis Factor Alpha as an Adjuvant in Cryosurgery of ELT-3 Uterine Leiomyoma (Fibroid) Tumor

Uterine leiomyoma (fibroid or myoma) is the most common indication for hysterectomy in premenopausal women. Cryomyolysis is a uterus sparing procedure in which a myoma is frozen by a cryoprobe, thereby causing tissue necrosis upon thawing and eventual reduction in myoma size. Unfortunately, although the iceball is readily visualized (by ultrasound-US or magnetic resonance-MR), the tissue at the periphery of the iceball is not completely destroyed. One potential solution to this problem is the use of cryosurgical adjuvants that increase cryosurgical image guidance and efficacy. Previous work in our lab has shown that TNF-$α$ (native or as the nanodrug, CYT-6091, Cytimmune Sciences, Inc.) can act synergistically with cryosurgery to destroy all prostate cancer within an iceball. Building on this work, the current study was designed to test TNF-$α$ as an adjuvant in an in vivo model of uterine fibroid (ELT-3) in a nude mouse. The aims of this study are to characterize in vivo: 1) the destruction of the uterine fibroid over time after cryosurgery; 2) the effect of TNF-$α$ pre-treatment on enhancement of cryosurgery; 3) the effect of TNF-$α$ dose, pre-treatment time and mode of delivery on the above and to note any toxicities. ELT–3 rat uterine fibroid cells were grown in the hind limb of female nude mice. TNF-$α$ at various dose (⁠$2μg$ and $5μg$⁠) was administered at 1, 2 and 4 hours before cryotreatment in native or CYT-6091. Native TNF-$α$ was injected either intra-tumorally or peri-tumorally. Injecting TNF-$α$ solution into the center of the tumor comprised the intra-tumoral approach. For peri-tumoral injection, TNF-$α$ solution was injected at each one of eight evenly distributed points spanning the circumference of the tumor base. CYT-6091 was administered by i.v. injection only. Cryosurgery was performed with a modified 1 mm diameter cryoprobe tip $(−120°C)$⁠. Freezing was allowed to continue to the visible edge of the tumor. Injury was assessed by measuring tumor-growth delay. Baseline tumor size was measured on day 0; fold-changes in tumor size are reported relative to size at day 0. Toxicity was evaluated by survival rate. Groups were 4–6 animals in each group. The data suggests that pre-treatment with TNF-$α$ before cryosurgery significantly enhances visually guided destruction of uterine leiomyoma, and that the dose, timing and mode of delivery are important variables in optimization of this combination treatment. First, it was observed that at least four hours pretreatment with TNF-$α$ is required to obtain the synergistic effect of TNF-$α$ and cryoinjury. Second, peri-tumoral injection of native TNF-$α$⁠, was the most effective delivery method to enhance cryoinjury at low dose $(2μg)$⁠, however it was also the most toxic method at high dose $(5μg)$⁠. On the other hand, CYT-6091, although less effective than peri-tumoral injection at $2μg$⁠, was the safest delivery mode (0% lethality at $2μg$⁠; 33% at $5μg$⁠). Finally, CYT-6091 delivery at $5μg$ with cryosurgery resulted in a dramatic tumor growth delay compared with cryosurgery alone. Therefore, i.v. injection of CYT-6091 followed by cryosurgery allowed the highest dose of TNF-$α$⁠, the least toxicity and the best overall myoma reduction. Funding: R01 CA075284, American Medical Systems, Inc. TNF-$α$ and CYT-6091: Cytimmune Sciences, Inc.