MONOTHERAPY                                                                   

In Vitro Efficacy

Radioreceptor binding assays show Prolanta has a high affinity for the human prolactin receptor (PRLR). As Prolanta is nearly identical to prolactin (PRL), it is anticipated that the ADME (absorption, distribution, metabolism, excretion) profile for Prolanta will be very similar to PRL.

PRL signaling via STAT5 causes significant up-regulation of Bcl-2, known to block apoptosis in tumor cells. Prolanta blocks PRL up-regulation of Bcl-2, thereby removing this impediment, resulting in apoptosis.

The Bcl-2/BAX ratio within any cell determines its apoptotic fate. High Bcl-2/BAX ratios allow continued proliferation and inhibit apoptosis while low Bcl-2/BAX ratios trigger apoptosis. While PRL signaling causes a high Bcl-2/BAX ratio, Prolanta (G129R) triggers the opposite scenario, a low Bcl-2/BAX ratio. Studies have also shown that not only does Prolanta prevent signal cell proliferation via STAT5 but this molecule also inhibits/blocks this signal transduction pathway, even in the presence of PRL. Prolanta's mechanism of action is through blocking signal transduction by multiple routes: STAT3, STAT5 and to a lesser extent through pMAPK and pAKT.

In Vivo Efficacy

Transgenic mice that constitutively over-express PRL have a high incidence of breast cancer. These mice generate high Bcl-2/BAX ratios in breast tissue and low or no cytochrome c release from mitochondria (a measure of apoptosis). Prolanta transgenic mice generate a lower than normal Bcl-2/BAX ratio and cause the appearance of cytochrome c in breast tissue showing a stimulation of apoptosis as compared to normal non-transgenic mice.

Thirty 6 week old nude mice were implanted subcutaneously with slow-releasing estrogen pellets. Five million T47D breast cancer cells mixed with Matrigel were injected into the mammary fat pad. One week after tumor cell inoculation, the mice were randomized into three groups and treated 5 times/week with either Matrigel control, hPRL/Matrigel or G129R/Matrigel for seven consecutive weeks.

The phosphorylation of the human proto-oncogone, HER2 in breast cancer cells directly determines its oncogenic potential. Inhibitory effects of Prolanta on the phosphorylation of HER2/neu has been demonstrated in natural tumors developed in HER2/neu transgenic mice. Tumor-bearing mice were treated with 200 micrograms of Prolanta for 5 or 10 days. The tumors were removed 24 hours after the last treatment. Lysates of the tumor cells were evaluated for levels of phosphorylated HER2/neu (pHER2/neu), by Western blotting. Results indicated that of the 14 mice that received a 5 day treatment, 5 mice were high responders with a drastic reduction in the level of pHER2/neu 4 were moderate responders and 5 were non-responders. Of the 10 mice receiving a 10 day treatment, 3 mice were high responders with a drastic reduction in pHER2/neu level, 5 were moderate responders and only 2 were non-responders.

Prolanta also has inhibitory effects on the growth of tumors in the HER2/neu transgenic mice. Tumors were isolated from female transgenic mice, sectioned into equivalent sized pieces and implanted into recipient age-matched female transgenic mice. Ten days after implantation, mice were randomized into either control (n=3-4) or Prolanta-treated groups (n=4, 10mg/kg, i.p. daily).  Tumor volume and tumor weight were greatly reduced in the Prolanta treated mice.

Prolanta has also been shown to reduce the number of cancer stem cells in the HER2/neu transgenic mouse tumor. The number of these stem cells is estimated by determining the activity of an enzyme, aldehyde dehydrogenase (ALDH), that is selectively expressed in these cells. Cancer stem cells are postulated to be the cells responsible for initiating and sustaining the growth of the tumor. They comprise a rare, phenotypically distinct subset of cells within the tumor that can proliferate and form new tumors very efficiently.

Tumors were removed from HER2/neu mice following control treatment or Prolanta treatment (200 ug/day, i.p. for 5 or 10 day)

Tumor tissues were isolated at the end of the treatment, digested to single cell suspension and the level of ALDH enzyme activity was measured. Treatment with Prolanta for either 5 or 10 days dramatically reduced the number of cancer stem cells present in the cell suspension.