By David N. Leff
Just as you can't change a sow's ear into a silk purse, so you can't change a mouse into a man — or a woman, for that matter.
The closest that cancer researchers have come to this murine-human conversion is implanting human tumor cells into nude mice, which lack the immune defenses to reject them as does a non-mouse.
Now, a group of Italian oncologists has teamed with a U.S. biotech company to narrow this human-subhuman gap by treating their animal models with therapeutic regimens that more closely approximate the multi-drug anticancer treatments that humans get.
At the University of Naples, medical oncologists Giampaolo Tortora and Fortunato Ciardiello run what they call a "translational research laboratory." Its aim, Ciardiello told BioWorld Today, "is to translate basic bench science into clinical practice, by way of in vitro and in vivo research."
At Hybridon Inc., in Cambridge, Mass., biochemist Sudhir Agrawal, the company's chief scientific officer and senior vice-president/discovery, offered the Neopolitans the latest versions of his antisense oligonucleotides, for testing in their anticancer strategies.
The target of these RNA sequence blockers is protein kinase A (PKA), a powerful enzyme that promotes cell division and growth. That capacity is a plus in normal, healthy tissues, but a decided minus in malignant cells, which never stop multiplying.
Actually, the Hybridon antisense oligos that the Italians are testing in vitro and in nude mice do not target the entire PKA molecule, but a small regulatory subunit of it, R1-alpha. In healthy cells, R1-alpha and its twin subunit R1-beta, co-exist in equal amounts, balancing cell proliferation against cell growth arrest.
But alpha is over-expressed in some tumor cell types, which makes it legitimate prey for blockage by antisense oligos.
"Those two catalytic subunits of PKA," Agrawal told BioWorld Today, "form the complex to become an active PKA enzyme, which has a cell-cycle function of signal transduction, involving many pathways. So our idea is to suppress the R1-alpha, and see what the changes are."
An article in the current issue of the Proceedings of the National Academy of Sciences (PNAS), dated Nov. 11, 1997, reports on the Italian antitumor experience testing Hybridon's antisense oligos alone, and in combination with virtually the entire arsenal of standard cancer chemotherapy drugs. Their paper bears the title: "Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A." Its joint senior authors are Torotora and Ciardiello; Agrawal is a co-author.
After extensive in vitro trials, the Italian team xenografted one million human colon cancer tumor cells under the skin of nude mice. Ten days later these inocula had grown into visible, palpable tumors 0.4 cubic cm in volume.
Score Card: Plus/Minus Antisense Oligos, Anticancer Drugs
At that point, four groups of seven animals each received high-dose shots of paclitaxel — a potent cytotoxic drug — alone, or with one of two antisense oligos, HYB 190 or HYB 239. Prior in vitro testing had shown 190 to be a potent antitumor agent, while 239 was scarcely more effective than a placebo. Doses of these oligos went into one or another of the animal test cohorts, for five consecutive days following the paclitaxel.
Untreated, control mice died within 40 days; those that got either 239 alone, paclitaxel alone, or the two together, were dead by 60 days. But the lucky animal models that received the cytotoxic chemotherapy combined with HYB 190, Ciardiello told BioWorld Today, "were still alive up to 67 days after xenografting. On that day," he continued, "we stopped the experiment and sacrificed those mice in order to examine the pathology of their tumors." These were half the size of the cancers growing in the other cohorts.
"What we are doing now, in our present ongoing series of experiments," Ciardiello went on, "is to keep the animals alive as long as possible, compatible with life with the tumors. What I expect, since the previous 67-day animal trials involved only a single cycle of treatment, if we keep the animals for a longer period, sooner or later — maybe a little later — the tumor will start to grow again, and eventually kill them. This is the reason we are now doing two or three or four cycles of chemotherapy, each followed by antisense oligos."
Agrawal added: "In this case, as reported in PNAS, "we wanted to see how long tumor suppression would last. The paper reported that a single dose of paclitaxel delays tumor growth. But after that, with five daily doses of HYB190, we can further delay tumor growth for quite a long time, and with no toxic side effects, compared to paclitaxel's considerable toxicity."
Clinical Trials Next Year
Ciardiello observed: "Our purpose is just to obtain a model that can be translated to cancer clinics."
When this can happen, he added, "depends mostly on the work that Dr. Agrawal and his colleagues are doing at Hybridon, in terms of bringing these or other anti-R1-alpha antisenses to the clinic. I guess they are starting right now," he went on, "with Phase I trials of antisense alone, before synergizing these with chemotherapy."
So they are, confirmed Hybridon's executive director of clinical research, William Slichenmyer. "We have an improved oligo design, which we call GEM 231. It has the same sequence as HYB 190, but with both ends capped by segments of modified RNA, to improve metabolic stability and the safety profile."
Hybridon filed an IND (investigational new drug application) with the FDA last week for GEM 231, Slichenmyer revealed. He told BioWorld Today, "We do expect our Phase I trial of GEM 231 to start enrolling patients with refractory solid tumors in January. Then, if all goes according to plan, a Phase II clinical study, combining the new oligo with another antitumor agent, will begin in mid-1998."
Asked to describe the mechanism of such synergism between antisense and cytotoxic compounds, Ciardiello replied: "This is really the best question you could ask. We do not have the answer yet, because, as suggested in our PNAS paper, there is potentiation of apoptosis. But apoptosis is a terminal event, so we don't know what happens upstream of that. But this is all in progress in our lab." *