Cancer & HIV Controlled With ONCONASE, A Novel RNase Therapeutic
Carmine M. Zingarino
gn686 at cleveland.Freenet.Edu
Sat Apr 15 22:22:47 EST 1995
The article is appended to this message.
Cancer & HIV Controlled With ONCONASE, A Novel RNase Therapeutic
by Carmine M. Zingarino
1 INTRODUCTION - What is ONCONASE? (1-8,11-19)
The word "ONCONASE" is a contraction of the words ONCOLOGY and
RIBONUCLEASE (RNASE). It is used to identify a protein substance that
possesses anti-cancer properties and RNase specific enzymatic
In fact, Onconase is a ribonuclease protein (RNase) derived from the
eggs (oocytes) and early embryos of the leopard frog "Rana pipiens." It
is a novel p
rotein based on its comparison with over 10,000 other
proteins registered with the National Biomedical Research Foundation
Protein Identification Resource. Though the scope of its anti-cancer
activity embraces a wide variety of tumor cell types, it is especially
active against carcinomas (i.e. solid tumor cancers), which may account
for about 90% of all cancers. Alfacell Corporation - a
biopharmaceutical company located in Bloomfield, New Jersey - is
credited with the discovery of this RNase prote
In addition to the anti-tumor properties just noted Onconase also
possesses anti-viral activity. In particular, scientists at the
National Institutes of Health (NIH) have shown that Onconase is active
against the HIV-1 virus, the causative agent of AIDS. NIH studies
demonstrate that Onconase inhibits replication of the HIV-1 virus up to
99.9% "in vitro" within 4 days.
The scientific data clearly demonstrates that Onconase has
considerable potential in the treatment of cancer and HIV inf
These considerations convinced the Food and Drug Administration (FDA) in
January 1995 to approve Onconase for a Phase III clinical trial
involving pancreatic cancer. In fact, Onconase is the first RNase drug
to reach this stage of clinical testing. Consequently, it represents an
entirely new approach to the treatment of cancer and viral infections.
This article summarizes the scientific research and developmental
history of ONCONASE (P-30 protein), a novel RNase therapeutic.
1 Introduction - What is ONCONASE?
3 Basic Science Research
3.1 Basic Chemistry & Physical Characteristics
3.1.1 Primary Structure
3.1.2 Secondary & Tertiary Structure (X-ray Crystallographic Studies)
3.1.3 Enzymatic Properties of the RNase Subunit
3.2 Biological Activity Profile
3.2.1 Anti-cancer Activity
126.96.36.199 "In vitro" anti-tumor activity
188.8.131.52.1 Early "in vitro" studies
tic Activity Observed)
184.108.40.206.2 Hypothesized Cytotoxic Mechanism of Action
220.127.116.11 Synergism with other chemotherapeutic agents
18.104.22.168 "In vivo" anti-tumor activity in mice
3.2.2 Anti-HIV Activity
4 Clinical Studies
4.1 Phase I Clinical Trial - Toxicity Profile /
Objective Clinical Activity Noted
4.2 Phase II/III Clinical Trial
4.2.1 Clinical Response in Pancreatic Cancer Patients
4.3 A Clinical Trial for HIV?
5 Uniqueness of the Onconase Protein Molecule
6.1 National Institutes of Health (NIH)
6.2 National Cancer Institute (NCI)
6.3 Other Biologically Active Proteins
3 BASIC SCIENCE RESEARCH
3.1 Basic Chemistry & Physical Characteristics
3.1.1 Primary Structure (4,17)
The Onconase protein molecule is a single chain of 104 amino acid
residues. It has a calculated molecular mass of 11,834 Daltons, and is
rich in the basic amino acid
lysine. A total of twelve lysine residues
or approximately 11.54% of the molecule is composed of lysine. It is a
novel protein based on the comparison of its amino acid sequence with
over 10,000 protein sequences registered with the National Biomedical
Research Foundation Protein Identification Resource.
The sequence homology demonstrates that it belongs to the pancreatic
RNase superfamily. In fact, it is the smallest member of this
superfamily known to date. Onconase demonstrates 30% identit
RNase A and 27% identity with bovine seminal RNase (BS-RNase). Both
Onconase and BS-RNase exert growth-inhibitory activity on neoplastic
cell lines (9). These considerations support the notion that proteins
belonging to the RNase superfamily exhibit tumor growth-inhibitory
3.1.2 Secondary & Tertiary Structure
(X-ray Crystallographic Studies) - (7,19)
The secondary structure reveals 40.4% antiparallel Beta strand, 20.2%
helix, 39.4% turns and aperiodic segments (loops).
Beta strands are manifested as two three-stranded Beta sheets. This
gives the molecule a bilobate appearance. These strands are designated
Beta-1, Beta-3, Beta-4 in the first Beta sheet and Beta-2, Beta-5,
Beta-6 in the second Beta sheet. The residues assigned a helical
conformation form three helices Alpha-1, Alpha-2 and Alpha-3. The
Alpha-1 helix is entirely alpha-helical in nature. The other two alpha-
helices are distorted and contain mixtures of alpha-helix and 3(10)
x. Nevertheless, the structural roles of the Alpha-2 and Alpha-3
helices are similar. Both pack against one of the three stranded
antiparallel Beta sheets. In each the helix axis is almost
perpendicular to the strand direction. The balance of the molecule
includes four reverse turn segments and several aperiodic segments.
Additionally, Onconase is a disulfide rich protein molecule. It
contains eight cysteine residues which form four disulfide bonds. Three
of these four bonds have counterpart
s in RNase A. The fourth disulfide
bond is unique to the amphibian members of the RNase superfamily.
The active site is a long cleft formed at the junction of helix
Alpha-1, strand Beta-1 and strand Beta-6. It runs almost parallel to
strand Beta-6. Seven amino acid residues contribute to the active site.
They include: an N-terminal pyroglutamyl residue (Pyr1), Lys9, His10,
Lys31, Thr35, His97 and Phe98.
3.1.3 Enzymatic Properties of the RNase Subunit (4,19)
Onconase's RNase subunit is esp
ecially active against highly
polymerized ribosomal RNA. This activity is essential to its
antiproliferative effect. Though Onconase and RNase A have a similar
pyrimidine sensitive endoribonucleolytic activity, Onconase is
specifically active against uridylyl-3',5'-guanosine (UpG) and
cytidylyl-3',5'-guanosine (CpG). RNase A by contrast hydrolyzes all
diribonucleotide combinations that begin with a pyrimidine (UpX, CpX
where X = A,C,G,U). In either case ribonucleolytic activity proceeds in
steps (i.e. an intramolecular transesterfication followed by
hydrolytic cleavage of the cyclic phosphate intermediate). The result
is a polyribonucleotide that ends with a 3'-phosphoester of cytidine or
uridine. This reaction is catalyzed in both Onconase and RNase A by
three homologous amino acid residues (i.e. two histidine residues and
one lysine residue). With Onconase these are His10, Lys31 and His97.
3.2 Biological Activity Profile
3.2.1 Anti-Cancer Activity
22.214.171.124 "In vitro" anti-tum
126.96.36.199.1 Early "in vitro" studies (Cytotoxic/Cytostatic
Activity Observed) (1,2,3,17)
Onconase exerts a powerful effect on the cell proliferative cycle in
tumor cell cultures. Flow cytometry measurements demonstrate that an
accumulation of G1 phase cells, and a decrease in both S phase (DNA
synthesis) and G2+M (mitosis) fractions occurs in treated cell cultures.
The increase in the proportion of G1 phase and decrease in S phase cells
suggests that Onconase either arres
ts a portion of the cells in G1 or
prolongs duration of G1 phase of all cells (cytostatic effect).
Alternatively, Onconase may be selectively toxic to S phase cells
(cytotoxic effect). Cells with low RNA content also were observed in G1
phase. This is a feature typical of quiescent cells, and suggests that
Onconase may trigger transition of cells from the cell cycle to
quiescence. Such an event in tumor cells is short lived and can result
in loss of cell viability (1).
"In vitro" studies on h
uman colorectal carcinoma (COLO 320DM) have
been performed by Dr. Vincent Allfrey at Rockefeller University in New
York. Based on these studies Dr. Allfrey concludes that Onconase (P-30)
contributes to the down regulation of the c-myc oncogene and, at higher
doses, contributes to increased expression of the c-fos gene. Dr.
Allfrey claims this combination of events may be consistent with a
differentiation inducing effect(s) (2,3). As already noted Onconase
inhibits cell cycle progression from G1 t
o S phase. Since the degree of
c-myc expression is inversely correlated with the duration of G1 phase,
this observation concurs with the decreased c-myc expression observed by
Dr. Allfrey (3).
Nevertheless, tumor cells are ostensibly more susceptible to Onconase
than are normal cells. Some scientists contend that the malignant
phenotype of tumor cells may mimic characteristics of early embryonal
cells. Since Onconase is derived from oocytes and early embryos of
"Rana pipiens," it could exploit
this feature and act preferentially
against tumor cells. Alternatively, it could be substituting for a
specific anti-growth factor deficiency in tumor cells (3).
These considerations suggest that Onconase may play a role in the
determination of cell fate. In particular, it may induce tumor cell
apoptosis (i.e. programmed cell death) (15).
Onconase is unique in its pattern of anti-tumor activity against a
variety of cultured human tumor cell lines "in vitro." Additional
research studies demo
nstrate that it halts cell cycle progression in
either the G1 or G2 phase of the cell cycle. This was determined by MTT
assay (Microculture tetrazolium assay), clonogenicity, and flow
cytometry studies in four human tumor cell lines (i.e. HL-60 human
leukemia cells, COLO 320DM human colon adenocarcinoma, A-253 human
submaxillary carcinoma, and A-549 human lung adenocarcinoma). With
HL-60, COLO 320DM and A253 cells Onconase inhibited cell cycle
progression in the G1 phase. A-549 cells, however, w
ere inhibited in
the G2 phase (1,2,16).
Note: Flow cytometry studies were performed by Zbigniew Darzynkiewicz,
M.D., Ph.D. and his associates. Dr. Darzynkiewicz is director of the
Cancer Research Institute at the New York Medical College and professor
of medicine at New York Medical College. The flow cytometry studies
were performed during his tenure with Sloan-Kettering Institute for
Cancer Research in Rye, New York.
188.8.131.52.2 Hypothesized Cytotox
ic Mechanism of Action (13)
Based on studies performed by Dr. Richard Youle at the NIH Onconase's
cytotoxic mechanism of action has been partially delineated. Here is a
summary of the events involved in this cytotoxic mechanism.
Onconase Cytotoxic Mechanism of Action
1) The Onconase molecule appears to have a distinct affinity for
receptors on tumor cells. As a result, Onconase molecules bind
preferentially to these tumor cell surface receptors. This also
demonstrates that the molecule
possesses a receptor binding moiety in
addition to a RNase enzymatic subunit.
2) The binding event ostensibly induces a conformational change in the
cell membrane cytoskeleton that leads to endocytosis with the bound
Onconase molecule. Consequently, cytosolic internalization of the
molecule may occur by endocytosis.
3) Cytosolic routing of the internalized molecule is a subject of
current investigation. However, once entry to the cytosol is
accomplished, ribonucleolytic degrad
ation of RNAs occurs. In
particular, the enzymatic subunit hydrolyzes the 18S and 28S
subunits of the ribosomal RNA (rRNA).
Recall that cells having low RNA content in G1 phase were revealed by
flow cytometry measurements. This observation, therefore, is
consistent with the degradation of rRNA.
Note that the ribosomes are the infrastructure upon which cell
protein synthesis occurs. Once the ribosomes have been degraded through
Onconase hydrolysis of rRNA, protein synthesis is i
nhibited. This may
result in cell death.
Also note, Onconase is an amphibian RNase. Consequently, mammalian
RNase inhibitors have no effect on its activity. Its RNase activity,
therefore, is unrestrained in mammalian tumor cells. This makes
Onconase an effective cytotoxin in extremely small concentrations
(e.g. 1 x 10 -6 M).
184.108.40.206 Synergism with other chemotherapeutic agents (2,6,8,11,17)
Onconase as a single agent is an exceptional anti-cancer compound. It
can either inhibit tumor cel
l growth or kill tumor cells (i.e. is
cytotoxic). Nevertheless, its anti-cancer activity can be potentiated
when used in combination with other chemotherapeutic drugs. Onconase
acts in a synergistic fashion with these other drugs. Its cytotoxic
effect was especially pronounced when used with anti-estrogen compounds
(e.g. Tamoxifen). Besides Tamoxifen, Onconase has been studied with
Trifluoperazine (Stelazine), Lovastatin, and Cisplatin. The performance
of these therapeutic combinations were eva
luated on a variety of human
cancer cell lines. Researchers have concluded that Onconase and
Tamoxifen work effectively on ASPC-1 pancreatic adenocarcinoma and
OVCAR-3 ovarian carcinoma. Onconase and Trifluoperazine work
effectively on A-549 lung adenocarcinoma. Onconase and Lovastatin are
effective against ASPC-1 pancreatic adenocarcinoma, OVCAR-3 ovarian
carcinoma, A-549 lung adenocarcinoma and HT-520 lung carcinoma.
Onconase and Cisplatin are effective against OVCAR-3 ovarian carcinoma.
.1.3 "In vivo" anti-tumor activity and
primary toxicology studies (3)
The success of the "in vitro" studies motivated several researchers
to examine Onconase's activity "in vivo." Accordingly, "in vivo"
studies of Onconase's toxicity and efficacy were performed on mice, rats
and dogs. The results of these studies were extremely encouraging.
For example, BALB/c mice with M109 mouse Madison lung carcinoma had
an ILS (Increased Life Span) of over 180% when treated with Onconase.
is is in contrast with control groups that were untreated. Animals
treated on a weekly bolus schedule showed less toxicity and greater
antitumor response than those treated on a daily schedule. In fact, the
majority of the long term survivors were treated on the weekly schedule.
All twelve survivors of this study were apparently cured of cancer.
A dose dependent toxicity was noted that consisted of weight loss,
diminished vigor, reduced food intake and ruffled coat. However, the
ies disappeared when treatment was withdrawn.
3.2.2 Anti-HIV Activity (16,18)
Studies performed by Dr. Richard Youle at the NIH demonstrate that
Onconase inhibits HIV-1 viral replication in H9 Leukemia cells
"in vitro" by 90 to 99.9% within 4 days. Moreover, the Onconase
concentrations used in the study were not toxic to uninfected H9 cells.
The research data intimates that Onconase's entry to the white blood
cell cytosol is viral assisted. The research team believes that
become associated with either the viral particle or
the area of the cell membrane through which the virus enters.
Consequently, Onconase molecules may enter the cell with the HIV-1
Four members of the RNase superfamily were studied for anti-HIV
activity by the research team. These four RNases are eosinophil derived
neurotoxin (EDN), bovine pancreatic RNase A, Onconase and bovine seminal
RNase (BS-RNase). Only Onconase and BS-RNase demonstrated anti-viral
activity. However, the anti-v
iral activity displayed by Onconase was
significantly greater than the activity observed with BS-RNase.
When white blood cells become infected with the HIV-1 virus, an
exponential increase in the production of certain viral proteins occurs
(e.g. p24 antigen and reverse transcriptase). Therefore, we can measure
the degree of viral replication by examining the levels of these
proteins. As a result, the research team observed one such protein
throughout the study after treatment with each of the fo
ur RNases. The
viral p24 antigen was selected for this purpose.
Onconase inhibited p24 expression by 99 to 99.9% at a concentration
one order of magnitude less than the concentration required for
cytotoxicity in cancer cells. Moreover, p24 expression was reduced
significantly the first day after viral infection. Even with
concentrations that were two orders of magnitude less than the cytotoxic
amount Onconase inhibited viral replication. About 90% inhibition was
achieved within four days at t
hese concentrations. So, the study
concludes that subtoxic concentrations of Onconase are very effective at
inhibiting viral replication.
The research study also demonstrates that Onconase does not operate
on the virus outside the cell. It must enter the cell with the virus to
inhibit viral replication.
Besides the increase in viral proteins, the formation of syncytial
cell aggregates is observed in HIV-1 infected cells. However, when
these infected cells are treated with Onconase, the forma
syncytial cell aggregates is abrogated. In fact, the cells are
indistinguishable from uninfected cells. This histological view
correlates perfectly with the biochemical data on reduced viral p24
Anti-Viral Mechanism of Action
The research team hypothesizes that the mechanism of Onconase's anti-
viral activity may be similar to its anti-cancer activity. Once inside
the white blood cell, Onconase may hydrolyze the cell's ribosomal RNA.
Since viral protein production dep
ends on the integrity of the host
cell's ribosomes, any degradation of the ribosomes will have an impact
on viral replication.
Note, HIV-1 is a RNA virus. Its genetic material is composed of RNA
not DNA as in most other life forms. Accordingly, the HIV-1 viral RNA
could be vulnerable to hydrolytic attack by Onconase. This also would
inhibit viral replication. However, further study is needed to confirm
A primary concern of medical professionals is controlling the virus's
ability to r
einfect neighboring white blood cells (i.e. lateral
transmission). Consequently, any activity that degrades either the
ribosomes or the viral RNA could help slow the rate of this infection.
Augmentation of Anti-HIV Activity
The research team also suggests that Onconase conjugates could be
engineered to target HIV infected cells. These conjugates may increase
Onconase's endogenous anti-viral activity. The research team considers
CD4 and monoclonal antibodies that bind HIV infected cells as pote
candidates for conjugation with Onconase. With this technique the
Onconase conjugate would compete with the virus for available CD4
receptor binding sites. Moreover, the rate of Onconase's entry to the
cell cytosol would increase. This also would help slow the rate of
reinfection in neighboring white blood cells.
4 CLINICAL STUDIES
The FDA officially recognized Onconase as a drug substance possessing
anti-tumor activity in October 1987. Having cleared this major
regulatory hurdle, On
conase began Phase I clinical trials in April 1989.
This section reviews some clinical trial results.
4.1 Phase I Clinical Trial - Toxicity Profile /
Objective Clinical Activity Noted (5,12)
Twenty - eight patients were administered a weekly intravenous (IV)
bolus of Onconase (ONC) in the Phase I study. The goal of the Phase I
study was to assess the maximum tolerated dose (MTD), and the safety and
toxicity of Onconase in these patients. This study demonstrated t
Onconase was well tolerated by the majority of Phase I patients. It
exhibited a consistent and reversible clinical toxicity pattern, and did
not induce most of the toxicities usually associated with other
chemotherapeutic agents such as alopecia (hair loss) and myelosuppression
(bone marrow suppression). A study of the clinical symptoms demonstrated
that peripheral edema (i.e. swelling of the appendages due to water
retention) and asthenia (i.e. weakness and fatigue) were the two main
fects of Onconase treatment.
The high degree of homology to known low-immunogenicity proteins
suggests that Onconase may be weakly immunogenic if at all to humans.
Nevertheless, there was no clinical evidence of a true immunological
sensitization to the drug. In fact, Onconase served to boost the human
body's natural defense mechanisms by contributing to an increase in the
total white blood cell count. Consequently, the research team
concluded that the drug is safe to humans. The maximum tolera
IV bolus dose (MTD) of Onconase was 960 ug/m2 (16X), with the dose
limiting toxicities being renal, (i.e. dependent on kidney function).
The anticipated human dose is 60 ug/m2 (1X). Dose levels are stated as
micrograms per square millimeter of human body surface area (ug/m2).
Objective clinical responses were observed in Phase I patients with
advanced and resistant to therapy tumors. Six of the twenty-eight
patients treated with Onconase responded to treatment. This represents
ponse rate of 21.43% in the Phase I trial. The responses ranged
from stabilization of disease to partial remission. Stabilization of
disease (SD) was observed in three patients. One of these patients had
a malignant thymoma, another had a colorectal carcinoma, and the third
had a non-small cell carcinoma (NSC) of the lung. A minor response (MR)
was observed in a patient with colorectal carcinoma. In fact, this tumor
had metastasized to the lungs. Two partial remissions (PR) of the
also observed. One was seen in a patient with an
adenocarcinoma of the lung and one in a patient with esophageal carcinoma.
The tumor in the patient with esophageal carcinoma also had metastasized
to the lungs. Recall that the goal of the Phase I study was to assess
the maximum tolerated dosage, safety, and toxicity of Onconase.
Nevertheless, the clinical responses just noted demonstrate that some
efficacy was observed.
Criteria For Clinical Response (12)
This section lists the criteria used by
the clinical research team to
evaluate clinical response.
CR: Complete Remission Disappearance of all clinical evidence of active
tumor, and patient free of any symptoms related
to cancer for at least one month.
PR: Partial Remission 50% or greater decrease in the sum of the
products of the bidimensionally measured diameters
of all measurable tumor lesions; no simultaneous
increase in the size of any lesion or appearance
of any new lesions.
MR: Minor Response Response less than PR but more than SD,
i.e. between 25 to 50%.
SD: Stable Disease Steady state or response less than 25%.
PD: Progressive Disease Unequivocal increase of at least 25% in the size
of any measurable lesion, or the development of
new metastatic lesions.
Relapse: The appea
rance of new lesions; the re-appearance
of old lesions in patients who were in CR.
For patients in PR, an increase of 25% or more
in the sum of the products of the diameters of
all measured tumors over that which was obtained
at the time of maximum regression.
4.2 Phase II/III Clinical Trial
Phase II clinical trials establish safety and efficacy. Onconase began
Phase II clini
cal trials in January 1991. A total of 245 patients with a
variety of tumor types have been studied in this trial. Among these are:
51 pancreatic carcinoma patients, 43 non-small cell (NSC) lung cancer
patients, 11 mesothelioma patients, 17 breast cancer patients, 18 melanoma
patients, 50 colorectal cancer patients and 55 patients with other cancers.
In pancreatic cancer patients Onconase is being studied in combination
Analysis of the drug's safety on all 245 patients has been
and the safety profile is excellent. Analysis of efficacy has been
completed for the pancreatic carcinoma patients and the NSC lung patients.
These results have been submitted for publication. Efficacy evaluation of
the remaining patients and cancer types is in progress.
4.2.1 Clinical Response In Pancreatic Cancer Patients
Pancreatic cancer claims the lives of more than 25,000 Americans a
year. It has a median survival rate of 90 days, and no standard
effective treatment current
ly exists for this disease. Moreover, new
chemotherapeutic compounds have been tested in these patients
unsuccessfully. For example, Tamoxifen as a single agent has been
completely ineffective in the treatment of this disease (10).
On the other hand, Onconase as a single agent is moderately effective
in the treatment of pancreatic adenocarcinoma. Moreover, as the
"in vitro" studies have established, the synergism between Onconase and
anti-estrogen compounds (e.g. Tamoxifen) can produce a potent
tumor response. Accordingly, a combination therapy of Onconase and
Tamoxifen was administered to fifty-one advanced and resistant to therapy
pancreatic carcinoma patients. The recently completed Phase I/II study
demonstrates that this combination therapy is active against advanced
Forty-six of the patients, or 90% were diagnosed as having Stage 4
disease. This means that the cancer had spread beyond the pancreas to
distant sites in these patients. Thirty-
two patients were evaluable
(i.e. they were administered at least 3 doses of Onconase). As of
October 31, 1994 the median survival of these thirty-two patients was
3.7 months. In eight of these patients (i.e. 25%) objective clinical
activity was observed. With one individual achieving complete remission
(CR). As of October 31, 1994 this individual was alive and cancer-free
more than 3.6 years after entering the study. Seven other patients
achieved stabilization of disease (SD) with a median sur
vival of 1.6
years. Median survival for these eight patients was 2.2 years. This
survival is considerably longer than the median survival range of 7.5
months to 12 months for the best responders (CR and PR only) in the
leading pancreatic cancer trials conducted over the past two decades
(i.e. approximately 2 to 3.5 times longer). As of October 31, 1994 five
patients were alive, with survivals ranging from 1.6 years to 3.6 years
after study entry. All survivors had Stage 4 disease upon study entr
Understandably, the FDA approved Onconase for a Phase III clinical trial
involving pancreatic cancer in January 1995.
Directors of the Clinical Trials Program
Clinical studies are under the direction of John J. Costanzi, M.D.;
David N. Mesches, M.D. and Abraham Mittelman, M.D.
John J. Costanzi, M.D. has served as a principal investigator in the
Onconase clinical trials program since its inception. He is currently
in the practice of oncology a
nd hematology in Austin, Texas. Dr.
Costanzi formerly served as medical director of the Thompson Cancer
Survival Center in Knoxville, Tennessee. He also was director of the
cancer center for the University of Texas Medical Branch in Galveston.
David N. Mesches, M.D., is professor and chairman of the department of
family medicine at New York Medical College. The original Onconase
Phase I clinical trials were initiated and completed under his direction.
Abraham Mittelman, M.D., is assistant profess
or of medicine and director
of experimental oncology at New York Medical College in Valhalla, New
York. Dr. Mittelman is an oncologist and hematologist who has been the
principal investigator of numerous cancer trials.
4.3 A Clinical Trial for HIV?
In consideration of the "in vitro" studies performed by the NIH on
HIV-1 infected H9 Leukemia cells an Onconase clinical trial for HIV is
likely. In fact, it is an issue that is being actively discussed.
The Case For Early Human HIV Clinical Trials
Given the current data a compelling argument can be made for initiating
a Phase I/II human HIV clinical trial with Onconase.
Consider the following:
Since the "in vitro" study demonstrates that Onconase has significant
anti-viral activity against HIV-1, we would expect "in vivo" animal
testing to follow this study. However, a favorable human safety profile
for Onconase has been established by the Phase I cancer clinical trial.
This would obviate the need for further "in vivo" animal testing
another Phase I human clinical trial. Moreover, subtoxic concentrations
are required for anti-viral activity compared with the concentrations
required for anti-cancer activity.
Besides, a favorable regulatory environment exists. The approval
process has been shortened for drugs that demonstrate potential in
treating life threatening illnesses. AIDS is among the group of
illnesses that have been classified by the FDA as life threatening (20).
These arguments make Onconase a superb candida
te for a human HIV
5 UNIQUENESS OF THE ONCONASE PROTEIN MOLECULE
Onconase is a novel protein molecule with a unique bioactivity profile.
Some attributes of the molecule that make it structurally novel and
functionally unique are listed here.
1) It is a structurally novel protein based on its comparison with over
10,000 other proteins registered with the National Biomedical Research
Foundation Protein Identification Resource.
2) The protein preferentially binds to membran
e receptors on tumor
cells. This is followed by cytosolic internalization presumably by the
process of endocytosis.
3) The ribonucleolytic activity of the molecule is not inhibited by
mammalian RNase inhibitors. As a result, the mammalian ribosomal RNA
is degraded. This leads to the inhibition of protein synthesis and
4) Attributes 2 and 3 make Onconase selectively cytotoxic to tumor cells.
5) Entry to the cytosol of viral infected cells may be facilitated due to
molecule's small size. This characteristic may allow it to become
trapped within a viral endosome, and internalized with a viral particle.
Consequently, its ribonucleolytic activity can inhibit viral replication
6) Onconase serves to boost the human body's natural defense mechanisms.
For example, it contributes to an increase in white blood cell counts.
7) Protein molecules that are foreign to the human body are usually
immunogenic in humans. Although the onconase protein is
the human body, it has a high degree of homology (i.e. similar
molecular structure) to known low-immunogenicity proteins (e.g.
RNase A, a human pancreatic RNase and angiogenin). Its molecular
structure has been well conserved over evolution. Therefore, it may
be only weakly immunogenic if at all in humans.
8) The major dose dependent toxicities are peripheral edema and asthenia.
Nevertheless, the observed toxicities are reversible upon withdrawal of
6.1 National Institutes of Health (NIH)
The NIH is engaged in the development of Onconase conjugates and
recombinant Onconase. Areas of research at the NIH include studies of
anti-HIV activity, the study of the molecular mechanism(s) of action of
Onconase at the cellular and subcellular levels, tests of the anti-tumor
activities of Onconase conjugates (14), Onconase gene therapy, and
investigation of anti-tumor activity of Onconase against primary brain
6.2 National C
ancer Institute (NCI)
Onconase also is being tested at the National Cancer Institute's
Biological Response Modifier and Developmental Therapeutics Programs.
Areas of research include studies of Onconase as a single agent against
various tumor cell lines (including the NCI Cancer Screen), and Onconase
incorporated into liposomes both as a single agent and in combination with
other drugs. In particular, the NCI is examining Onconase's ability to
overcome multiple drug resistance (MDR) in cancer t
herapy (e.g. colorectal
cancer). The NCI also is studying Onconase's anti-HIV activity.
6.3 Other Biologically Active Proteins
In addition to Onconase, a series of biologically active proteins from
the same amphibian natural source material have been discovered. These
proteins appear to be involved in the regulation of early embryonic and
malignant cell growth. So far two additional proteins have been purified
and characterized. These proteins have been tentatively named P-31 and
Onconase is a novel protein molecule in both biological structure and
function. For example, it exhibits antiproliferative activity against a
wide variety of tumor cell types. Moreover, it has demonstrated activity
against the HIV-1 virus, the causative agent of AIDS.
The molecule's potential as both an anti-cancer and anti-viral
therapeutic has been well documented in the scientific literature.
Furthermore, safety and efficacy as an anti-cancer agent in humans have
. As the first RNase therapeutic to reach Phase III
clinical trials, Onconase represents a completely new approach to the
treatment of cancer and viral infections.
(1) "Cytostatic and Cytotoxic Effects of Pannon (P-30), a Novel
Anticancer Agent." Z. Darzynkiewicz, S. Carter, S. Mikulski,
W. Ardelt, K. Shogen. Cell Tissue Kinet. 21:169-182, 1988.
(2) "Tamoxifen and Trifluoroperazine (Stelazine) Potentiate Cytostatic/
Cytotoxic Effects of P-30 Protein, a Novel Protein P
Tumor Activity." S. Mikulski, A. Viera, W. Ardelt, H. Menduke, K.
Shogen. Cell Tissue Kinet. 23: 237-246, 1990.
(3) "Striking Increase of Survival of Mice Bearing M109 Madison
Carcinoma Treated With a Novel Protein From Amphibian Embryos."
S. Mikulski, W. Ardelt, K. Shogen, E. Bernstein, H. Menduke.
J Natl Cancer Inst. 82(2): 151, Jan. 17, 1990.
(4) "Amino Acid Sequence of an Anti-tumor Protein from Rana pipiens
Oocytes and Early Embryos." W. Ardelt,
S. Mikulski, K. Shogen.
J Biol Chem. 266(1): 245-251, Jan 5, 1991.
(5) "A Phase I Study of P-30 Protein (PP) Administered Intravenously on
a Weekly Schedule." J. J. Costanzi, A. Grossman, P. Carter,
A. Ebenezer, W. Hanna, K. Shogen, S. M. Mikulski. The Thompson
Cancer Survival Center, Knoxville, IN and Alfacell Corporation.
Proc. Amer. Soc. Clin. Oncol., Houston, TX. Abstract #286, 1991.
(6) "Synergism Between a Novel Amphibian Oocyte Ribonuclease and
Inducing Cytostatic and Cytotoxic Effects in Human
Lung and Pancreatic Carcinoma Cell Lines." S. Mikulski, A. Viera,
Z. Darzynkiewicz, K. Shogen. Br J Cancer. 66: 304-310, 1992.
(7) "Comparative Molecular Modeling and Crystallization of P-30 Protein:
A Novel Antitumor Protein of Rana pipiens Oocytes and Early Embryos."
S. Mosimann, K. Johns, W. Ardelt, S. Mikulski, K. Shogen, M. James.
Proteins Struct Funct Genet. 14: 392-400, 1992.
(8) "In Vitro Synergism Between a Nove
l Amphibian Oocytic Ribonuclease
(ONCONASE) and Tamoxifen, Lovastatin and Cisplatin, in Human OVCAR-3
Ovarian Carcinoma Cell Line." S. Mikulski, A. Viera, K. Shogen.
Int J Oncol. 1: 779-785, 1992.
(9) "In Vivo and in Vitro Growth-inhibitory Effect of Bovine Seminal
Ribonuclease on a System of Rat Thyroid Epithelial Transformed Cells
and Tumors." P. Laccetti, G. Portella, M.R. Mastronicola, A. Russo,
R. Piccoli, G. D'Alessio and G. Vecchio.
Cancer Res. 52: 4582-
(10) "Clinical trial of tamoxifen in patients with irresectable
pancreatic adenocarcinoma." O. M. Taylor, E. A. Benson,
M. J. McMahon. Br J Surg. 80(3): 384-6, Mar 1993.
(11) "Human tumor cell growth modulatory effects of the AEBS/H IC-binding
drugs used as single agents and in combination with a novel
amphibian oocyte RNase." S. Mikulski, A. Viera and K. Shogen.
Int J Oncol. 2: 807-812, 1993.
(12) "Phase I human clinical trial of ONCONASE (P-30 protein)
administered intravenously on a weekly schedule in cancer patients
with solid tumors." S. M. Mikulski, A. M. Grossman, P. W. Carter,
K. Shogen and J. J. Costanzi. Int J Oncol. 3: 57-64, 1993.
(13) "A Cytotoxic Ribonuclease - Study of the Mechanism of ONCONASE
Cytotoxicity." Y-N. Wu, S. Mikulski, W. Ardelt, S. Rybak and
R. Youle. J Biol Chem. 268(14): 10686-10693, May 15 1993.
(14) "Cytotoxic Onconase and Ribonuclease A Chimeras: Comparison and in
Vitro Characterization." S.
Rybak, D. Newton, S. Mikulski, A. Viera,
R. Youle. Drug Targeting Delivery. 1: 3-10, 1993.
(15) "Pathogenesis of cancer in view of mutually opposing apoptotic and
anti-apoptotic growth signals (Review)." S. Mikulski.
Int J Oncol. 4: 1257-1263, 1994.
(16) "RNase Inhibition of HIV Infection of H9 Cells." R. Youle, Y-N Wu,
S. Mikulski, K. Shogen, R. Hamilton, D. Newton, G. D'Alessio and
M. Gravell. Abstract: A-2. International Cytometry Symposium,
San Francisco, CA. Janu
ary 18-21, 1994.
(17) "Anti-tumor Properties of ONCONASE, A Novel Ribonuclease From The
Eggs And Early Embryos Of Leopard Frog (Rana pipiens)." S. Mikulski,
Z. Darzynkiewicz, W. Ardelt and K. Shogen. Abstract: J-2.
International Cytometry Symposium, San Francisco, CA.
January 18-21, 1994.
(18) "RNase inhibition of human immunodeficiency virus infection of H9
cells." R. Youle, Y-N. Wu, S. Mikulski, K. Shogen, R. Hamilton,
D. Newton, G. D'Alessio and M. Gravell.
roc Natl Acad Sci. 91: 6012-6016, June 1994.
(19) "Refined 1.7 A X-ray Crystallographic Structure of P-30 Protein, an
Amphibian Ribonuclease with Anti-tumor Activity." S.C. Mosimann,
W. Ardelt and M.N.G. James. J. Mol. Biol. 236: 1141-1153, 1994.
(20) "Faster Evaluation of Vital Drugs." D.A. Kessler and K.L. Feiden.
Scientific American 272(3): 48-54, March 1995.
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