Hepatitus-C
Project
History
and Background of Hepatitis C Virus
Go To Top
Human hepatitis
C virus (HCV) was identified in 1989 using molecular biological techniques.
The explosion of information following the description of the genomic complexity,
nucleic acid sequences, of HCV has greatly enhanced our understanding of
the epidemiology of the majority of non-A non-B hepatitis (NANBH) and some
biology of this agent.
HCV exists as
a highly mutating, heterogeneous group of viruses sharing approximately
70% homology. HCV is an RNA virus similar to the virus of human flavivirus
and animal pestivirus families. The flaviviruses are the causative agents
of dangue fever and yellow fever in man while the pestiviruses cause bovine
diarrhea.
Several genotypes
of HCV, their subtypes and sero-types, roughly correlate with geographic
distribution of hepatitis. The significance of this heterogeneity is currently
not well understood. The natural history of chronic infection with HCV
is not known due to the absence of an in vitro system with self replicating
HCV. The acquisition of HCV may be due to several sources of exposure
such as infected blood and blood products (e.g., platelets, packed cells,
plasma, clotting factor, and drug abuse paraphernalia). In addition,
it can be acquired sporadically from unknown sources or from the community.
Roughly, half of all patients with HCV have no known source of infection,
and the remainder includes intravenous drug abusers (42%), blood transfusion
patients (6%), individuals with history of hemodialysis, and health care
workers exposed to blood (2%).
In May 1990, serologic
tests that detect anti-HCV by EIA were licensed and commercially available
in the United States. Studies have shown that HCV is the etiologic
agent of the majority of parenterally transmitted or blood born non-A,
non-B hepatitis worldwide. This type of non-A, non-B hepatitis was
first identified and characterized in studies of post transfusion hepatitis
conducted in early 1970s. Epidemiological and experimental studies
indicate that the parenteral route transmits HCV. Persons at increased
risk of acquiring hepatitis C include parenteral drug users; health-care
workers with occupational exposure to blood; hemodialysis patients, and
recipient of whole blood, cellular blood components and/or plasma.
Approximately 4 million Americans are considered to be chronically infected
by HCV.
An average of
50% of patients with either post-transfusion or community-acquired hepatitis
C followed for at least twelve months develop biochemical evidence of chronic
liver disease. Of patients with transfusion-associated chronic non-A,
non-B hepatitis who undergo biopsy within 5 years after onset, greater
than or equal to 40% have histologic evidence of chronic active hepatitis
and 10-20% have evidence of cirrhosis; many of these patients have no clinical
manifestations of their disease. In contrast, biopsy specimens from
patients with community-acquired chronic non-A, non-B hepatitis, less than
20% have evidence of chronic active hepatitis and 3% have evidence of cirrhosis
within 4 years after onset of disease.
Prevalence
of Hepatitis C Infection
Go To Top
HCV infection
is prevalent in almost every country and all races. Approximately
four million Americans are infected with HCV and the same number of Europeans
is also infected. Worldwide it is estimated that approximately 200
million individuals are infected. The prevalence rate ranges from
0.5% to 8%. HCV becomes a chronic infection in more than 60% of infected
persons. Chronic hepatitis due to HCV is a commonly progressive viral
disease and an important public health problem. A large percentage
of chronic HCV infections lead progressively to liver cirrhosis and also
to primary hepatocellular carcinoma.
By way of comparison,
the number of individuals infected with HCV is, conservatively, at least
four times the number infected with HIV / AIDS.
Drawbacks
of Existing Therapies and Research Methods
Go To Top
Currently available
treatments for HCV infections have drawbacks and limited efficacy.
Currently, guidelines
issued by the National Institutes of Health’s consensus Development Conference
Panel recommends that treatment for hepatitis C infection be limited to
those patients who have histological evidence of progressive disease.
The panel recommends treatment of all patients with hepatic fibrosis or
moderate to severe inflammation and necrosis on liver biopsy. It
is estimated that fibrosis will develop in 1 out of 3 cases of hepatitis
C within about 3 years of its onset. The patients with less severe
microscopic disease may be managed on an individual basis. It is
their consensus that patients should not be treated, outside of controlled
trials, who have clinical decompensated cirrhosis, normal aminotransferase
levels, either kidney, liver, heart or other solid organ transplant, or
specific contraindications to either monotherapy currently approved or
to combination therapy.
Current therapeutic
regimens include use of alpha interferon, ribavirin, or a combination of
these two agents. It is difficult to determine which patients will
respond to this therapy, and relief is often transient. The use of
alpha interferon in children is not yet established, and the dosage and
safety of ribavirin in children with hepatitis C has not yet been determined.
To date, only mono therapy is recommended in these cases.
Adverse effects
of alpha-interferon are numerous and include nausea and vomiting, mild
bone marrow suppression, irritability and weight loss. Alpha-interferon
may also cause neuropsychiatric side effects including anxiety, depression,
personality changes and even acute psychosis.
Ribavirin may
cause anemia, itching, skin rash, mild hemolysis of red blood cells, fatigue,
and irritability.
In instances where
patients do not respond to therapy, or have a relapse, a 24-week course
of combination therapy may be tried especially if they became and remained
HCV RNA negative during the monotherapy treatment period.
Peglylated interferons,
and other experimental treatments such a recombinant IL-10 are under study.
Research into
therapies for HCV have been hampered by the absence (except at CIMM) of
a method for isolating a strain of the virus that will replicate in vitro
(in a culture) as opposed to in an infected host animal. Studies
to date, so far as CIMM is aware, have been confined to studying the virus
in an infected host animal (human or simian) or cell. The usual study
is of infected chimpanzees.
These studies
are expensive and complicated because of the cost of test subjects, and
because of the difficulty in separating other causes and effects in the
test animal. By the time chimpanzees are available for HCV infection
and study, the animals have usually undergone other studies and have a
variety of residual effects from those studies. The number of animals
is also limited, so statistically significant numbers are hard to obtain.
These studies
are also limited in that, strictly speaking, what are being studied are
not the virus itself but the effects and manifestations of the virus in
an infected host. It is a little like studying a tapeworm by examining
an infected animal, instead of taking the tapeworm out and studying it
directly.
A model system
for in vitro experiments with HCV that would be reasonable in terms of
time, resources, and reproducibility is in urgent need. For this
model a population of infectious and replicating HCV particles are needed.
Potential drugs or vaccines can be introduced directly into the culture
and studied in vitro. At a minimum, potential therapeutics can be
screened efficiently, with only the most promising drugs tested further
in live subjects. The virus itself can also be analyzed.
CIMM's
Self-Replicating HCV Strain
Go To Top
CIMM has developed
a method to isolate and culture, in the laboratory, what testing indicates
to be a self-replicating strain of hepatitis-C virus.
Specifically,
at CIMM we have been successful in transmitting HCV from patients with
chronic active hepatitis C disease. These isolates have been maintained
in a cloned cell line for over eighteen months. They replicate at
varying rates for unknown reasons. However, the viral RNA is detected
in cell free supernatants by polymerase chain reaction (PCR) technique,
indicating the presence of HCV. This is the first long-term human
HCV producing system known to this day. The isolates are currently
being analyzed further for patent and publication purposes.
CIMM's isolate
is the first of its kind for hepatitis C virus research and should be easily
useful for the following:
1. Testing therapeutic
agents for toxicity and efficacy;
2. Studying the
life cycle of the virus;
3. Development
of immunological assays for a vaccination program; and
4. Development
of reagents for scientific research.
Commercial
Potential of CIMM's HCV Strain
Go To Top
Utilizing CIMM's
self-replicating HCV, if further testing is successful and approvals obtained,
CIMM can offer, as a service, to test proposed HCV drugs and vaccines against
CIMM's virus in vitro. Such a test would be immeasurably faster and
more efficient than existing live-subject testing. CIMM could offer
the test in return for a fee and/or a percentage of any income derived
from successful tested drugs
The CIMM assay
system uses the virus produced in the cell free supernatant from the continuous
cell culture system at programmed time intervals. RNA extracted from
the supernatant is tested by PCR using several different primer pairs for
the presence of HCV. The comparative levels of RNA are seen in a
stained gel. The relative levels of viral RNA on a gel reflect effect
of a drug on HCV production. Concentration of the drug under test
is titrated and positive and negative controls are always present in the
test system. Cell viability and concentration of cellular RNA help
in the interpretation of test results.
CIMM's
Own HCV Drug Research
Go To Top
CIMM has already
utilized its own HCV strain to test CIMM's own proposed anti-HCV drugs.
CIMM has identified several formulations with varying degrees of inhibitory
effect on HCV. One of the formulations, called XHC, was shown to
shut down the HCV replication within 7-10 days of treatment of virus producing
cells in vitro in CIMM's test.
Kupffer
Cell Therapy Project
Kupffer’s
Cells (KCs)
Go To Top
Karl Wilhelm von
Kupffer, a German anatomist, first described Kupffer’s cells of the liver
in 1800. KCs are large fixed macrophages. Morphologically,
they are usually star shaped or pyramidal/cuboidal cells with a large oval
nucleus and a small prominent nucleolus. In vitro cultured cells
morphologically resemble both forms. KCs play a major role in the
physiological maintenance of hepatic architecture and wound healing process
when a liver is chronically injured. These cells line the walls of
the sinusoids of the liver and hence form a part of the reticuloendothelial
system that removes impurities from the circulating blood. The activated
form of KCs is implicated in liver fibrosis.
KCs exhibit vigorous
phagocytosis, and produce many kinds of soluble mediators e.g., prostanoids,
oxygen radicals, proteases, and cytokines. Activated KCs are the
source of prostaglandins (PGE). When KCs are activated by ethanol,
obstructive jaundice results causing impaired phagocytic function of the
KCs. Chemokines (IL-12, IL-18, etc.) produced by KCs are also implicated
in the pathogenesis of alcoholic liver disease. KCs are highly responsive
to the effects of bacterial stimuli (endotoxin, for example lipopolysaccharide,
and superantigens).
Kupffer cells
in patients with liver disease, such as HCV or cirrhosis show damage and
impaired function. If the Kupffer cells could be replaced or augmented
with healthy cells, the patient's liver function could be prolonged while
the patient awaits a liver transplant or other treatment.
CIMM's
Kupffer’s Cell Therapy
Go To Top
CIMM has developed
a proprietary process for replicating Kupffer cells in vitro. Briefly,
cells are extracted from a liver patient through biopsy. CIMM's process
then replicates the healthy Kupffer cells in the sample. Healthy
cells are then returned to the liver through the portal vein. This could
seed the failing liver with freshly grown KCs, which could take up residence
in available areas, potentially resulting in restoration of varying levels
of metabolic, synthetic, and other liver functions. In theory rejection
will not be an issue because the implanted cells were grown from the patient's
own healthy cells. Assuming successful testing, clinical trials and
regulatory approvals, the process suggests a method to prolong liver function
while patients await transplants or other treatment.
Testing thus far
by CIMM indicates that CIMM's process of KC replication in vitro is reproducible
(>95% efficiency) from individual patients. At 120 days significant
accumulations of live KCs are achieved at a concentration of 2x107 per
ml. Morphologically, they form monolayer colonies of stelate or cuboidal
cells and require 7 to 10 days to achieve >90% confluency. The results
depend upon many factors, including the presence of living cells in the
biopsy specimen arriving in sterile condition.
Benefits
and Potential of CIMM's Kupffer’s Cell Therapy
Go To Top
Liver transplant
is the established therapy of choice for end stage acute and chronic liver
disease of various etiologies. On average, liver transplantation
achieves a five-year survival rate. It is estimated that while there
are approximately 15,000 patients waiting for liver transplant in the United
States, only 4,500 donor livers appropriate for transplant become available
every year. The liver transplant surgery is not only risky for the
patient, but is also very expensive. The average cost of for a liver
transplant surgery is $300,000.
The ability to
grow KCs from individual patients will make it possible to return these
cultured cells through the portal vein into the failing liver of the patients.
Candidates for the risky surgical procedure of heterologous organ transplant
could potentially receive an autologous transplant of freshly grown KCs
instead. As a result, this may extend the survival time for patients
who are candidates for liver transplants, but who do not have a suitable
organ donor available.
Implantation of
the Kupffer cells might even, potentially; defer the need for a transplant.
Only testing can demonstrate the full potential of CIMM's process.
In short, the
opportunity to transplant in vitro grown KCs from the impaired livers of
patients could reduce the need for organ transplants and provide a painless,
durable treatment for some patients. Kupffer’s cell transplant via
the portal vein is relatively simple, safe, and uses a commonly available
inexpensive procedure. This procedure will also be the only choice
for the patients that do not qualify for a liver transplant due to their
age, frailty, or for any other reason. Safety should be easy to establish
from the perspective of incompatibility of tissue antigens (HLA) and the
consequent immunological complications, because it is an autologous transplant
of cells.
After further
clinical testing and upon approval of the therapeutic by the Food and Drug
Administration, the Kupffer’ Cell Therapy could potentially generate significant
revenue for CIMM. Since the average liver transplant cost approximately
$300,000 per procedure, CIMM’s hope is to be able to grow Kupffer’s cells
to generate revenues of $40,000 to $60,000 per patient. This would
represent significant savings as compared to a liver transplant.
If testing is successful, and assuming FDA approval and once the technology
is in full production, it is projected that CIMM will create the capacity
to grow 750 to 2,000 patient samples annually. The projected revenue
is $40,000 to $60,000 per patient resulting in projected annual revenues
of $30 M to $120 M.
A patent application
to protect CIMM’s proprietary technique for culturing KCs was filed in
December 2000. After the close of this round of financing the Kupffer’s
Cell Therapy will be one of CIMM’s major research initiatives in hopes
of gaining FDA approval as quickly possible.
Feline
Luekemia Drug Project section
FeLV
Overview and Prevalence
Go To Top
FeLV is one of
the endogenous retrovirus to cats. It is the most important infectious
disease agent producing fatal illness in domestic cats today. Most
cats carry this virus in circulation in varying levels. The feline
leukemia virus is excreted in saliva, milk, tears, semen, and most likely
the urine and feces of infected cats. Cat-to-cat contact is required
for the spread of the virus as the virus is rapidly inactivated by warmth
and drying. Kittens may become infected with FeLV in utero.
A cat with the disease will only live a few weeks to a few months, as death
is almost always inevitable. The prevalence of FeLV in single-cat
households is about 3% and can be as high as 11% in stray cat populations.
In large multi-cat households and in households where cats roam freely
outdoors, the prevalence can reach as high as 70%.
Exposure to FeLV
may result in:
1. No persistent
viremia (never virus positive in the blood despite exposure);
2. Transient
viremia (become virus positive but revert to negative usually within three
months), no significant disease.
3. Persistent
viremia:
a. Immunosuppression
(30% of all exposures result in this).
b. Leukemia/lymphoma
c. Bone marrow
suppression/non-regenerative anemia
70% of persistently
viremic cats die from complications related to their viremia within three
years of diagnosis.
Current
FeLV Therapeutics for Cure -- None
Go To Top
To date there
is no cure for FeLV infection. There are a variety of chemotherapeutic
regiments that have been developed, but at best those drugs can only produce
temporary remission in some cats depending on the physical condition of
the cat when the therapy is started. Chemotherapeutic drugs are very
potent and their effects must be monitored carefully, to avoid overdosing
the cat. The cost is obviously high, usually prohibitively high for
pet owners.
Additionally,
various antiviral compounds may be used to treat infected cats. These
agents may reduce the amount of virus present in the blood of the cat,
and may extend the remission of the disease, but they do not produce a
permanent cure, and death results eventually.
Regarding prevention,
there are several vaccines available to aid in the protection of a cat
against FeLV infection. The vaccines are produced by various methods
and either contains the inactivated, killed, whole virus, or a subunit
protein of the virus. The principle is the same for each of the different
types of vaccines. Vaccination is not fully effective even under
the best of conditions. Moreover, to reach the maximum level of effectiveness
cats must be vaccinated twice as a kitten and then once a year for the
rest of their life. Most cat owners do not follow-up with the yearly
vaccination, therefore rendering the majority of the cat population susceptible
to the disease. Even if the schedule is strictly followed, the cat
can still become infected.
CIMM's
FeLV Therapeutic Development
Go To Top
CIMM has developed
a compound, XFL, which has demonstrated efficacy against feline leukemia
virus in preliminary in vitro testing. Funds will be used to advance
the development of the XFL compound and to conduct testing in live cats
with active FeLV disease. This will be a high priority for CIMM.
The process for animal drug approval is much shorter and less expensive
than that for human drugs, yet the market remains substantial. The
feline leukemia virus represents an area where, if the drug proves successful,
CIMM can obtain revenues in short order and with much less investment than
for human therapies.
|
