Note: Descriptions are shown in the official language in which they were submitted.
CA 02501751 1998-04-15
TITLE OF INVENTION
A COMBINATION THERAPY FOR HIV INFECTIONS
Reference to Related Application
This application is a division of
copending Canadian Patent Application Serial No.
2,285,463 filed April 15, 1998.
Field of the Invention
The present invention relates to a method
for using Product R as hereinafter defined in
combination with other anti- human immunodeficiency
virus (HIV) to treat patients having HIV infections.
Description of the Related Art
A retrovirus designated human
immunodeficiency virus (HIV) is the etiological
agent of the complex disease that includes
progressive destruction of the immune system
(acquired immune deficiency syndrome; AIDS) and
degeneration of the central and peripheral nervous
system. This virus was previously known as LAV, HTL-
III, or ARV. The virus specifically attacks T-4
helper lymphocytes, a subgroup of T-lymphocytes that
plays a major role in defending the body against
infectious diseases. Depletion of this subset of
lymphocytes is manifested by an increased incidence
of opportunistic infections like ' pneumocystis
carinii and certain cancers. More specifically, the
virus enters the T-lymphocyte and incorporates viral
encoded DNA into the DNA of the host T-lymphocyte.
As long as the infected T-lymphocyte remains
inactivated, the virus will quietly remain in the
DNA of the host cell. This will not kill the cell
but may impair its function. When the infected
T-1?'mphocytes are activated by stimuli such as a
specific antigen, the viral DNA in the host DNA is
expressed and produces new viral particles. The host
CA 02501751 1998-04-15
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T-lymphocyte is then killed and lysed, releasing new
viral particles that can invade and kill other
T-lymphocytes. The loss of T-4 lymphocytes is
profound and occurs even faster than can be
accounted for by direct viral killing of the cells.
This has led some investigators to postulate that
the infection somehow shuts off the production of
T-4 lymphocytes. In any case, the normal thymus is
no longer functioning and the killed T-lymphocytes
cannot be replaced leaving the patient vulnerable to
subsequent infections. Especially striking are
recent studies of the thymuses of deceased AIDS
patients ranging in age from 10 months to 42 years.
AIDS victims have profound thymic involution; much
more extensive than in age-matched patients who died
of other causes.
The cure of a person with AIDS will
probably require one agent to eliminate the virus
and other agents to cause the body to replace T
cells that have been killed by the virus. The first
step is to eliminate the AIDS virus from the
patient. This will have to be supported by other
therapies to induce restoration of immune function.
Studies to date with macrophage activating agents,
interferon inducers and lymphokines have been
disappointing, possibly because their targets,
T-lymphocytes, do not exist in sufficient numbers.
Interleukin 2 restores the function of one subset of
non T-cells (natural killer cells) but has no effect
on a host of other serious defects. More drastic.
measures can be performed. One potential method of
restoring the immune system is by transplanting bone
marrow from healthy donors. However, this is a
dangerous procedure. It may produce lethal graft
CA 02501751 1998-04-15
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versus host disease unless the patient' s donor is an
identical twin.
A common feature of retrovirus replication
is the extensive post-translational processing of
precursor polypeptides by a vitally encoded protease
to generate mature vital proteins required for virus
assembly and function. Inhibition of this processing
prevents the production of normally infectious
virus. It has been demonstrated that genetic
inactivation of the HIV encoded protease resulted in
the production of immature, non-infectious virus
particles. These results indicate that inhibition of
the HIV protease represents a viable method for the
treatment of AIDS and the prevention or treatment of
infection by HIV. However, administration of a HSV
protease inhibitor sometimes cause side effects
including nausea, nephrolithiasis, increased
bilirubin, or gastro-intestinal upset.
Zidovudine tAZT) is a synthetic pyrimidine
analog that differs from thymidine in having an
azido substituent instead of a hydroxyl group at the
3' position of the deoxyribose ring. It was
initially developed as an anticancer agent and
subsequently found to inhibit the reverse
transcriptase fRT) of Friend leukemia virus. Soon
after the identification of a human retrovirus as
the etiologic agent of AIDS, zidovudine was shown to
have anti-HIV activity in vitro. Zidovudine
selectivity is due to the preferential interaction
of AZT-TP with the RT. Phosphorylation of zidovudine
to its active form, AZT-TP, is accomplished by
cellular enzymes. Zidovvdine is an efficient
substrate for the cellular thymidine kinase which
converts it to AZT-MP in both infected and
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uninfected cells. AZT-MP accumulates in cells
because of slow phosphorylation to AZT-DP by host-
cell thymidylate kinase which is the rate-limiting
step in AZT-TP formation. AZT-MP is a competitive
inhibitor or thymidylate kinase and reduces the
conversion of dTMP to dTDP which leads to decreased
formation of dTTP. Other nucleoside analogs
including ddI, ddC and ddA also have activity
against HIV through a similar mechanism to that
1.0 described for AZT.
However, the major toxicity of 2idovudine
is on the bone marrow, with macrocytic anemia and
granulocytopenia common occurrences. The mechanisms
of these toxic effects are uncertain. Rare instances
of pancytopenia with hypocellular marrow have been
described, and patients with poor bone marrow
reserve, secondary to opportunistic infections or
vitamin Blz deficiency, have more toxicity than
patients with sufficient marrow reserve. Nausea,
myalgia, insomnia, fever, rash, nail pigmentation,
and severe headaches may also be observed.
Product R' emerged as an antiviral product
in the 1930'x. while it was originally believed to
be a product composed of peptone, peptides and
nucleic acids (fully defined hereafter), the precise
composition remains unidentified. Nevertheless,
Product R has demonstrated an ability to inhibit
rapidly the course of several viral diseases. It is
nontoxic, miscible with tissue fluids and blood sera
and free from anaphylactogenic properties. Recent
studies demonstrated that Product R can also
The agent is known under the trademark "Reticulose",
a trademark of Advanced Viral Research Core.
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stimulate the immune system and red blood cell production,
suggesting that Product R is an immune system modulator.
Insofar as the applicant knows, Product R has
never been used, nor suggested for treating AIDS patients in
combination with other anti-AIDS agents. It is now
discovered that a combination of Product R and other
antiviral agents useful for treating HIV infections or AIDS
presents an advantageous treatment for AIDS patients.
SUMMARY OF THE INVENTION
The claims of the parent application No. 2,285,463
are directed to the use of Product R in a sterile injectable
formulation with at least one antiviral agent or nucleoside
analog useful for treating AIDS or HIV infections, for
treating a patient having HIV infections.
In the present invention, there are provided
methods of producing Product R and Product R produced by
such methods.
Accordingly, in one aspect of the present
invention, there is provided a method for producing Product
R comprising the steps of a) suspending about 35.0 grams of
casein, about 17.1 grams of beef peptone, about 22.0 grams
of ribonucleic acid (RNA), and about 3.25 grams of bovine
serum albumin in about 2.5 liters of water; b) adding about
16.5 grams of sodium hydroxide to the mixture from step a;
c) autoclaving the product from step b at about 9 lbs
pressure and 200 to 230°F until the RNA is completely
digested; d) cooling the product from step c to about 3 to
8°C; e) sequentially filtering the product from step d
through a 2 micron filter, a 0.45 micron filter and a 0.2
micron filter; f) diluting the product from step a with
water to yield a final volume of about 5 liters; g)
adjusting the pH of the product from step f to a range of
about 7.3 to 7.6; h) filtering the product from step g
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through a second 0.2 micron filter; and i) autoclaving the
product from step h at 240°F and 20 to 30 pounds pressure
for about 30 minutes.
In another aspect of the present invention, there
is provided a method for producing Product R comprising the
steps of a) suspending about 35.0 grams of casein, about
17.1 grams of beef peptone, about 22.0 grams of ribonucleic
acid (RNA) , and about 3 .25 grams of bovine serum albumin in
about 2.5 liters of water; b) adding about 11.75 ml of
hydrochloric acid to the mixture from step a; c) autoclaving
the product from step b at about 9 lbs pressure and 200 to
230°F until the RNA is completely digested; d) cooling the
product from step c to about 3 to 8°C; e) sequentially
filtering the product from step d through a 2 micron filter,
a 0.45 micron filter and a 0.2 micron filter; f) diluting
the product from step a with water to yield a final volume
of about 5 liters; g) adjusting the pH of the product from
step f to a range of about 7.3 to 7.6; h) filtering the
product from step g through a second 0.2 micron filter; and
i) autoclaving the product from step h at 240°F and 20 to 30
pounds pressure for about 30 minutes.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
As used herein, Product R is the product produced
according to either of the following methods.
Method I for Preparing Product R
Suspend about 35.0 g of casein, about 17.1 g of beef
peptone, about 22.0 g of nucleic acid (RNA), about 3.25 g
bovine serum albumin in about 2.5 liters of water for
injection USP at about 3 to 7°C in a suitable container and
gently stir until all the ingredients have been properly
wet. Carefully add while stirring about 16.5 g of sodium
hydroxide (reagent grade ACS) and continue stirring until
sodium hydroxide completely dissolved. Autoclave at about 9
CA 02501751 1998-04-15
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lbs pressure and 200 to 230°F for a period of time until RNA
is complete digested, for example about 4 hours. At the end
of the period, the autoclave is stopped and the reaction
flask and contents are permitted to slowly cool to ambient
temperature. Then cool for at least six hours at about 3 to
8°C. The resulting solution is filtered through 2 micron and
0.45 micron filters using inert gas such as nitrogen or
argon at low pressure (1 to 6 psi). In a similar manner the
solution is filtered again through 0.2 micron pyrogen
retention filters. The resulting filtrate is sampled and
assayed for total nitrogen. A calculation is then performed
to determine the quantity of cooled water for injection to
be added to the filtrate to yield a diluted filtrate with a
nitrogen content between about 165-210 mg/m1, the final
volume is approximately 5 liters. The pH is then adjusted
with either concentrated HCL (reagent grade ACS) or 1.0
normal NaOH to about 7.3-7.6 range. The diluted solution is
then filtered again through 0.2 micron filters with inert
gas at low pressure. The final filtrate is then filled and
sealed into 2 ml glass ampules while in an inert gas
atmosphere. The ampules are collected and autoclave for
final sterilization at 240°F and 20 to 30 pounds pressure
for about 30 minutes. Following the sterilization cycle, the
ampules with Product R are cooled and washed.
All quantities are subject to plus or minus 2.5%
variation for pH, volume, and analytical adjustments.
Method II for Preparing Product R
Suspend about 35.0 g of casein, about 17.1 g of
beef peptone, about 22.0 g of nucleic acid (RNA), about 3.25
g bovine serum albumin in about 2.5 liters of water for
injection USP at about 3 to 7°C in a suitable container and
gently stir until all the ingredients have been properly
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wet. Slowly add while stirring about 11.75 ml of
hydrochloric acid (reagent grade ACS) and continue stirring
until hydrochloric acid is completely dissolved. Autoclave
at about 9 lbs pressure and 200-230°F for a period of time
until RNA is completely digested, for example, about 4
hours. At the end of the period, the autoclave is stopped
and the reaction flask and contents are permitted to slowly
cool to ambient temperature. Then cool for at least six
hours at about 3-8°C. The resulting solution is filtered
through 2 micron and 0.45 micron filters using inert gas
such as nitrogen or argon at low pressure (1-6 psi). In a
similar manner the solution is filtered again through 0.2
micron pyrogen retention filters. The resulting filtrate is
sampled and assayed for total nitrogen. A calculation is
then performed to determine the quantity of cooled water for
injection to be added to the filtrate to yield a diluted
filtrate with a nitrogen content between about 165-210
mg/ml, the final volume is approximately 5 liters. The pH is
then adjusted with either concentrated HCL (reagent grade
ACS) or 35~ (w/v) of NaOH to about 7.3-7.6 range. The
diluted solution is then filtered again through 0.2 micron
filters with inert gas at low pressure. The final filtrate
is then filled and sealed into 2 ml glass ampules while in
an inert gas atmosphere. The ampules are collected and
autoclave for final sterilization at 240°F and 20 to 30
pounds pressure for about 30 minutes. Following the
sterilization cycle, the ampules with Product R are cooled
and washed.
All quantities are subject to plus or minus 2.5~
variation for pH, volume, and analytical adjustments.
HIV protease inhibitors include oligopeptide
analogs, such as saquinavir (Roche Laboratories), indinavir
(Merck) or ritonavir (Abbott Laboratories), which are fully
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described in detail in U. S. patent Nos. 5,413,999 and
5,476,874.
The antiviral agents useful for a combination
therapy of AIDS or HIV infections other than HIV protease
inhibitors is listed in Table I.
TABLE I
Dru4 Name Manufacturer
Indication
AL-721 Ethigen
PGL
(Los Angeles, CA) HIV
positive, AIDS
Recombinant Human Triton Hiosciences AIDS,
Kaposi's
Interferon Beta (Almeda, CA)
sarcoma, ARC
Acemannan Carrington Labs ARC
(Irving, TX) (See
also
immunom
odulato
rs)
Cytovene Syntex sight
threatening CMV
Ganciclovir (Palo Alto, CA)
peripheral CMV
retinitis
d4T Bristol-Myers . AIDS,
ARC
Didehydrodeoxy- (New York, NY)
thymidine
ddI Bristol-Myers AIDS,
ARC
Dideoxyinosine (New York, NY)
EL10 Elan Corp, PLC HIV
infection
(Gainesville, GA)
Trisodium Astra Pharm. C
retinitis, HIV
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Phosphonoformate Products, Inc.
infection, other CMV infections
(Westborough, MA)
Dideoxycytidine; Hoffman-La Roche AIDS,
ARC
ddC (Nutley, NJ)
Novapren Novaferon Labs, HIV
inhibitor
Inc., Akron, OH)
Diapren, Inc.
(Roseville, MN,
marketer)
Peptide T Peninsula Labs AIDS
Octapeptide (Helmont, CA)
Sequence
Zidowdine; AZT Hurroughs AIDS,
adv, ARC
Wellcome (Rsch.
pediatric AIDS,
Triangle Park, NC) Kaposi'
s
sarcoma
asympto
matic
HIV
infecti
on,
~ less
severe
HIV
disease
neurolo
gical
involve
ment,
in
combing
tion
with
other
therapi
es.
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Ansamycin LM 427 Adria Laboratories ARC
(Dublin, OH)
Erbamont
(Stamford, CT)
Dextran Sulfate Ueno Fine Chern. AIDS,
ARC, HIV
Ind. Ltd.
positive, asymptomat ic
(Osaka, Japan)
Virazole Viratek/ICN
asymptomatic HIV
Ribavirin (Costa Mesa, CA)
positive, LAS, ARC
Alpha Interferon 8urroughs
Kaposi's sarcoma,
Wellcome (Rsch. HIV in
combination
Triangle Park, NC) with
Retrovir
Acyclovir Hurroughs AIDS,
ARC,
wellcome asymati
c HIV
positiv
e, in
combina
tion
with
AZT.
Antibody which Advanced Hio- ~ AIDS,
ARC
neutralizes pH therapy Concepts
labile alpha abet- (Rockville, MD)
rant Interferon
in an immuno-
adsorption column
L-697,661 Merck AIDS,
ARC,
(Rahway, NJ) asympto
matic
HIV
positiv
e, also
~ in
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combina
tion
with
AZT.
L-696,229 Merck AIDS,
ARC,
(Rahway, NJ) asympto
matic
HIV
gositiv
also
in
combina
tion
with
AZT.
Lamivudine Glaxo HIV
(Research Triangle
Park; NC)
Nevirapine Roxane HIV
(Columbus, OH)
It will be understood that the scope of
combinations of Product R with antiviral agents is
not limited to the list in the above Table I or
described above, but includes in principle any
combination with any pharmaceutical composition
useful for treating patients having AIDS or HIV infections.
Preferred combinations are concurrent or
alternating treatments of one or more HIV protease
inhibitors or antiviral agents useful for treating
AIDS or HIV in forms of nucleoside analogs, such as
AZT, ddI or ddC, and Product R. Preferably, the HIV
protease inhibitors or antiviral agents useful for
treating AIDS or HIV infections are administered
first, e.g. for one week, to quickly decrease the
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viral -load; Product R then is administered to
stimulate the immune system to eradicate the HIV
infections. For patients who are very ill with
opportunistic infections, Product R may be
administered to stimulate the immune system first
before and then after the indicated antiviral agents
are applied.
Alternatively, two antiviral agents useful
for treating AIDS or HTV such as lamivudine and AZT,
together with one protease inhibitor such as
ritonavir, may be administered concurrently first
until the viral load is no longer measurable, then
Product R is administered to eradicate the HIV
infections.
Or, Product R may be administered
concurrently with two antiviral agents such as
lamivudine and nevirapone, one protease inhibitor
such as indinavir.
For the patient having. HIV infections,
whether exhibiting AIDS symptoms or having
antibodies against HIV or having asymptomatic
infections, a suitable effective dose of Product R
may be in the range of from about 5 microliters to
about 40 microliters per kilogram of body weight per
day, preferably in the range of about 10 microliters
to about 25 microliters per kilogram of body weight
per day. Most preferably Product R is administered
in an amount of about 30 microliters per kilogram of
body weight per day for about one week, followed by
about 15 microliters per kilogram of body weight per
day in a sterile injectable formulation. The
desired dose may be administered as two, three or
more sub-doses at appropriate intervals, generally
equally spread in time throughout the day.
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Preferably, the full daily dose is administered in
one administration.
The dose of the antiviral agents or HIV
protease inhibitors to be co-administered with
Product R can be readily determined by those skilled
in the art, based on the usual patient symptoms, and
severity of the diseases.
Product R may be administered by any
suitable injection route including, but not~limited
to, intravenously, intraperitoneally,
subcutaneously, intramuscularly, and intradermally,
etc. The presently preferred route of administration
is intramuscularly. It will be appreciated that the
preferred route may vary with, for example, the
condition and age of the recipient.
while it is possible for Product R to be
administered as part of a pharmaceutical
formulation, it is preferable to present it alone,
although it may be administered at about the same
time as one or more other pharmaceuticals are
independently administered. If Product R is
administered as part of a pharmaceutical
formulation, the formulations of the present
invention comprise at least one ' administered
ingredient, as above defined, together with one or
more acceptable carriers thereof and optionally
other therapeutic ingredients. The carriers) must
be "acceptablep in the sense of being compatible
with the other ingredients of the formulation and
not deleterious to the recipient thereof.
The formulations may conveniently be
presented in unit-dose or multi-dose containers,
e.g. sealed ampules and vials.
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Thus, while there have been shown and
described and pointed out fundamental novel features
of the invention as applied to preferred embodiments
thereof, it will be understood that various
S omissions and substitutions and changes in the form
and details of the devices illustrated, and in their
operation, may be made by those skilled in the art
without departing from the spirit of the invention.
For example, it is expressly intended that all
combinations of those elements and/or method steps
which perform substantially the same function in
substantially the same way to achieve the same
results are within the scope of the invention. It
is the intention, therefore, to be limited only as
indicated by the scope of the claims appended
hereto.
