U.S. patent application number 12/613189 was filed with the patent office on 2010-03-04 for chemically induced intracellular hyperthermia.
This patent application is currently assigned to ST. JUDE PHARMACEUTICALS, INC.. Invention is credited to Nicholas Bachynsky, Woodie Roy.
Application Number | 20100056643 12/613189 |
Document ID | / |
Family ID | 22244263 |
Filed Date | 2010-03-04 |
United States Patent
Application |
20100056643 |
Kind Code |
A1 |
Bachynsky; Nicholas ; et
al. |
March 4, 2010 |
CHEMICALLY INDUCED INTRACELLULAR HYPERTHERMIA
Abstract
An invention relating to therapeutic pharmacological agents and
methods to chemically induce intracellular hyperthermia and/or free
radicals for the diagnosis and treatment of infections, malignancy
and other medical conditions. The invention relates to a process
and composition for the diagnosis or killing of cancer cells and
inactivation of susceptible bacterial, parasitic, fungal, and viral
pathogens by chemically generating heat, and/or free radicals
and/or hyperthermia-inducible immunogenic determinants by using
mitochondrial uncoupling agents, especially 2,4 dinitrophenol and,
their conjugates, either alone or in combination with other drugs,
hormones, cytokines and radiation.
Inventors: |
Bachynsky; Nicholas;
(Parkland, FL) ; Roy; Woodie; (Parkland,
FL) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY, SUITE 5100
HOUSTON
TX
77010-3095
US
|
Assignee: |
ST. JUDE PHARMACEUTICALS,
INC.
|
Family ID: |
22244263 |
Appl. No.: |
12/613189 |
Filed: |
November 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09744622 |
May 7, 2002 |
7635722 |
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PCT/US99/16940 |
Jul 27, 1999 |
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12613189 |
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60094286 |
Jul 27, 1998 |
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Current U.S.
Class: |
514/728 |
Current CPC
Class: |
Y02A 50/409 20180101;
A61K 38/26 20130101; A61K 31/06 20130101; A61K 45/06 20130101; Y02A
50/401 20180101; A61P 33/00 20180101; A61K 41/0052 20130101; A61P
31/00 20180101; Y02A 50/30 20180101; A61K 31/277 20130101; Y02A
50/411 20180101; A61K 31/00 20130101; A61K 31/06 20130101; A61K
31/00 20130101; A61K 31/06 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/728 |
International
Class: |
A61K 31/045 20060101
A61K031/045 |
Claims
1-54. (canceled)
55. A method for inducing intracellular hyperthermia in a subject
comprising the step of administering an amount of a mitochondrial
uncoupling agent sufficient to the subject to thereby induce whole
body intracellular hyperthermia in the subject, wherein the induced
intracellular hyperthermia in the subject is sufficient to treat a
cancer in the subject
56. The method of claim 55 wherein the cancer is selected from the
group consisting of prostate carcinoma, glioblastoma multiform,
Kaposi's sarcoma, peritoneal carcinomatosis, and glioma and
combinations thereof.
57. The method of claim 55, wherein the mitochondrial uncoupling
agent is 2,4-dinitrophenol and/or a conjugate thereof.
58. The method of claim 56, wherein the mitochondrial uncoupling
agent is 2,4-dinitrophenol and/or a conjugate thereof.
59. The method of claim 55, further comprising the step of
administering a second medication, wherein the second medication
increases a) the overall metabolic rate of the subject, b) the
metabolic rate of a specific target tissue in the subject, and/or
c) free radical flux in the subject.
60. The method of claim 59, wherein the second medication is
selected from the group consisting of glucagon, arbutamine,
dobutamine, vasopressin, glutamine, proline, octanoate, methylene
blue (tetramethylthionine), ubiquinone, menadione, hematoprophyrin,
polyunsaturated fatty acids, monounsaturated fatty acids and
combinations thereof.
61. The method of claim 55, wherein the induced intracellular
hyperthermia is sufficient to cause the induction of heat shock
proteins in a cell of the subject.
62. The method of claim 55, further comprising the step of
administering to the subject an anti-cancer agent selected from the
group consisting of metholtrexate, mercaptopuorine, fluorouracil,
cytarabine, thioguanine, azacitidine, etoposide (VP-16) and
teniposide (VM-26), vincristine, vinblastine, paclitaxel, busulfan,
cyclophosphamide, mechlorethamine, melphalan, altaretarnine,
ifosfamide, cisplatin, dacarbazine, procarbazine, lomustine,
carmustine, lomustine, semustine, chlorambucil, thiotepa,
carboplatin; flutamide, prednisone, ethinyl estradiol,
diethylstilbestrol, hydroxyprogesterone caproate,
medroxyprogesterone, megestrolacetate, testosterone,
fluoxymesterone, diiodothyroidine, triiodothyroidine,
tetraiodothyroidine, aromatase inhibitor, amino glutethimide,
octreotide, goserilin acetate, leuprolide, interferon alpha-2a,
interferon alpha-2b, interferon-gamma, interferon-beta,
interleukin-1, interleukin-2, interleukin-4, interleukin-10,
anti-HER-2/neu humanized antibody, tumor necrosis factor,
granulocyte-macrophage colony-stimulating factor,
macrophage-colony-stimulating factor, phenylacetates, retinoic
acids, leukotrienes, thromboxanes, and combinations thereof.
63. The method of claim 55, further comprising administering
radiation to the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S.
provisional patent application Ser. No. 60/094,286, filed Jul. 27,
1998, which is hereby incorporated by reference in its
entirety.
FIELD OF INVENTION
[0002] This invention relates to therapeutic pharmacological agents
and methods to chemically induce intracellular hyperthermia and/or
free radicals for the diagnosis and treatment of infections,
malignancy and other medical conditions. This invention further
relates to a process and composition for the diagnosis or killing
of cancer cells and inactivation of susceptible bacterial,
parasitic, fungal, and viral pathogens by chemically generating
heat, free radicals and hyperthermia-inducible immunogenic
determinants. Such pathogens, infected or transformed cells are
inactivated or killed without irreparable injury to
non-transformed, uninfected, normal cells. More specifically, this
invention relates to the diagnosis and treatment of cancer;
treatment of AIDS; and, other diseases and conditions using
mitochondrial uncoupling agents, especially 2,4 dinitrophenol and,
their conjugates, either alone or in combination with other drugs,
hormones, cytokines and radiation.
GENERAL BACKGROUND
[0003] Local heat, systemic hyperthermia and fever therapy have
been empirically used as effective treatments for malignant,
infectious and other diseases since antiquity. Therapeutic
hyperthermia was first documented in the Edwin Smith surgical
papyrus in the 17th century B.C. Coley's toxin extracts of
Streptococcus erysipelatis (group A streptococcus) and Bacillus
prodigiosus (Serratia marcescens) were used to induce fever for the
treatment of patients with advanced cancer. The Nobel Prize was
awarded for using fever therapy in the treatment of neurosyphilis
with the injection of malarial blood. As late as 1955, the Mayo
Clinic advocated using malariotherapy or heat therapy for cases of
tertiary syphilis "resistant to penicillin". Long term remissions
in patients with inoperable carcinomas that were treated with hot
baths and local heat applications have also been reported.
Published observations on the disappearance of malignancies such as
a soft tissue sarcoma in a patient experiencing high fever due to
erysipelas and tumor lysis of Burkitt's lymphomas following
malignant hyperthermia during surgical anesthesia are known. A
comprehensive historical review on anecdotal observations and
intuitive rational for the empirical use of therapeutic
hyperthermia has been published by Myer, J. L.
[0004] The temperature of a body can be intentionally increased
either by pyrogens to produce fever (fever therapy) or, by the
induction of hyperthermia (therapeutic hyperthermia). Fever raises
body temperature by elevating the thermoregulatory "set point"
located in the preoptic region of the anterior hypothalamus. In so
doing, the body physiologically works to maintain the higher
temperature setting. The elevated core body temperature increased
by fever may or may not be raised above the higher set point value.
In contrast, induced hyperthermia always raises the body
temperature above the hypothalamic thermoregulatory set point and
the physiologically intact body attempts to lower it's core
temperature back to the set point baseline.
[0005] Renewed clinical interest in hyperthermia has occurred over
the past 35 years due to continued failure of standard therapies to
treat various forms of cancer and emerging infections. Except for a
few exceedingly rare forms of cancer like childhood leukemias and
testicular cancer or immune responsive infections, chemotherapy,
radiation or drug therapy often do very little except briefly
extend survival. One of the major obstacles to "cure" disseminated
cancer and infections has been the innate or acquired resistance of
tumor cells and emerging microbes to antibiotics, drugs and
treatments given in tolerable doses. Escalation of treatments, or
use of multiple drugs to overcome resistance is invariably
prevented by concomitant toxicities or development of multi-drug
resistance. Further, in contrast to drugs, which represent a single
molecular species that biochemically interact with specific enzymes
or receptors of viruses, prokaryotes and eukaryotes, the action of
hyperthermia is biophysical and global. Hyperthermia has no
specific heat receptors. Therefore, the possibility of a point
mutation causing a functional change in a receptor and conferring
resistance to hyperthermia is unlikely, and would be equivalent to
the development of resistance to the in vitro process of
Pasteurization. Among pathogenic bacteria, it has been reported
that only one variant in 1.times.10.sup.6 cells of an original
population is resistant to hyperthermia.
[0006] Hyperthermia has been used alone or in conjunction with
radiation and chemotherapy in the treatment of a variety of
malignancies. Overgaard et al., reported that a combination of heat
and radiation results in complete control of twice as many melanoma
lesions compared to radiation alone. Maeda, M., Watanabe, N. et
al., published in Gastroenterologia Japonica, that hyperthermia
with tumor necrosis factor resulted in successful treatment of
hepatocellular carcinoma. Prospective randomized studies of
hyperthermia combined with chemoradiotherapy for esophageal
carcinoma demonstrated the cumulative three year survival rates to
be more than doubled with the addition of hyperthermia to
chemoradiotherapy. Combination chemotherapy with hyperthermia in
metastatic breast cancer refractory to common therapies, i.e.,
failed prior hormonal therapy and chemotherapy, resulted in 39%
complete remissions and 23% partial remissions: relief of bone pain
was striking. Fujimoto, S., Takahashi, M. et al., demonstrated that
the 5 year survival rate of patients with peritoneal carcinomatosis
from gastric carcinoma treated with intraperitoneal hyperthermic
chemoperfusion was 41.6%, whereas the 50% survival duration of the
group that did not receive intraperitoneal hyperthermia was 110
days. Preoperative hyperthermia with chemotherapy and radiation is
also known to improve long-term results in patients with carcinoma
of the rectum, especially those with advanced disease. It is
clinically known that regional, i.e., limb, hyperthermic perfusions
with chemotherapy is useful for the treatment of melanoma.
Combination therapy with hyperthermia and radiation has been
successful in the treatment of non-Hodgkins lymphomas. More
recently, a survival benefit of hyperthermia was shown in a
prospective randomized trial for patients with glioblastoma
multiforme undergoing radiotherapy. However, rigorous clinical
prospective randomized trials with hyperthermia alone or, in
combination with agents outside its use with radiation therapy have
not been performed.
[0007] The scientific rationale for therapeutic hyperthermia in
cancer therapy rests on-known data from pre-clinical, in vitro and
animal studies. Tumor cells in tissue culture have been
demonstrated to be directly more sensitive to heat as compared to
their non-malignant counterparts. Cells undergoing mitosis,
synthesizing DNA in the `S-phase`, are especially more sensitive to
hyperthermia. Human leukemic progenitor cells have been shown to be
selectively killed by hyperthermia and, such in vitro use has been
shown to purge bone marrow of residual tumor cells before
autologous bone marrow transplantation. Microcalorimetric
measurements confirm that timorous tissues produce more heat and
are "hotter" than their non-tumorous counterparts. As a
consequence, they are less able to tolerate additional heat
loads.
[0008] Tumor cells are also killed by heat indirectly. Tumor
angiogenesis is inhibited by heat. Hyperthermia causes tumors to
have increased heat retention with increased cytoxicity due to
tumor neovasculature lacking smooth muscle and vessel wall
precursors needed for cooling by vasodilation. Increased hypoxia,
acidity, Fos gene death signaling, decreased nutrient supply and
enhanced immunologic cytotoxicity have also been reported to be
caused by hyperthermia and contribute to enhanced tumor cell death.
Further, the combination of hyperthermia with chemotherapy and/or
radiation has been shown to be supraadditive or synergistic on
killing of tumors. Human gastric carcinoma cells have been shown to
be selectively killed by a combination of cisplatin, tumor necrosis
factor and hyperthermia: a 40% increase in cisplatin DNA damage was
noted in the presence of the three agent combination over cisplatin
alone or either dual combination. Numerous animal studies,
including the initial publication by Crile, show that neoplasms
transplanted into mice regress when treated with hyperthermia
without irreparable damage to adjacent tissues.
[0009] Body temperature is a critical factor in determining host
susceptibility, location of lesions, and the natural history of
many infectious diseases. Temperature has direct effects on the
growth of all microorganisms, including those that are pathogenic.
Almost all of the bacteria that cause disease in humans grow
optimally within the range of 33-41.degree. C. and, their
temperature growth characteristics are not easily altered in vitro.
By example, the lesions of Hansen's disease (leprosy) caused by
Mycobacterium leprae, characteristically grow and destroy the most
acral, coolest parts of the body such as fingers, toes, external
ear, the air-stream cooled nasal alae and larynx. Leprosy organisms
proliferate and follow the coolest temperature gradients in the
body, 25-33.degree. C. In animals, the leprae organisms can only be
grown in the armadillo or foot pads of mice were the in situ lesion
temperatures are 27-30.degree. C. Spontaneous improvement in
leprosy lesions have been reported in patients following febrile
illness. Fever therapy, hot baths and local heat therapy were
formerly utilized in treating this disease. Hyperthermia is also
known to destroy Treponema pallidum, the causative agent of
syphilis, by heating five hours at 39.degree. C., three hours at
40.degree. C., two hours at 41.degree. C. or one hour at
41.5.degree. C. The spirochetes responsible for yaws, bejel, pinta
and Lyme disease show similar temperature sensitivity.
[0010] Other bacteria that predominately cause lesions at cool
sites and are susceptible to heat inactivation include, Neisseria
gonorrhea, Hemophillus ducrei (chancroid), Mycobacterium ulcerans,
Mycobacterium marinum ("swimming pool" granuloma), Diptheria, etc.
Further, hyperthermia has been reported to be synergistic with
antibiotic and chemotherapy in the treatment of various bacterial
diseases. Elevated body temperature potentiates the effect of
penicillin on stapholococci and syphilis. Hyperthermia makes
sulfadiazene bactericidal for streptococci. Moreover, recent
controlled studies show that when antipyretics are used in animals
with severe experimentally induced infections, there is increased
mortality. Nonetheless, systemic hyperthermia has generally been
abandoned as a treatment for bacterial infections with the advent
of antibiotics.
[0011] Hyperthermia has remained an effective treatment for many
fungal infections. Superficial dermatophytosis flourish in cooler
regions of the body and heat treatment is oftentimes the only
viable therapy for their chronic granulomatous lesions. By example,
Sporothrix schenkii, the causative agent of sporotrichosis, has a
temperature growth optimum well below 37.degree. C. and is
successfully eliminated by local hyperthermia. Similarly, patients
with pseudallescheriosis unresponsive to antifungal antibiotics are
healed with hyperthermic treatments. In Japan, pocket warmers, hot
water and infrared heating remain current and effective treatments
for various fungal infections. Systemic hyperthermia, utilizing a
Liebel-Flarsheim (Kettering) Hypertherm Fever Cabinet, dramatically
treated a case of disseminated sporotrichosis with recurrent
iridocyclitis, repeated post-treatment cultures from the patient
remained negative.
[0012] The role of hyperthermia in modulating the clinical course
of other fungal infections, including histoplasmosis, North
American blastomycosis, chromomycosis, cryptococcosis,
paracoccidioidomycosis, Lobos' disease and candidiasis has been
described. Fungi, such as Nocardia, Actinomyces and Aspergillus
also proliferate in cooler regions of the body causing mandible
(lumpy jaw) and foot lesions (Madura foot) respectively. In vitro
heat sensitivity data for many of the above and other pathogenic
fungi have been reported by Mackinnon et al., Silva and others.
[0013] The effect of temperature and hyperthermia on the
pathogenesis of parasitic disease is also well known.
Leishmaniasis, a wide spread parasitic disease transmitted by the
bite of a sandfly, clinically infects 12 million people worldwide.
The cutaneous and mucocutaneous lesions, i.e., Oriental sore,
Baghdad boil, Delhi boil, Chiclero's ulcer and espundia, are often
very destructive and permanently disfiguring. Hyperthermia with
moist heat of 39.degree. to 41.degree. C. applied for 20 hours over
several days has proven to be an effective treatment. In vitro,
human macrophages infected with Leishmania mexicana are completely
destroyed by heating at 39.degree. C. for 3 days. All
muco-cutaneous Leishmania strains, regardless of subspecies,
demonstrate a growth optimum of 35.degree. C. with only the L.
tropica and L. donovani strains surviving temperatures of
39.degree. C. Clinical observations have shown that hyperthermic
treatment of one Leishmania lesion often invokes an immune response
and results in the healing of other lesions over a 5-6 week period.
The effect of hyperthermia on other parasites, including
Trypanosoma cruzi, malaria, microfilaria, acanthamoeba, trematodes
and cestodes has been published.
[0014] Increased body temperature is also recognized as a major
factor in recovery from viral infections. Many viruses multiply
better at temperatures below 37.degree. C. and their multiplication
is inhibited or stopped if the body temperatures exceeds 39.degree.
C. In vitro Rhinovirus replication, for example, falls off by
10.sup.6 log units with an upward temperature shift of 2.degree. C.
(37.degree. to 39.degree. C.). Herpes virus replication, as well as
the intracellular and extracellular herpes virus concentration,
markedly decrease when the incubation temperature is elevated to
40.degree. C. Varicella virus production in human fibroblastic cell
culture is optimal at 37.degree. C. and ceases at 39.degree. C.
[0015] Beneficial effects of hyperthermia on the outcome of viral
disease in laboratory animals infected with myxomatosis,
encephalomyocarditis, herpes, gastroenteritis, rabies and the
common cold in man have been documented. Influenza and viruses,
causing upper respiratory infections, such as the common cold,
thrive in a cool body milieu of 30.degree.-35.degree. C.
Temperature gradients in this range exist in the fall and winter
within the oral, nasal, tracheal and laryngeal mucosa and lead to
flu and influenza epidemics. Live respiratory-virus vaccines for
influenza have been developed by use of heat-sensitive mutants that
cannot reduplicate or cause clinical disease at
36.degree.-37.degree. C. It is known that even as little as a
0.5.degree. C. difference in the ceiling replication temperature of
a virus can have a dramatic effect on virulence and
pathogenicity.
[0016] Other animal viruses such as Newcastle disease in chickens,
rabbit papilloma, feline leukemia, rabbitpox, hoof-and-mouth
disease in cattle, hand, foot, and mouth disease, human plantar
warts, and the "grease" of horses, due to horsepox involvement of
the colder acral extremities above the fetlocks, are known to be
very sensitive to inhibition by heat. Heat treatment of cells
infected with human immunodeficiency virus (HIV-1) at 39.degree. C.
for 2 days has been documented to significantly decrease viral
production and reduce reverse transcriptase enzyme marker activity
30 fold. In vitro hyperthermia of 42.0.degree. C. for 1 hour, 4
days apart selectively lowers HIV RNA loads in chronic (latent)
infected T lymphocytes. Hyperthermia of 42.degree. C. for 3 hours
combined with tumor necrosis factor has been published to
selectively kill all acute and chronically infected HIV cells in
tissue culture.
[0017] Use of whole body hyperthermia has been reported to cause
regression of Kaposis' sarcoma, clear oral candidiasis, eliminate
hepatitis C, cause remission of Varicella-zoster, increase weight
gain and improve CD4 lymphocytes counts in patients with acquired
immunodeficiency syndrome (AIDS). Dramatic improvement with
hyperthermia therapy has been documented in a patient infected with
a debilitating Verruca vulgaris and HIV. The FDA has approved
clinical trials involving hyperthermia for the treatment of AIDS
with a patented extracorporeal blood heating machine to induce
whole body hyperthermia. The FDA has recently expanded the
extracorporeal heating machine trials to permit treatment of 40 HIV
infected patients.
[0018] Hyperthermia can augment cytotoxicity and reverse drug
resistance to many chemotherapeutic agents. Moreover, hyperthermia
has also been shown to enhance the delivery of many novel cancer
therapeutic agents, i.e., monoclonal antibodies to neoplasms with
resultant improvement in antitumor effect; enhance the delivery of
gene therapy with use of viral vectors; and, augment drug delivery
and antitumor effects when using drug containing liposomes. In
addition to increasing the rate of extravasation of liposomes from
the vascular compartment by a factor of 40-50, hyperthermia can
also be used to selectively release chemotherapeutic agents from
liposomes designed to be thermosensitive. Thermosensitive liposomes
are small vesicles composed of lipid phosphatidylcholine moieties
constructed to contain and transport a variety of drugs. The
liposomes are designed to remain stable in the blood and tissues at
physiologic temperatures. When passing through an area of heated
tissue however, they dissolve and effectively release their
encapsulated contents. Thermosensitive liposomes are used to entrap
and carry drugs whose systemic toxicity is desired to be limited to
a particular heated tumor, organ or tissue. Examples of drugs that
have been encapsulated into liposomes include methotrexate,
doxorubicin, amphotericin B, cisplatin and others. Liposomes can be
designed so as to release their contents at pre-determined
temperatures.
[0019] Hyperthermia has also been an effective solution for the
treatment of a variety of heat labile toxin or poisonous
envenomations. For example, an easy treatment for Scorpaenidae and
Siganidae envenomation is the local application of heat. The major
poisonous component of this and many other venoms from lionfish,
weever fish, bullrout, sculpin, surgeon fish, scorpion fish,
stonefish, butterfly cod, etc., is a very heat labile,
non-dialyzable protein. As opposed to the nuances of using specific
anti-venom, emmersing the envenomated area or patient in hot water,
or applying other forms of hyperthermia, is a simple and prompt
treatment.
[0020] Standard clinical methods of inducing hyperthermia are
dependent on the deposition of exogenous heat to that normally
produced by the metabolism. All current deliberate and controlled
methods of heating require an external source of energy.
Non-surgical methods of heating include: hot air, ultrasound,
microwaves, paraffin wax baths, hot water blankets, radiant heat
devices, high temperature hydrotherapy and combinations thereof.
Invasive means of inducing hyperthermia include surgical insertion
of various heating devices, infusion of heated solutions into the
peritoneal cavity through catheters or heating the blood
extracorporeally through a heat exchanger. The later method,
developed by Parks et al., involves the surgical placement of a
femoral arterio-venous shunt for the removal, heating and
replacement of blood to induce whole body hyperthermia. A more
recent experimental improvement on this method has been the
induction of whole body hyperthermia with veno-venous shunt
perfusions. Several machines have been patented for extracorporeal
heating of blood to induce hyperthermia (see U.S. Pat. Nos.
5,391,142 and 5, 74,190).
[0021] Endogenous heating by creating fevers induced with toxins,
pyrogens and microorganisms have been used in the past and have
recently been re-attempted. Heimlich has been reported to use
Malaria therapy for the treatment of Lyme disease, AIDS and
malignancy. Pontiggia et al, treated AIDS patients by combining
fever, induced by parenteral injections of a streptococcal lysate
preparations, with hyperthermia generated by an infrared heating
bed.
[0022] Another way that the prior art has dealt with inducing
hyperthermia has been by introducing micron size magnetic particles
and subjecting them to either magnetic fields or hyperbaric oxygen
(see U.S. Pat. No. 4,569,836). This method was designed for the
treatment of cancer based on the belief that cancer cells would
engulf the particles and concentrate them intracellularly. A
magnetic field would then be applied to heat the particles and
generate lethal hyperthermia within the cancer cells. A
modification of this technology is the use of magnetic cationic
liposomes to induce intracellular hyperthermia. This technology was
based on the observation that glioma cells have a greater affinity
for positively charged rather than `neutral` magnetic lipsomes. A
more recent variation on this science has been developed in Germany
using `targeted` magnetoliposomes. This methodology has been
developed in an attempt to treat AIDS by using magnetic
nanoparticles coupled to either CD4 lymphocyte or anti-gp120 HIV
antibodies. The magnetic nanoparticles are intended to selectively
bind to either the HIV protein envelope or the HIV infected cells
and then be heated by external high-frequency alternating magnetic
fields.
[0023] Whether invasive or non-invasive, all current methods of
inducing hyperthermia depend on an external energy source and
cannot safely deliver adequate power to result in therapeutic
heating. Delivery of heat to obtain the actual desired temperature
to deep target tissues has not been possible because of the actual
physics involved in the thermodynamic, conductive transfer of heat
from the outside into the cell. Heating tissues deeper than five
centimeters below the skin with microwave, radio frequency or
ultrasound devices is difficult because energy absorption is not
uniform or focused. Radiant heat, hot water, molten wax and other
methods cause excessive heating of subcutaneous fat which acts as a
barrier to body heat gain. Common adverse effects of such external
heating methods include surface skin burns, blistering,
ulcerations, secondary opportunistic infections and pain.
Additionally, many tumors have high blood flow cooling which
nullifies any potential therapeutic gain achievable through the use
of such extracellular, systemic hyperthermia devices. Also,
insufficient heating power prolongs the induction time required to
reach the actual therapeutic temperature. This promotes resistance
to heat treatment through the development of the heat shock
response and thermotolerance.
[0024] High frequency electromagnetic devices used to heat
intracellular magnetic particles invariably induce eddy currents
within the body making it difficult to provide uniform, controlled
and safe heating without toxic effects to normal cells. Further,
not all tumors possess characteristics that cause them to
selectively take up magnetic particles or have an affinity for
positively charged magnetic liposomes. Also, magnetic cationic
liposome particles are subject to various neutralizing interactions
with anions, giving them a short charged half-life. Moreover, the
complexity of using specific anti-HIV antibodies bound to
electromagnetic particles also assumes a non-mutating HIV genome
with stable antigenic determinants. To the contrary, a high
mutation rate in the HIV genome and it's protein antigenic
determinants is known to exist and is the main obstacle to the
development of an effective vaccine. Such treatments therefore, do
not selectively heat transformed cells without heating and injuring
normal cells.
[0025] Extracorporeal blood heating methods require surgery and
anesthesia. Further, as with all external heating methods,
temperature variances and toxic conductive thermogradients from the
point of initial heating to the target tissue cannot be avoided. By
example, bone marrow temperatures are consistently known to be
1.degree.-2.degree. C. below the average body core temperature
achieved by extracorporeal blood hyperthermia. This is a major
problem in systemic hyperthermic therapy since the marrow is a
common repository of metastatic cancer cells and infectious
microorganisms. Therapeutic bone marrow temperatures are not
achievable due to the fact that the intermediate tissues between
the blood and the marrow create a temperature gradient cooling the
blood before it reaches the bone marrow. Since efficacy and
toxicity of hyperthermia depend on both the actual temperature and
duration of heating, delivering the desired
temperature-and-duration of heating (thermal dose) to the bone
marrow would require the blood and intermediate tissues to be
heated beyond that which is safe for normal, healthy cells. A
multicentre European trial documented that only 14% of all
protocols achieve required target temperatures. Further, current
extracorporeal heating methodology and equipment is labor
intensive, time-consuming and expensive.
[0026] Use of fever inducing agents such as live microorganisms,
pyrogens and toxin lysates is clinically uncontrollable,
unpredictable or insufficient as to both the degree and duration of
temperature increase.
[0027] Further reasons why hyperthermia has not yet become more
widely accepted as a mode of therapy is because current heating
machines are not compatible with noninvasive temperature
measurement technology. Measurement of the actual temperatures
reached in target tissues is critical for heating efficacy, i.e.,
determining the thermal dose. Recently, noninvasive thermometry
with Magnetic Resonance Imaging (MRI), ultrasound backscatter,
electrical impedance, electromagnetic adaptive feedback and
advanced, high-precision pixel infrared temperature imaging have
been developed. To use MRI or other equipment to monitor real time
hyperthermia however, it is necessary to combine a hyperthermia
device with an MRI unit. This has proven to be difficult and costly
since each device is functionally disturbed, if not damaged, by the
presence of the other.
[0028] The exact molecular and cellular mechanism by which heat
kills or inactivates tumor cells and microorganisms is unknown.
Heat is an entropic agent and acts globally on every molecule
constituting the cell. Heating is known to cause conformational
changes in proteins, denature enzymes and affect cell membrane
fluidity. By example, herpes simplex virus (type 1) thymidine
kinase has a shortened half-life at 40.degree. C. of only 30
minutes. The transforming gene product-enzyme of Rous sarcoma virus
(protein phosphatase), a critical protein for cellular regulation,
is totally inactivated in 30 minutes at 41.degree. C. Hyperthermia
is known to increase the formation of oxygen free radicals,
including superoxide, hydroxyl, hydroperoxyl, hydrogen peroxide and
lipid peroxides. These reactive oxygen species react
indiscriminately and oxidize many organic molecules causing DNA
damage, protein denaturation, lipid peroxidation and other
destructive chain reactions. Acid microenvironments, known to exist
in tumors and microorganisms with high rates of glycolysis
(Embden-Meyerhof Pathway) and lactic acid production, favor
protonation of the superoxide radical to form the highly reactive
and toxic hydroperoxyl radical. Thus, thermal sensitivity of many
tumors increases with decreasing intracellular pH. As compared to
normal cells, many malignant and virally transformed cells have a
reduced total functional capacity to withstand the increase flux of
oxygen free radicals produced by hyperthermia.
[0029] On the intracellular level, moderate heating is known to
activate phospholipase A2, which increases the formation of
pro-inflammatory mediators such as the leukotrienes, prostaglandins
and eicosanoids. Heat also increases release of intracellular
calcium through the stimulation of phospholipase C. Calcium cycling
across the mitochondrial membrane appears critical to the increased
production of oxygen free radicals. Increased intracellular calcium
also inhibits the mitochondrial, anti-apoptotic Bcl-2 protein and
induces the production of heat shock proteins, mediating
thermotolerance. Heat injury to the intracellular tubulin network,
lysosomes, Golgi bodies, mitochondria, and control of RNA splicing
are some of the many known subcellular systems affected by heat.
While the initial primary event leading to cell death by
hyperthermia is unknown, a decrease in mitochondrial membrane
potential followed by uncoupling of oxidative phosphorylation and
generation of reactive oxygen species on the uncoupled respiratory
chain are the first biochemical alterations detectable in cells
irreversibly committed to apoptosis. The cytotoxic effect of
hyperthermia is thus believed to be caused by numerous changes and
complex damage to multiple vital cell functions. Those biochemicals
altered by heat and essential to the function or viability of the
cell are the pivotal targets of therapeutic heating.
[0030] The mode of hyperthermic cell injury is dependent on the
severity of the heat stress, temperature and duration of heating.
Moderate heating of 39.degree.-42.degree. C. is used
therapeutically and is known to promote programmed cell death
through apoptosis, an active process of selectively eliminating
heat sensitive cells without inflammation, bystander-cell death or
subsequent tissue fibrosis. Malignant and other transformed cells
undergo apoptosis by suppression or activation of one or more genes
such as bcl-2, c-myc, p53, TRPM-2, RP-2, RP-8, raf, abl, APO-11FAS,
ced-3, ced-4, ced-9, etc. Drugs (methotrexate, cisplatin,
colchicine, etc.), hormones (glucocorticoids), cytokines (tumor
necrosis factor-alpha), radiation (free radicals) and hyperthermia
can all initiate apoptosis. Increasing the temperature or duration
of heating, or both, leads to cell death via necrosis. This
physical process of indiscriminate cell killing is associated with
inflammation and causes significant injury to normal, healthy
cells.
[0031] For purposes of systemic hyperthermia, apoptosis of target
cells is the therapy of choice. In the clinical setting it must be
controlled under conditions of moderate heating so as to
selectively differentiate and eliminate target cells with minimum
toxicity to normal cells. Such controlled conductive heating by
external technologies is inherently not possible. The thermal
physical and thermophysiologic properties of cells vary and are
dependent on their thermal conductivity, specific heat, density and
blood perfusion among the various organs and tissues. Based on
these properties, the actual temperatures at some of these sites
are often `partitioned`, independent of one another and do not
represent the monitored, mean "core" temperature achieved during
therapy. Additionally, it is well recognized that it is the actual
intracellular temperature increase, with it's associated internal
physical and chemical changes, that is critical to the successful
use of hyperthermia in exploiting the fundamental biochemical
differences between normal and heat susceptible cells.
Unfortunately, the initial cellular targets of all extracorporeal
heating methods are the cell membrane and it's integrated proteins.
The cell's internal contents, including mitochondria,
compartmentalized enzymes, other organelles and any intracellular
pathogens, etc., are progressively heated in sequence by thermal
conduction from the outside-in. Thus, to sufficiently heat the
interior of the cell, the external temperature must overcome the
cellular and mitochondrial membranes, each composed of a lipid
bilayer that acts as an effective thermal barrier.
[0032] By necessity, therefore, prior art heating methods require
high external temperatures to establish a sufficient gradient to
overcome the nonisotropic and non-homogeneous conductive heat loss
between internal tissues and the insulating barrier of the cellular
and mitochondrial membranes. For example, the Organetics PSI.RTM.
(now First Circle Medical Inc.) device has to heat blood externally
to 48.degree. C. (118.4.degree. F.) before returning it directly
into the vascular system of the patient. Other extracorporeal
circuit perfusion devices need to achieve ex vivo temperatures of
49.degree. C. (120.2.degree. F.). Animal studies require
temperatures of 54.degree. C. (129.1.degree. F.) during the
induction phase to achieve adequate target tissue temperatures.
Safety in such prior art is therefore limited by the incipient
destruction of surrounding tissues at the sites of the high
temperature phases of heating. When lesser temperatures are
attempted, effectiveness is compromised by either inadequate
temperatures or duration of heating or development of
thermotolerance. As a result, only regional hyperthermia has been
widely used clinically and only in combination with more
traditional techniques such as radiation and chemotherapy.
Presently, none of the known heating technologies provide
clinically safe and effective hyperthermia to treat systemic or
disseminated disease. In order for systemic hyperthermia to become
more widely used clinically, current heating methods must also
overcome the use of labor intensive, complex equipment, including
invasive extracorporeal infusion and it's related toxicity problems
to interposed tissues. Further, new hyperthermic technology must be
compatible with noninvasive, real time thermometry.
[0033] The present invention avoids the problems of heat toxicity,
inadequate target tissue heating, excessive cost, surgery,
anesthesia and incompatibility with noninvasive temperature
measuring devices: problems that are inherent to all therapeutic
methods that deliver heat extracellularly, from the outside-in.
This invention is an intracellular, therefore, an intracorporeal
heating system which has additional distinct advantages. First, the
human body is biochemically and physiologically designed to
tolerate higher temperatures when heated from the inside-out as
opposed from the outside-in. By example, in comparison to
extracorporeal heating, which can safely generate a maximum body
core temperature of 42.degree. C. (107.6.degree. F.),
intracorporeal hyperthermia caused by strenuous exercise induces
physiologic temperatures of up to 45.degree. C. (113.0.degree. F.)
in muscle and liver with body core temperatures of up to 44.degree.
C. (111.2.degree. F.). Exertional heat stroke patients have
survived rectal body temperatures as high as 46.5.degree. C.
(115.2.degree. F.) without any permanent clinical sequela. While
the critical maximum temperature humans can tolerate is unknown,
physiologic hyperthermic temperature induced under controlled
conditions with adequate hydration have not shown any permanent
untoward effects. Liver biopsies from subjects with such
temperatures have not shown any significant microscopic
abnormalities. Second, since heating with the present invention is
chemically induced from within the cell, the actual intracellular
therapeutic temperature will be higher than the measured core
temperatures. As a result, intracellular organelles, including
mitochondria, are heated at higher temperatures, undergo greater
uncoupling and generate an increased flux of reactive oxygen
species. Since oxygen free radicals, including superoxide, enhance
and probably mediate the effects of hyperthermia, an improved
therapeutic gain will be obtained at lower body core temperatures.
Further, it is known that for each 0.5 degree Celsius increase in
body temperature the metabolic rate and oxygen consumption increase
7%. Such an increase will assist heating the body in itself. Third,
safety and control of temperatures with the present invention is
far superior to that of exogenous methods. The body is naturally
designed to dissipate heat from the inside-out. This is evident
from the fact that a temperature gradient of
3.5.degree.-4.5.degree. C. exists between the visceral core and the
skin. This gradient represents the transfer of heat from regions of
high temperature to regions of low temperature, with ultimate heat
loss from the skin to the environment through conduction,
convection, radiation and sweat induced evaporation. The margin of
safety and control represented by the `feedback gain` of this
intact physiologic heat dissipating system is extremely high,
approximating 27-33. This rate of cooling can balance an influx of
heat in a naked human body in a dry room at about 120.degree. C.
(248.0.degree. F.). Thus, the human heat flow system permits the
body to rid itself of excess endogenous heat very quickly and
effectively. As a result, there is a wide margin of safety in case
the target temperature is exceeded. In contrast, exogenous heating
contravenes the natural physiologic flow of heat and its
dissipating mechanisms. The natural heat dissipating mechanisms are
overwhelmed and compromised. Control and safety over hyperthermia
induced by extracellular means is thus fragile, with little room
for error.
SUMMARY OF THE INVENTION
[0034] The present invention encompasses a composition and method
using mitochondrial uncoupling agents, especially DNP, DNP with
free radical producing drugs, DNP with liposomes, DNP conjugated to
free radical formers, and DNP with other therapeutic pharmaceutical
agents which are activated intracellularly by heat or reaction with
mitochondrial electrons or free radicals to cause release of active
medications for the treatment of cancer, HIV, other viruses,
parasites, bacteria, fungi and other diseases. While not being
bound by theory, it is submitted that the use of mitochondrial
uncoupling agents, to increase intracellular heat and free
radicals, as treatment for non-related cancers, viruses and other
pathogens presupposes that the mechanism of action is non-specific
for enzymes and receptors but is specific for interference with
cellular and pathogen viability and induction of programmed cell
death. The degree of intracellular heating, free radical formation,
whole body hyperthermia and release of active drug molecules is
controlled by the dose of DNP. Based on the quantity of oxygen
consumed, the dose of DNP is adjusted to achieve the desired degree
of hyperthermia. Safety and effectiveness is further controlled by
manipulating metabolic rates of target tissues, duration of
treatment and permissiveness of body cooling. In accordance with
the present invention, intracellular, mitochondrial heat is
generated by the use of DNP, other uncouplers, their conjugates,
either alone or in combination with other drugs for the treatment
of thermosensitive cancers such as non-Hodgkins lymphoma, prostate
carcinoma, glioblastoma multiforme, Kaposi's sarcoma, etc; bacteria
such as Borrelia burgdorferi, Mycobacterium leprae, Treponema
pallidum, etc.; viruses such as HIV, hepatitis C, herpes viruses,
papillomavirus, etc.; fungi such as Candida, Sporothrix schenkii,
Histoplasma, Paracoccidiodes, Aspergillus, etc.: and, parasites
such as Leishmania, malaria, acanthomoeba, cestodes, etc.
2,4-dinitrophenol was selected as the uncoupler of choice because
it can be used at relatively high concentrations, permitting
uniform distribution in organs and tissues. This invention also
encompasses the use of DNP to selectively augment energy metabolism
and heat production in inchoate malignant tumors for the purpose of
increasing sensitivity of diagnostic positron emission tomography,
temperature-sensitive magnetic resonance, and high-precision pixel
temperature infrared imaging in differentiating normal from
aberrant cell metabolisms. An additional object of the invention is
the use of DNP to increase transcription of heat shock proteins,
especially HSP 72, as a form of cellular pre-conditioning to
decrease post-angioplasty restenosis, increase successful outcome
of other surgeries, and facilitate antigen processing and
presentation of immunogenic determinants on infectious agents,
virally transformed cells and tumors so as to increase the natural
or biologically activated immunological response.
[0035] In accordance with another aspect of the present invention,
controlled thermogenesis with DNP is combined with other agents
used to treat infectious, malignancy and other diseases. Examples
of other agents include antifungal, antiviral, antibacterial,
antiparasitic and antineoplastic drugs. Such drugs, including
angiogenesis inhibitors and radiation have increased synergistic or
additive activity when combined with hyperthermia in the treatment
of cancer.
[0036] The method can be used for enhancing the sensitivity of
positron emission tomography, nuclear magnetic resonance
spectroscopy and infrared thermography in the diagnosis and
monitoring of treatment of various diseases, including cancer.
Similarly, the method can be used for enhancing the identification
of unstable "hot" coronary and carotid artery plaques predisposed
to rupture or undergo thrombosis. Such diagnostic and treatment
monitoring methodology is based on the fact that most tumors have
higher metabolic rates and generate more heat than normal tissues.
Likewise, unstable atherosclerotic plaques are presumed to rupture
because they have a dense infiltration of macrophages which have
high metabolic rates and generate excessive enzymes and heat,
causing the plaque to degrade and loosen. In both instances,
controlled doses of DNP or other uncouplers can further increase
metabolic rates and heat production to increase diagnostic
sensitivity. Controlled heating with DNP and fibrinolytic
recombinant tissue-type plasminogen activators can also be used
therapeutically to accelerate fibrinolysis of clotted arteries.
[0037] In another aspect of the invention, DNP is administered in
controlled and timed dosages to provide physiologic stress,
"chemical exercise", so as to induce synthesis of autologous heat
shock proteins (HSPs). Intracellular heat exposure associated with
autologous HSP induction has a significant cytoprotective effect
against ischemia and cellular trauma and acts as a form of cellular
thermal preconditioning in patients about to undergo surgery.
Induction of HSPs by DNP in patients some 8 to 24 hours prior to
angioplasty, coronary bypass surgery, organ transplantation and
other forms of high risk surgery, would provide for improved
clinical outcome with decreased post-angioplasty intimal thickening
or restenosis, increased myocardial protection from infarction,
improved musculocutaneous flap survival in plastic reconstruction
and reduced ischemia/reperfusion injury in organ transplantation
cases.
[0038] Another aspect of the invention provides for controlled
dosages of DNP to induce long duration (6 to 8 hour), mild whole
body hyperthermia (39.0 to 40.0.degree. C.) to afford maximum
expression of immunogenic HSPs or peptides associated with HSPs.
The antigenic properties of HSPs and HSP-peptide complexes, induced
by DNP in infectious agents, especially those located
intracellularly, or on tumors can be exploited to enhance the
immune response. This aspect of the present invention provides a
process for modulating the immune system of a patient with other
therapies, comprising the steps of: (1) increasing the expression
of HSPs by the process described above, and (2) administering
humanized monoclonal or polyclonal antibodies, or (3) administering
recombinant cytokines, lymphokines, interferons, etc., or (4)
administering standard anti-infectious or anti-neoplastic
therapy.
[0039] Additional objects and advantages of the invention will be
set forth in part in the description of drawings that follows, and
in part, will be obvious from the description, or may be learned by
practice of the invention. The objects and advantages can be
realized and obtained by means of the uses and compositions
particularly pointed out in the detailed description of the
preferred embodiments and in the appended claims.
BRIEF DESCRIPTION OF DRAWINGS
[0040] FIG. 1 shows features of glycolysis with formation of
pyruvic acid and release of energy as heat.
[0041] FIG. 2 depicts the conversion of pyruvic acid into acetyl
CoA and the 2 carbon fragments entering the TCA cycle.
[0042] FIG. 3 shows the transfer of electrons down the electron
transport chain during the process of oxidative phosphorylation
[0043] FIG. 4 shows oxidative phosphorylation as a coupling of two
distinct processes, oxidation of reducing equivalents and formation
of ATP. Both processes are "coupled" by an electrochemical membrane
potential created by electrons passing down the electron transport
chain.
[0044] FIG. 5 shows the process of chemiosmosis. Electrons passing
down the electron transport chain create energy to pump H.sup.+
outside the inner mitochondrial membrane. This process creates a
protonmotive force that causes formation of ATP by protons
re-entering the membrane through ATP-synthase.
[0045] FIG. 6 depicts the uncoupling of oxidative phosphorylation
through injury of the inner mitochondrial membrane. FIG. 6(a) shows
how oxidative phosphorylation is uncoupled by DNP in intact and
uninjured mitochondrial membranes.
[0046] FIG. 7 shows the initial formation of superoxide radicals by
the univalent reduction of oxygen in the electron transport chain.
FIG. 7(a) depicts the formation of hydrogen peroxide and hydroxyl
radicals through the Haber-Weiss Reaction. FIG. 7(b) shows an
overview of mitochondrial oxygen utilization and free radical
formation.
[0047] FIG. 8 depicts the effects of heating on mitochondrial
uncoupling and correlation of uncoupling to superoxide free radical
formation.
[0048] FIG. 9 depicts the increased formation of oxygen free
radicals after cessation of DNP uncoupling and normalization of
oxygen consumption.
[0049] FIG. 10 shows the global intracellular effects of DNP,
including the dominant foci of increased heat generation.
[0050] FIG. 11 shows the relative potencies of various
uncouplers.
[0051] FIG. 11(a) shows the effect of body temperature on metabolic
rate.
[0052] FIG. 12 shows six of the Hottest organs in the human body
and their relative blood flow.
[0053] FIG. 13 shows the effect of successive doses of 2,4-DNP on
oxygen consumption.
[0054] FIG. 14 shows a typical DNP induced hyperthermia patient
monitored flow chart.
[0055] FIG. 15 shows a monitored patient flow chart after
successive infusions of DNP and glucagon for treatment of parasitic
disease of the liver.
[0056] FIG. 16 shows killing of chronically HIV infected HUT-78
cells with varying concentrations of DNP.
[0057] FIG. 17 shows a patient flow chart after infusion of
norepinephrine and successive intravenous doses of DNP for
treatment of HIV disease. FIG. 17(a) depicts surrogate parameters
relating to HIV disease before and after DNP treatment.
[0058] FIG. 18 shows a monitored patient flow chart after
successive infusion of DNP for treatment of Lyme disease.
[0059] FIG. 19 shows a monitored patient flow chart using an
alpha-1 adrenergic agonist with DNP to induce hyperthermia in a
patient with disseminated cancer.
[0060] FIG. 20 shows survival studies of tumor growth-regressed
animals treated with DNP and a thermosensitive liposome
encapsulated drug.
[0061] FIG. 21 shows the protective effects of DNP pretreatment on
arterial catheter balloon induced injury.
[0062] FIG. 22 shows the protective effects of DNP pretreatment on
survival after prolonged hepatic eschemic induced by Pringle's
maneuver.
[0063] FIG. 23 shows the improved effect of musculocutaneous flap
skin survival after DNP pretreatment.
[0064] FIG. 24 shows the effects of oral DNP on oxygen consumption
prior to a patient undergoing a PET scan.
[0065] FIG. 25 shows a monitored DNP flow chart with incremental
increases in oxygen consumption prior to a patient undergoing
diagnostic thermography.
[0066] FIG. 26 shows a monitored patient flow chart using
dinitrophenol and methylene blue for the treatment of prostate
carcinoma.
[0067] FIG. 27 shows biochemical and clinical response of
androgen-independent prostatic carcinoma to dinitrophenol and
methylene blue treatment.
[0068] FIG. 28 shows a monitored patient flow chart using
interferon-alpha and dinitrophenol for the treatment of chronic
hepatitis C infection.
[0069] FIG. 29 shows the effects of dinitrophenol and
interferon-alpha treatment on liver enzymes and hepatitis C viral
loads.
[0070] FIG. 30 shows an exemplary method of synthesis of novel
2,4-dinitrophenol conjugates and derivatives.
[0071] FIG. 31 shows synthesis of an expanded combinatorial library
of uncoupling agents.
DETAILED DESCRIPTION OF THE INVENTION
[0072] Electron transferring, transporting and energy converting
elements are ubiquitous and are necessary for life. All eukaryotic
and prokaryotic organisms depend on electron transferring and
transporting elements such as metal containing hemes and nonmetal
moieties such as flavins and adenine nucleotides. These biochemical
entities convert the energy stored in chemical bonds of foodstuffs
into cellular and organelle membrane potentials, high energy
containing molecules such as adenosine triphosphate (ATP),
creatinine phosphate, and other forms of chemical energy needed to
maintain the highly negative entropic state of life.
[0073] The most common form of biologic energy is adenosine
triphosphate (ATP). ATP is produced either anaerobically through
the Embden-Myerhoff Pathway (glycolysis) or through oxidative
phosphorylation. The latter, an oxygen dependent chemical energy
conversion process, is generally associated with the Tricarboxylic
Acid Cycle [(TCA), Krebs Cycle or Citric Acid Cycle]. The TCA cycle
links the products of glycolysis to a multi-enzyme coupled series
of electron carriers called an electron transport chain (ETS). The
electron transport chain is coupled to production of ATP. The
entire TCA cycle and oxidative phosphorylation process is located
in intracellular organelles known as mitochondria.
[0074] While release of energy from foodstuffs can come about
through a variety of biochemical means, the most important means by
which energy release is initiated is by splitting glucose into two
molecules of pyruvic acid. This occurs through the non-oxygen
dependent process of glycolysis in a series of ten chemical steps
depicted in FIG. 1. The overall efficiency of trapping energy in
the form of ATP through this anaerobic process is 43%. The
remaining released energy (57%) is discharged in the form of
heat.
[0075] Pyruvic acid molecules derived from glucose, as well as end
products of fat and protein breakdown, are transported into the
mitochondrial matrix were they are converted into 2 carbon
fragments of acetylcoenzyme A, FIG. 2. As depicted, these acetyl
fragments enter the TCA cycle were their hydrogen atoms are removed
and released as either hydrogen ions (H.sup.+) or combined with
nicotinamide and flavin adenine dinucleotides (NAD.sup.+ and FADH)
to produce large quantities of usable reducing equivalents (NADH
and FADH.sub.2). The carbon skeleton is converted to carbon dioxide
(CO.sub.2) which becomes dissolved in body fluids. Ultimately the
dissolved CO.sub.2 is transported to the lungs and expired from the
body. As noted in FIG. 2, the flux of reactants in the TCA cycle is
always in the same direction because NADH and FADH.sub.2 is
constantly removed as hydrogen is oxidized by the mitochondrial
electron transport chain.
[0076] It is the electron transport chain that provides
approximately 90% of the total ATP formed by glucose catabolism.
During this process, known as oxidative phosphorylation, hydrogen
atoms that were released during glycolysis, the TCA cycle, and
converted to NADH and FADH.sub.2, are oxidized by a series of
enzymatic redox complexes (electron transport chain) located in the
inner mitochondrial membrane, FIG. 3. Energy released in these
steps is captured by a chemiosmotic mechanism that is dependent on
the ultimate reduction of O.sub.2 to form H.sub.2O. As depicted in
FIG. 4, oxidative phosphorylation is two distinct processes: (1)
oxidation of NADH and FADH.sub.2; and, (2) formation of ATP. Both
processes are interdependent or "coupled" by a high energy linked
proton (H.sup.+, pH) gradient and membrane potential across the
inner mitochondrial membrane provided by electrons as they pass
through the electron transport chain. Energy released by the
electrons pumps hydrogen ions (H.sup.+) from the inner matrix of
the mitochondrion into the outer inter-membrane space, FIG. 5. This
process is known as chemiosmosis and creates a high concentration
of H.sup.+ outside the inner mitochondrial membrane and a powerful
negative electrical potential in the inner matrix. This
transmembrane proton gradient (protonmotive force) causes hydrogen
ions to flow back into the mitochondrial matrix through an integral
membrane protein (ATP synthase) to form ATP from ADP and free ionic
phosphate. The efficiency of oxidative phosphorylation in capturing
energy as ATP is about 69%. The remaining (31%) liberated energy is
dissipated as heat. The overall efficiency of energy transfer to
ATP from glucose via glycolysis, the TCA cycle and oxidative
phosphorylation is 66% with about 34% of the energy being released
as heat.
[0077] Heat is continually produced by the body as a byproduct of
metabolism and eventually all energy expended by the body is
converted to heat. On a thermodynamic basis, total body heat
production is the algebraic sum of the enthalpy changes of all
biologic processes in the body. The pathways are irrelevant, even
though in the body oxidation involves numerous enzyme catalyzed
reactions taking place at 37.degree. C. Biochemically,
approximately 95% of all the oxygen (O.sub.2) consumed is used by
mitochondria to stoichiometrically couple oxygen reduction to ATP
and heat production via oxidative phosphorylation. The rate of
O.sub.2 consumption (VO.sub.2) can be measured by indirect
calorimetry and thus related to body heat production. Although this
method does not include anaerobic processes such as glycolysis,
indirect calorimetry is in close agreement with direct body heat
measurements and it is generally accepted that 1 liter of VO.sub.2
generates 4.825 Kcal (kilocalorie of energy), .sup.ths of which can
be detected as heat.
[0078] In human adults, increased VO.sub.2 and endogenous heat
production can occur via muscular (work or shivering) and/or
chemical [(catecholamines, thyroid, etc.) non-shivering]
thermogenesis. Whereas muscular activity can increase heat
production 4-10 fold, non-shivering thermogenesis can only increase
heat production by a maximum of 15%. However, oxygen consumption
and non-shivering thermogenesis can dramatically increase when even
mild injury to the inner mitochondrial membrane occurs so that it
is no longer intact and protons leak or reenter the mitochondrion,
uncoupled to ATP synthesis. Heating, endotoxin, osmotic imbalance,
etc., can cause such injury, i.e., loss of coupling, with resulting
respiration and ATP metabolism proceeding independently and
maximally--respiration forward, phosphorylation in reverse. FIG. 6
compares normal coupled respiration and ATP formation to that which
occurs when there has been injury to the inner mitochondrial
membrane. The increased reduction of oxygen results in increased
heat production.
[0079] Additionally, certain chemicals, including biologicals, can
selectively increase the transport of protons across uninjured,
intact inner mitochondrial membranes and dramatically increase
VO.sub.2 and heat production. These compounds dissipate the
electrochemical-protonmotive transmembrane potential of
mitochondria and uncouple the electron transport chain from ATP
synthesis. FIG. 6(a) depicts one such uncoupling agent, DNP,
cycling protons across an intact mitochondrial membrane. DNP and
other uncouplers permit each of the two distinct processes involved
in oxidative phosphorylation to "unlink" and increase their rates
according to their own separate kinetic and thermodynamic signals,
FIG. 6(b). Uncouplers increase respiratory rates, electron
transport, VO.sub.2, heat production and increased utilization of
foodstuff substrates through glycolysis and the TCA cycle.
Controlled doses of an uncoupler will increase 0.sub.2 consumption
and heat production with minimal or no decrease in ATP levels
because of intracellular equilibrium shifts in creatinine
phosphate, oxidative phosphorylation reactants and increased
production of ATP through the anaerobic, glycolytic pathway. Excess
or toxic doses of virtually all uncouplers however, will produce
secondary untoward effects, including decreased respiration,
decreased heat production and eventual cellular death.
[0080] In addition to heat being a byproduct of oxidative
phosphorylation, reactive oxygen species are also continuously
produced by the mitochondrial electron transport chain. Free
radicals of oxygen are produced during aerobic oxidation as
electrons are transported by the electron carriers to ultimately
reduce O.sub.2 to H.sub.2O. As depicted in FIG. 7, superoxide
(O.sub.2.sup.-) radicals are generated by leaked electrons through
the univalent reduction of oxygen. FIG. 7(a) shows that superoxide
dismutase then converts the superoxide radical to hydrogen
peroxide. Additional hydrogen peroxide (H.sub.2O.sub.2) and
hydroxyl (OH.) radicals are formed through the Haber-Weiss
Reaction, the hydroxyl radical being the most reactive species,
reacting with any biologic moiety instantly. FIG. 7(b) depicts the
overall scheme of oxygen metabolism and free radical formation at
the level of the mitochondrion.
[0081] As mitochondria become progressively heated, uncoupling
occurs with increased flux of oxygen free radicals. The effects of
heat on mitochondrial uncoupling and superoxide radical generation
are depicted in FIG. 8. A linear correlation of 0.98 (P<0.01) is
obtained for the relationship between percent uncoupling and
percent superoxide generation. Similar to exercise increased body
temperature and VO.sub.2, hyperthermia induced by uncoupling agents
appears to inhibit electron transport at the level of cytochrome c
in the redox chain. Normal rat liver, infused with DNP, increases
formation of reactive oxygen species threefold upon cessation of
uncoupling, FIG. 9.
[0082] Generally, uncouplers are agents that are hydrophobic
ionophores which bind protons and traverse biologic membranes to
dissipate transmembrane proton (pH) and membrane potential
gradients (.DELTA..PSI., Delta Psim). In so doing, uncouplers
increase the rate of metabolism (substrate utilization) in intact
animals and isolated tissues by increasing the rate of oxygen
reduction through increased availability of protons. 0.sub.2
consumption is increased and remains rapid as long as the
mitochondrial respiratory (electron transport) chain attempts to
overcome the effects of the uncoupler to maintain a pH gradient.
Energy is still used to pump protons across the mitochondrial
membrane, but the protons are carried back across the membrane by
the uncoupler as depicted in FIG. 6(a). This creates a futile cycle
and energy is released as heat. This chemical heat releasing
process is comparable to heating that occurs when an electrical
wire is "short circuited". Depending on the degree of external body
heat dissipation, body temperature rises some 30 to 60 minutes
after the increase in 0.sub.2 consumption. Onset of action is rapid
after an intravenous injection of an uncoupler. Depending on the
intravenous dosage, human oxygen consumption is increased in about
15-20 minutes and the intracellular heat production is increased
proportionately. Metabolic rates as high as 10 times normal have
been reported. Persistent increases in the metabolic rate can
continue as long as 12 to 36 hours because of the long hydrophobic
half-life of uncouplers in tissues. Temperature increases can be
seen within 10 to 15 minutes in subjects whose heat dissipation
mechanisms have been compromised. Heretofore, hyperthermia induced
by uncoupling compounds has not been reported to have any
therapeutic application.
[0083] While there are three general classes of uncoupling agents,
each containing specific uncouplers of oxidative phosphorylation,
the present invention utilizes 2,4-dinitrophenol (DNP) as the
preferred embodiment. This is because DNP has been extensively
studied. DNP was commonly used in food dyes in the late 1800' s and
in the munitions industry of World War I. Rapid increased
respiration and hyperthermia, up to 49.degree. C., was noted in man
and animals that were accidentally intoxicated. Such dramatic
physiologic effects by the dinitro-aromatic dyes, especially DNP,
caused them to be inextricably tied to early and later modern
studies of metabolism and bioenergetics. In the 1930's DNP was
introduced into clinical medicine for the purpose weight loss. It
was, however, sold as an over the counter secret nostrum and
seriously misused. Had its long half-life in tissues been
recognized and physician supervision implemented, it might have
become an accepted drug. DNP has been reported in countless,
different enzyme, cellular and metabolic studies. Review of such
vast published studies have documented DNP's very specific
mechanism of action as a proton ionophore, with all other effects a
direct pharmacologic extension thereof. DNP is not mutagenic by the
Ames and modified Ames tests; it has not been found to be
carcinogenic or teratogenic; and, DNP blood plasma levels can
easily be determined. DNP can be used at pharmacologic doses that
achieve therapeutic concentrations in tissues. Further, DNP is
stable, inexpensive and commercially available in reagent grade
purity. It is understood however, that other uncouplers and
combinations of other uncouplers with other drugs, hormones,
cytokines and radiation can potentially be used under appropriate
clinical settings and dosages to induce intracellular hyperthermia
and promote additive or synergistic effects.
[0084] FIG. 10 shows the overall intracellular mechanism of action
of DNP (and other uncouplers). Intracellular foci of increased heat
and oxygen free radical flux are highlighted. Circled numbers in
the figure indicate both direct and indirect effects of DNP:
circled 1 and 2 effects shows that upon its intercalation into the
inner mitochondrial membrane, DNP shuttles H.sup.+ (hydrogen ions)
across the membrane [see FIG. 6(a)]--this short circuits
(de-energizes) the proton gradient established by the H.sup.+
pumping action of the mitochondrial electron transport system (see
FIG. 5). As a consequence, the inner mitochondrial membrane
potential is lowered from -180 to -145 mV. Circled 3, 4, 5 and 6
effects shows that normal oxygen consumption and flux of NADH and
FADH.sub.2 (reducing equivalents) through the electron transport
system is coupled to H.sup.+ re-entry via mitochondrial
availability of ADP for re-synthesis of ATP (see FIG. 4). By freely
returning protons into the mitochondrial matrix without concomitant
dependency on ADP to ATP reformation, DNP increases oxygen
consumption proportionately to the degree of uncoupling. The rate
of oxygen consumption remains linked however, to the flux of
electrons provided by NADH and FADH.sub.2 through the electron
transport chain [see FIG. 6(a)]. NADH and FADH.sub.2 utilization
(re-oxidation) is concomitantly increased. Circled 7, 8, 9, and 10
effects show that oxygen use and electron transfer proceed at
increasing rates to accelerate proton pumping against the added
hydrogen ion load introduced by DNP. As a result, NADH and
FADH.sub.2 is continually depleted by re-oxidation to NAD.sup.+ and
FAD.sup.++. The high "oxidation pressure" of NAD.sup.+ and
FAD.sup.++ increases substrate oxidation and flux of 2 carbon
segments through the tricarboxylic acid cycle (TCA). Augmented
acetyl-CoA consumption in turn is maintained by an increased rate
of glycolysis by depletion of pyruvate. If oxygen delivery is
inadequate, or the dose of DNP excessive, the concentration of
reduced NADH increases, pyruvate oxidation through acetyl-CoA and
the TCA cycle is inhibited and lactic acid will accumulate. Lactate
is also overproduced when cellular hypoxia is not present per se
but glycolysis exceeds pyruvate oxidation. Such intracellular
lactic acidosis exists in neoplastic cells, when there is lack of
insulin, when fructose is infused and in other conditions or use of
drugs which augment glycolysis and/or inhibit the mitochondrial
electron transport system. While it is understood that the
intracellular heat generated by DNP is the algebraic sum of the
enthalpy changes from all the metabolic processes within the cell,
effects circled as 11, 12 and 13 depict the most significant
intracellular foci of heat generated by DNP. Intracellular and
total body hyperthermia results when DNP releases energy at a rate
faster than it can be dissipated. Heat is generated mainly at the
inner mitochondrial membrane (electron transport system), the TCA
cycle and sites of cytoplasmic glycolysis. Initially DNP generates
heat at the inner mitochondrial membrane by discharging a portion
of the energy stored in its electrochemical gradient.
Operationally, such heat is from the "chemical short circuit"
created by DNP shuttling protons to the negative (matrix) side of
the polarized inner mitochondrial membrane [see FIG. 6(a)]. By
usurping controlled proton re-entry and energy capture as ATP from
availability of ADP through ATP-synthase, DNP causes NADH and
FADH.sub.2 (higher concentrations of NAD.sup.+ and FAD.sup.++)
reoxidation to occur at rates much higher than necessary for
oxidative phosphorylation. This causes an increased fall of
electrons through the electron transport chain with rapid reduction
of oxygen to water (see FIG. 3). The resultant energy is released
as heat within the mitochondrial membrane. The rate of heat
production from the TCA cycle is increased as it operates at a
higher flux to maintain depleting amounts of reduced NADH and
FADH.sub.2 used to reduce molecular oxygen. Flux of acetyl-CoA and
all metabolites through the TCA cycle (see FIG. 2) is increased by
activation of enzymes which sequentially degrade the hydrogen
containing two carbon fragments to CO.sub.2, NADH, FADH.sub.2 and
heat.
[0085] Glycolysis and its associated heat production in the
cytoplasm is also increased by DNP. Glycolytic activity is
increased by reduced concentration ratios of ATP to ADP, activating
puruvate dehydrogenase and phosphofructokinase respectively (see
FIG. 1). These enzymes increase the rate of glucose catabolism to
pyruvate and its conversion to acetyl-CoA for entry into the TCA
cycle. Glycolysis is very "energy inefficient" in making up the
energy equilibrium shortfall created by DNP. Uncaptured energy from
the glycolytic exergonic reactions accelerated by DNP is released
as heat in the cytoplasm. DNP stimulated anaerobic heat production
through glycolysis can oftentimes be greater than that produced by
the mitochondria. By example, many tumors and normal fibroblasts
treated with DNP increase heat production by 83%, with only a 36%
increase in oxygen consumption. Glycolysis is known to contribute
greater than 62% of the total heat produced by human lymphocytes.
Circled effect 14 shows that the mitochondrial electron transport
chain normally produces reactive oxygen species through the
univalent reduction of oxygen [see FIGS. 7, 7(a) & 7(b)]. Under
physiologic conditions, 2 to 4% of mitochondrial oxygen is
converted to superoxide. DNP induced partial uncoupling and
mitochondrial heating increases reactive oxygen species production
manifold. Cytochrome oxidase and reductase is known to be inhibited
by heating of the electron transport system. As a result, heated
mitochondrial membranes produce increased amount of oxygen free
radicals when DNP induced uncoupling is stopped and oxygen
consumption is normalized (see FIG. 9). Reactive oxygen species act
in synergy with heat to alter proteins, induce membrane changes and
initiate apoptosis in susceptible cells. Circled effects 15 and 16
shows the effects of DNP on intracellular calcium homeostasis.
Normally calcium is stored in the mitochondrial matrix, being
pumped by the energized mitochondrial membrane. By DNP directly
de-energizing mitochondria, and indirectly inducing membrane
heating and prooxidant stress, inner mitochondrial membrane
permeability is non-specifically increased with calcium efflux and
cycling. This activates intramitochondrial dehydrogenses to produce
more reducing equivalents in the form of NADH and FADH.sub.2 to
match increased energy demands. Heat production is increased as a
byproduct from the augmented TCA cycle.
[0086] Other known uncouplers that are considered to be "classic",
in the same category and act as DNP include clofazimine,
albendazole, cambendazole, oxibendazole, triclabendazole (TCZ),
6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and
their sulfoxide and sulfone metabolites, thiobendazole, rafoxanide,
bithionol, niclosamide, eutypine, various lichen acids
(hydroxybenzoic acids) such as (+)usnic acid, vulpinic acid and
atranorin, 2',5-dichloro-3-t-butyl-4'-nitrosalicylanilide (S-13),
3,4',5-trichlorosalicylanilide (DCC), platanetin,
2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,
AU-1421,
3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradec-
in-1,7(8H)-dione (zearalenone),
N,N.sup.1-bis-(4-trifluoromethylphenyl)-urea, resorcylic, acid
lactones and their derivatives,
3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847), 2,2-bis
(hexafluoroacetonyl) acetone, triphenyl boron, carbonyl cyanide
4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA),
carbonyl cyanide 3-chlorophenylhydrazone (ClCCP),
1,3,6,8-tetranitrocarbazole, tetrachlorobenzotriazole,
4-iso-octyl-2,6-dinitrophenol(Octyl-DNP),
4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxal
bisguanylhydrazone), pentachlorophenol (PCP),
5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),
N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),
4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,
2-azido-4-nitrophenol, 5-nitrobenzotriazole,
5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,
methyl-o-phenylhydrazone, N-phenylanthranilic acid,
N-(3-nitrophenyl)panthranilic acid, N-(2,3-dimethylphenyl)
anthranilic acid, mefenamic acid, diflunisal, flufenamix acid,
N-(3-chlorophenyl) anthranilic acid, carbonyl cyanide
4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233 (Tirapazamine),
atovaquone, carbonyl cyanide
4-(6'-methyl-2'-benzothiazyl)-phenylhydrazone(BT-CCP), ellipticine,
olivacine, ellipticinium, isoellipticine and related isomers,
methyl-0-phenylhydrazonocyanoaceticacid,methyl-0-(3-chlorophenylhydrazono-
) cyanoacetic acid,
2-(3'-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic
acid,
2-(2',4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,
relanium, melipramine, and other diverse chemical entities
including unsaturated fatty acids (up to C.sub.14 optimum),
sulflaramid and its metabolite perfluorooctane sulfonamide (DESFA),
perfluorooctanoate, clofibrate, Wy-14, 643, ciprofibrate, and
fluoroalcohols. Additional unnamed classic uncouplers can include
any analog which generally has a weakly acidic, removable proton
and an electron withdrawing, lipophilic molecular body that is
capable of charge delocalization. Hydrophobicity and capacity to
exchange proton equivalents are integral features of classic DNP
types of uncouplers.
[0087] A second class of uncouplers are ionophorous antibiotics.
These molecules uncouple oxidative phosphorylation by inducing
cation or anion influx across the mitochondrial membranes and
diffusing back in a protonated form. As a result, chemical futile
cycling ensues to reestablish the initial membrane potential.
Liberated energy is dissipated as heat. Examples of ionophores that
shuttle potassium ions (K.sup.+) across membranes includes the
antibiotics gramicidin, nigericin, tyrothricin, tyrocidin, and
valinomycin. Nystatin shuttle sodium ions. The calcium ionophore,
compound A23187, is a lipid soluble ionophore which mediates the
electroneutral exchange of divalent cations for protons.
Alamethicins, harzianin HA V, saturnisporin SA IV, zervamicins,
magainin, cecropins, melittin, hypelcins, suzukacillins, monensins,
trichotoxins, antiamoebins, crystal violet, cyanine dyes, cadmium
ion, trichosporin-B and their derivatives are examples of
uncoupling ionophores that depend on shuttling inorganic phospate
(P0.sub.4.dbd.) across the mitochondrial membrane.
[0088] A third class of uncouplers is a group of heterogeneous
compounds that dissipate the proton gradient by attaching or
interacting with specific proteins in the inner mitchondrial
membrane. Examples of such compounds include desaspidin, ionized
calcium (Ca.sup.++), uncoupling proteins such as UCPI-1, UCP-2,
UCP-3, PUMP (Plant Uncoupling Mitochondrial Protein) histones,
polylysines, and A206668-a protein antibiotic that ties up
phosphoryl-transfer proteins. Examples and a potency comparison of
a few uncouplers are depicted in FIG. 11.
[0089] Various conjugates, adducts, analogs and derivatives of the
above mentioned agents can be formulated and synthesized to enhance
intracellular uncoupling and heat production. Further, various
covalent compounds of uncouplers may be synthesized as prodrugs,
which upon, redox or reaction with free radicals within the cell
will become activated to induce uncoupling, heat production and
free radical cycling. Such derivatives and formulations may be
desirable in the treatment of many tumors with higher mitochondrial
membrane potentials and increased total bioreductive capacity.
Uncoupling-free radical prodrug compounds may thus exert greater
selective killing of transformed cells by undergoing a higher flux
of reduction or electron acceptance in tumor cells. In this regard,
the contents of U.S. Pat. No. 5,428,163 and the published methods
of C-Alkylation of phenols and their derivatives by Hudgens, T. L.
and Turnbull, K. D. are hereby incorporated by reference
[0090] From a physico-chemical and thermodynamic standpoint, the
amount of heat produced by uncoupling is proportional to the
density and rate of flux of electrons through the mitochondrial
electron transport chains. Such electron flux is initially
reflected by the magnitude of the electrochemical proton gradient
across the inner mitochondrial membrane. Those cells, tissues,
organs and organisms that are metabolically more active will
generally have an increased membrane potential and will respond
with a greater amount of heat production for a given dose and type
of uncoupler. FIG. 12 lists the six most "hottest" organs in the
human body along with their rates of blood flow and rates of heat
production. The actual amount of intracellular hyperthermia
produced by an uncoupler is dependent on the uncoupler dose, its
relative potency and availability of substrate such as glucose,
glutamine, fatty acids or other substances that produce NADH or
FADH.sub.2. Oxygen and magnitude of the mitochondrial proton
electrochemical gradient (.DELTA..mu.H.sup.+) are additional
factors that determine the amount of heat that can potentially be
released by an uncoupler. Among all the constituents,
.DELTA..mu.H.sup.+ is the most clinically important.
.DELTA..mu.H.sup.+ is composed of the transmitochondrial membrane
potential [.DELTA..PSI. (charge difference)] and pH gradient
[.DELTA. pH (H.sup.+ concentration difference)],
.DELTA..mu.H.sup.+=F.DELTA..PSI.-2.3RT.DELTA.pH, where, F=Faraday
Constant, R=Gas Constant, and T=degrees Kelvin. Thus,
.DELTA..mu.H.sup.+ represents the potential amount of heat that can
be liberated by an uncoupler when 1 mole of H.sup.+ is dissipated
through the inner mitochondrial membrane. This potential heat
energy is normally expressed in units of millivolts (mV) and is
called the protonmotive force,
.DELTA.p=.DELTA..mu.H.sup.+/F=.DELTA..PSI.-2.3(RT/F).DELTA.pH. In
vivo, .DELTA.pH is generally 1 unit or less so that 75% or more of
the total .DELTA.p is comprised of .DELTA..PSI.. Consequently, the
intracellular heat produced by an uncoupler can be estimated by the
mitochondrial membrane potential (.DELTA..PSI.) alone.
[0091] Knowing the .DELTA..PSI. is of practical importance because
biopsy specimens may be incubated with cationic organic probes to
estimate the .DELTA..PSI. and the degree of differential heating
that will occur between normal and transformed tissues. Dyes such
as rhodamine 123, mitotracker green, calcein plus Co.sup.++,
3,3.sup.1-dihexyloxacarbocyanine, triphenylmethylphosphonium,
JC-1,5,5.sup.1,6,6.sup.1-tetrachloro-1,1.sup.1,3,3.sup.1-tetraethylbenzim-
idazolocarbocyanine, etc., all have an affinity for a negative
mitochondrial .DELTA..PSI.. Based on the amount of cationic dye
uptake, the membrane potential of specific tissue, tumors, and
cells may be determined through the Nernst equation:
.DELTA..PSI.=-(RT/F) ln(C.sub.in/C.sub.out). Which at physiologic
conditions and 37.degree. C. is =-61 log(C.sub.in/C.sub.out), where
C.sub.in/C.sub.out is the concentration of the probe inside or
outside the mitochondria and plasma membrane. By example, a 10 to 1
gradient=-60 mV, 100 to 1=-120 mV. Uncouplers dissipate the
.DELTA..PSI., generate heat and release or prevent uptake of
cationic dyes. Six years of systematic measurement of mitochondrial
membrane potentials have been performed on human and mammalian
cells, including some 200 cell types derived from human malignant
tumors of kidney, ovary, pancreas, lung, adrenal cortex, skin,
breast, prostate, cervix, vulva, colon, liver, testis, esophagus,
trachea and tongue. Based on this exhaustive study, a .DELTA..PSI.
difference of at least 60 mV is known to exist between normal
epithelial cells and carcinoma cells. This is significant for the
present invention in that uncoupling or "short circuiting" a 60 mV
potential across a 5-nm mitochondrial membrane would be equivalent
to the amount of heat generated by short circuiting 120,000 V
across 1 centimeter. By exploiting or increasing the membrane
potential between normal and transformed cells the rate of
intracellular heat production by an uncoupler can be selectively
increased in target tissues.
[0092] In order for uncoupler induced intracellular hyperthermia to
be of therapeutic benefit, the development of thermotolerance is
also taken into account in practicing this invention. Mammalian
cells and prokaryotes acclimate and acquire transient resistance or
thermotolerance to gradual or non-lethal hyperthermia. Such
adaptation is believed to occur through increased synthesis of
highly conserved groups of proteins known as heat shock proteins
(HSP). The amount of HSP present in tissues, cells and organisms
subjected to non-lethal heat, or other forms of prolonged metabolic
stress, is proportional to their survival at higher temperatures.
In general, thermotolerance develops after 3 to 4 hours of
continuous hyperthermia, peaks in 1 to 2 days and decays back to
normal thermosensitivy within 3 to 4 days. Thermotolerance is known
to alter lethality of hyperthermia by as much as 2.degree. C.
increase or double the heating time required to achieve the same
temperature-cytotoxic effect. Such adaptive thermoresistance by
human tumors is problematic for continuous or fractionated
cytotoxic treatment with hyperthermia. Induction heating times with
the present invention are therefore kept to a minimum of 1 to 2
hours. Further, the uncoupler induced cytotoxic hyperthermia in the
present invention induces relative tissue hypoxia, lowers
intracellular pH and limits the production of ATP, all of which
repress the development of thermotolerance. Low doses of uncoupler,
which produce gradual heating can be used to induce HSP synthesis
and promote thermotolerance.
[0093] Determining the amount of DNP in mg/kg of body weight
required to produce the desired level of cytotoxic hyperthermia in
a safe and efficacious manner is established from the thermal
equivalents (Kcal) of oxygen consumed (V0.sub.2), and the known
average specific heat capacity of the human body. It is known that
at standard temperature and barometric pressure, 1 liter of oxygen
consumed per minute (VO.sub.2) generates approximately 4.862 Kcal.
It is also known that the average specific heat capacity of humans
is about 0.83 of that required to raise 1 gm of H.sub.20 1.degree.
K=4.184 J, a heat capacity of 3.47 J g K.sup.-1. An initial
estimate of the total energy required to be generated by DNP to
induce 41.0.degree. C. hyperthermia in 1 hour may be very simply
determined from the above and customized for a specific patient as
outlined below:
TABLE-US-00001 Patient Characteristics Body weight 70 kg Resting
V0.sub.2 0.25 L/min Basal energy expenditure 73.1 Kcal/hr (1754.4
Kcal/24 hrs.) Basal core temperature 37.0.degree. C. Target
temperature 41.0.degree. C.
Required Energy to Raise Temperature to Target Level in 1 Hour
[0094] (Weight in grams=70.times.10.sup.3) (human specific
heat=3.47 J g K.sup.-1) (Temperature
increase=41.0.degree.-37.0.degree. C.).about.0.97.times.10.sup.6 J.
Since 1 J=4.184.times.10.sup.-4 Kcal, a total power input of about
232 Kcal would be required to raise the temperature of the patient
to the objective level in 1 hour less that amount of heat generated
by a heated metabolism outlined below.
Increase in Metabolic Rate/Heat Production with Increase in Body
Temperature
[0095] The basal metabolic rate (BMR) is known to increase in
patients with endogenous fevers by approximately 7% for each
0.5.degree. C. rise in temperature. This is graphically depicted in
FIG. 11a. As a result, the increase in BMR relative to the
temperature will in itself assist in achieving the objective level
during the induction phase by the following equation:
BMR.sub.Tcore=73.1.times.1.07(Tcore-37)/0.5
[0096] Thus, at 41.0.degree. C. the metabolic rate will be 134.4
Kcal/hr, 61.3 Kcal/hr above the basal energy expenditure level.
This increase in metabolic rate will therefore reduce the initial
energy required to heat the patient by approximately 61 Kcal over
the 1 hour timeframe.
Initial Net Energy Input Required to Reach Target Temperature in 1
Hour
[0097] 232 Kcal-61 Kcal(by increased BMR)=171 Kcal
Required Increase in Initial V0.sub.2 to Obtain 171 Kcal Heat
Input
[0098] Since the Kcal equivalent for 1 liter of oxygen consumed per
minute is 4.862, then the initial increase in VO.sub.2 required to
generate 171 Kcal can be calculated as follows: Heat in
Kcal/min=V0.sub.2.times.4.862. Since the individual patient has a
resting V0.sub.2 of 0.25 l/min which=73.1 Kcal/hour BMR, then
X(V0.sub.2)=171 Kcal, or
X=0.25.times.171/73.1
[0099] An initial minimal increase in V0.sub.2 to approximately
0.60 l/min is required.
DNP Dosage Required to Increase V0.sub.2 to 0.60 l/min
[0100] The individual DNP dosage (mg/kg) required to produce an
increase in oxygen consumption to 0.60 l/min so as to achieve a 171
K/cal heat output is accomplished in the following fashion: (1) DNP
is prepared in a 200 mg/100 ml sterile aqueous solution. If not
fully dissolved, it can be brought into solution by buffering with
1% NaHC0.sub.3, the pH must be kept below 8 to avoid hydrolysis;
(2) the dose of DNP for each intravenous infusion can vary from 0.5
to 4 mg/kg and will depend on the clinical situation, as well as
the initial and subsequent increases in the metabolic rate
(V0.sub.2). In an especially preferred embodiment, the patient is
given an initial dose of DNP no greater than 1 mg/kg intravenously,
infused over no less than a 2 minute period. Within approximately
10-15 minutes, a minimum of a 15% increase in V0.sub.2 will occur.
The V0.sub.2 will continue to increase until a plateau is reached
within an additional 5 to 10 minutes. After a 5 minute plateau in
V0.sub.2, a subsequent dose of either 0.5, 1, 2, 2.5, or 3.0 mg/kg
DNP is administered and V0.sub.2 is again increased until a desired
plateau is reached. Additional infusions of DNP or other
medications are administered under clinical parameters of V0.sub.2,
respiratory rate, pulse rate, blood pressure, urine output, cardiac
output, core temperature, and clinical status of the patient so as
to maintain safe and effective control of heating. If heat
dissipating mechanisms are neutralized, measurable increases in
core temperature will occur approximately 20 to 30 minutes after an
increase in the V0.sub.2. FIG. 13 illustrates the increases in
V0.sub.2 associated with repeated infusions of DNP.
[0101] Medications which increase the overall metabolic rate, or
that of specific target tissues, and have short half-lifer can be
utilized to increase the relative activity of DNP or other
uncouplers to further adjust V0.sub.2 and heat production. Examples
of such medications are almost limitless because any drug, hormone
or biologic response modifier that causes changes in enthalpy (heat
content) during the course of its intracellular chemical and
biophysical activity and interaction in the life cycle of
biological cells can be utilized. A few illustrative examples
include glucagon (half-life of 9 minutes in plasma), arbutamine
(half-life 10 minutes), dobutamine (half-life 2 minutes), and
vasopressin (half-life 5 minutes). Various amino acids and fatty
acids, e.g., glutamine, proline, octanoate, etc., increase V0.sub.2
by translocating reducing equivalents into the mitochondrial matrix
via the malate-aspartate shuttle, B-oxidation or proline
metabolism. Agents such as methylene blue (tetramethylthionine),
ubiquinone, menadione, hematoporphyrin, phenazine methosulfate,
2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2, or their analogs
duroquinone and decylubiquinone, etc., can increase heat and/or
free radical production by acting as artificial electron acceptors.
Such agents, and numerous others, can be co-administered with DNP
or other uncouplers to effectively increase the enthalpy changes in
the entire organism or specific targeted tissues.
Minimizing Heat Loss and Temperature Control
[0102] Increased radiative and evaporative heat loss from man are
the two most dominant thermoregulatory mechanisms for cooling the
body. The body's methods of adjusting heat loss are
vasoconstriction and vasodilation in the skins blood vessels.
Radiation can account for 60% of the heat loss generated by the
body, while evaporation by sweating at 1.0 liter/hour can represent
a potential heat loss of about 1,000 Kcal/hour. By far, sweating
and evaporation is the principal mechanism that dissipates heat
under conditions that induce large heat gains. Depending on the
clinical circumstances, heat loss due to evaporation, as well as
radiation, can be managed and controlled by a variety of methods
including, but not limited to, using vasoconstricting agents,
placing the patient in a scuba diving wet suit, humidified survival
suit, or enveloping the patient in a water soaked blanket covered
or containing a polyethylene lining to prevent evaporative heat
losses. Use of room ultrasonic nebulizers to induce continuous mist
and high humidity is also known to prevent evaporative heat losses.
Evaporative and radiant heat loss from the cranium is controlled by
appropriate head gear, shower caps and/or wet towels. Control of
local air velocities and management of surroundings as to
temperature, emissivity, drafts, and convection currents are
important to avoid large heat losses. In those clinical
circumstances where total body hyperthermia is required, failure to
adequately control body heat loss will necessitate using higher
doses of DNP and induce a greater metabolic stress upon the
patient.
[0103] If the core target temperature is exceeded or continues to
rise after the target temperature is achieved, exposure of an
extremity or body surface for a brief interval will permit
sufficient heat loss to lower the core temperature to the target
range. At target temperatures of 39-41.degree. C., residual
uncoupling by DNP will continue for approximately 3 hours. Heat
production as a byproduct of glycolysis, and heated metabolism
further maintains body heat content and compensates for any heat
loss. Therefore, target plateau temperatures can be regulated with
a large margin of safety and with little to no additional use of
uncoupler. Therapy is terminated by removing the vapor barrier from
the patient. Evaporative and radiant heat loss from the patient
generally produces a fall in core temperature of about
2-2.5.degree. C. in about 20-30 minutes. Obese patients and those
with compromised thermoregulatory systems experience a slower
falloff in temperatures.
Patient Monitoring, Fluid Support and Evaluation During
Treatment
[0104] Placement of physiologic monitoring sensors, intravenous
fluids, supplemental oxygen (41/min) and optional oral diazepam
sedation (5-10 mg) is initiated prior to treatment. Patients
receive 0.85 to 1.0 liter of intravenous (IV) 5% dextrose in 0.25
normal saline per hour alternated with 5% dextrose in 0.5 normal
saline plus 7.5 to 10 meq of KCl per liter to insure a urinary
output of no less than 1 ml/kg/hr. Oxygen consumption, caloric
expenditure, rectal core temperature, cardiac rhythm, blood
pressure, heart rate and respiratory rate are continuously
displayed, monitored by a trained member of the treatment staff.
The data is automatically downloaded into a computer every 20
seconds to 3 minutes for the entire procedure and immediately
re-displayed on computerized graphs and charts. Two hours after
treatment and 48 hours post-treatment, serum chemistries and
hematologic profiles are repeated. A typical patient flow chart is
depicted in FIG. 14.
Treatment of Excessive Heating and Antidotes
[0105] In those rare instances when too much uncoupler is
administered or the metabolic rate of the patient unexpectedly
increases and V0.sub.2, hyperthermia, pulse rate and patient
fatigue ensue, appropriate supportive measures of cooling,
intravenous hydration and administration of specific medication
should be instituted. Cooling should be instituted by uncovering
the patient, spraying with tepid water and fanning with an
industrial grade fan. If cooling is inadequate, surface, axillary
and groin ice packs and intravenous cold glucose solutions should
immediately be considered. Bicarbonate, 1-2 mEq/kg should be
administered in the absence of blood gas analysis. Urine output of
>1 ml/kg/hour should always be maintained to avoid pre-renal
azotemia and oliguria secondary to possible rhabdomyolysis and
myoglobinuria. Mannitol should be administered if urine output is
inadequate. Hypoglycemia should immediately be corrected with 50%
saturated intravenous glucose. If severe or persistent
hypermetabolism ensues, rectal propylthiouracil--1,000 mg,
hydrocortisone (100 mg q 6 h) or dexamethasone 2 mg q 6 h
intravenously and/or sodium iodide as 1 g sodium ipodate (contrast
agent) should be administered intravenously to induce iatrogenic
hypothyroidism. The decreased metabolic rate will dramatically
reduce the physiologic response to DNP. Patient agitation and
restlessness can be avoided by appropriate IV or IM dose of
diazepam. Salicylates are of no value and may contribute to further
uncoupling. Medications that reduce sweating, e.g., tricyclic
antidepressants, antihistamines, anticholinergics, phenothiazines,
or decrease vasodilation, e.g., sympathomimetics, .alpha.-agonists,
or decrease cardiac output, e.g., diuretics, beta-blockers or
induce hypothalamic depression, e.g., neuroleptics,
.alpha.-blockers, opiods, etc., should be avoided prior, during and
immediately after treatment with uncouplers.
[0106] The hypermetabolic and hyperthermic activity of DNP can
further specifically be reduced by using calcium channel blockers
such as nifedipine, verapamil and others, in intravenous doses that
do not cause a drop in blood pressure or induce cardiac
arrhythmias. Dihydrobenzperidol (a neuroleptic drug with
.alpha..sub.1-adrenergic properties) can also be used to cause
similar, significant reductions in DNP induced hypermetabolism and
hyperthermia. Dosages of these anti-DNP agents are titrated in 5 mg
to 30 mg increments and can be given either by mouth or
intravenously. In those cases where DNP appears to decrease
electrical conduction or cause EKG conduction abnormalities,
Coenzyme Q10, in doses of 50 mg/kg, can be used to restore normal
electrical activity.
Patient Selection and Pretreatment Evaluation
[0107] It is imperative that in the practice of this invention,
patients be selected and evaluated prior to treatment. Recommended
patient inclusion and exclusion criteria includes: (1) patients
have a definitive histopathologic or other laboratory confirmed
diagnosis of their disease; (2) the disease or condition should be
responsive to intracellular hyperthermia treatment; (3) patients
should have a Karnofsky score of 70% or greater; (4) not be
pregnant; (5) weight should be within 45% (+/-) of ideal body
weight and patients must weigh at least 35 kg; (6) there should be
no history or findings of anhidrosis, scleroderma, ectodermal
dysplasia, Riley-Day Syndrome, arthrogryposis multiplex, extensive
psoriasis, serious dysrhythmias, malignant hyperthermia or
neuroleptic malignant syndrome, pheochromocytoma, hypocalcemia,
repeated episodes of hypoglycemia, chronic or recurrent venous
thrombosis, alcoholism, renal failure, cirrhosis, untreated
hyperthyroidism, anaphylaxis associated with heat or
exercise-induced cholinergic type urticaria, exercise or heat
induced angioedema, schizophrenia, catatonia, seizure disorders,
emotional instability, Parkinson's disease, brain irradiation,
cystic fibrosis, unstable angina pectoris, congestive heart
failure, patients with cardiac pacemakers, severe cerebrovascular
disease, spinal cord injury, severe pulmonary impairment,
hereditary muscle disease such as Duchenne type muscular disease,
central core disease of muscle, myotonia congenita, King-Denborough
syndrome, Scwanry-Jampol syndrome, or osteogenesis imperfecta; (6)
no immediate use of drugs that impair the body's heat dissipation
mechanisms such as phenothiazines, anti cholinergics,
antihistamines, antiparkinsonians, glutethimide, hallucinogens,
lithium, cocaine or other illicit drug use, monamine oxidase
inhibitors, sympathomimetics, phencyclidine, opioids,
phenylephrine, INH, tricyclic antidepressants, withdrawal from
dopamine agonists, or cardiovascular drugs that clinically impair
cardiac output or thermoregulatory vasodilation such as high doses
of .beta.-blockers, vasodilators, or calcium channel blockers; and,
(7) the patient should not be anemic or otherwise have a reduced
oxygen absorbing, carrying or utilizing capacity.
[0108] Pretreatment evaluation should include a complete medical
history and physical examination focused on the selection criteria
listed above. Laboratory evaluation should include pulmonary
function tests-if indicated, full hematological survey with
hemastatic profile, EKG, liver function tests, serum biochemical
profile, thyroid panel, serum creatinine, calcium, phosphate, and
stress-EKG or exercise-multigated radionucleotide ejection scan on
patients whose cardiac ejection fraction is suspect not to be
greater than 45% with probable deterioration on exercise. While
clinical exceptions to entry laboratory values may exist, the
following laboratory data should be a benchmark guide for
initiation of treatment: hemoglobin >=11.0 g/dl for men and
>=10.0 g/dl for women, platelet count >=75.00
platelets/mm.sup.3, bilirubin <=2.times.ULN (ULN=upper limit of
normal), ALT (SGPT)<=2.times.ULN, AST (SGOT)<=2.times.ULN,
pancreatic amylase <1.5.times.ULN, neutrophil count >=1,000
cells/mm.sup.3. Serum electrolytes and K.sup.+ should be well
within normal limits, as hypokalemia decreases muscle blood flow,
cardiovascular performance, and sweat gland function.
[0109] More generally, the method outlined above is to be tailored
to an individual patient. As set forth above, the DNP may be
administered by intravenous infusion. Alternatively, the route of
administration may also be orally, rectally or topically. The
frequency and optimal time interval between administrations is
individualized and determined by measuring V0.sub.2, as well as
other parameters. For example, various laboratory, x-ray, CAT scan,
MRI, PET scan, HIV load, CD4+ lymphocyte counts, HSP expression,
prostatic specific antigen (PSA) and other surrogate markers of
clinical outcome can establish the VO.sub.2, frequency and duration
of therapy. One treatment, or treatments as frequent as every day,
or every other day, as far apart as 1 year or longer may be
required for sustained beneficial results.
[0110] The optimal VO.sub.2, temperature, duration, and frequency
between treatments will probably vary from patient to patient and
the specific disease or condition being treated. One skilled in the
art would be able to modify a protocol within the present
invention, in accordance with standard clinical practice, to obtain
optimal results. For example, the HIV relationships between viral
load, CD.sup.4+ lymphocyte counts, presence of opportunistic
infections and clinical status of the patient can be used to
develop more optimal regimes of DNP administration. Applicants'
studies have revealed that the methods of the present invention can
be effective in the diagnosis and treatment of a wide range of
disease states and conditions in which uncoupler induced
hypermetabolism, hyperthermia, oxidative stress and their sequela,
play a beneficial role. To those skilled in the art, it is also
encompassed that a variety of different veterinary, as well as
medical, applications for treatment and diagnosis can be practiced
with the present invention.
[0111] It is envisioned that DNP, or other uncouplers, may also be
administered with other compounds used to treat infectious,
malignant or other diseases. Examples of other agents include
antifungal, antibacterial, antiviral or anti-neoplastic drugs, cell
differentiating agents, and, various biologic response modifiers.
Examples of anti-fungal agents include Amphotericin B,
Griseofulvin, Fluconazole (Diflucan), Intraconazole, 5
fluoro-cytosine (Flutocytosine, 5-FC), Ketatoconazole and
Miconazole. Examples of anti-bacterial agents include antibiotics,
such as those represented from the following classifications: beta
lactam rings (penicillins), macrocyclic lactone rings (macrolides),
polycyclic derivatives of napthacenecarboxamide (tetracyclines),
amino sugars in glycosidic linkages (aminoglycosides), peptides
(bacitracin, gramicedin, polymixins, etc.), nitrobenzene
derivatives of dichloroacedic acid, large ring compounds with
conjugated double bond systems (polyenes), various sulfa drugs
including those derived from sulfanilamide (sulfonamides,
5-nitro-2-furianyl compounds (nitrofurans), quinolone carboxylic
acids (nalidixic acid), fluorinated quinilones (ciprofloxan,
enoxacin, ofloxacin, etc.), nitroimidazoles (metroindazole) and
numerous others. These antibiotic groups are examples of preferred
antibiotics, and examples within such groups include: peptide
antibiotics, such as bacitracin, bleomycin, cactinomycin,
capreomycin, colistin, dactinomycin, gramacidin A, enduracitin,
amphomycin, gramicidin J, mikamycins, polymyxins, stendomycin,
actinomycin; aminoglycosides represented by streptomycin, neomycin,
paromycin, gentamycin ribostamycin, tobramycin, amikacin;
lividomycin beta lactams represented by benzylpenicillin,
methicillin, oxacillin, hetacillin, piperacillin, amoxicillin and
carbenacillin; lincosaminides represented by clindamycin,
lincomycin, celesticetin, desalicetin; chloramphenicol; macrolides
represented by erythromycins, lankamycin, leucomycin, picromycin;
nucleosides such as 5-azacytidine, puromycin, septacidin and
amicetin; phenazines represented by myxin, lomofungin, iodin;
oligosaccharides represented by curamycin and everninomycin;
sulfonamides represented by sulfathiazole, sulfadiazine,
sulfanilimide, sulfapyrazine; polyenes represented by
amphotericins, candicidin and nystatin; polyethers; tetracyclines
represented by doxycyclines, minocyclines, methacylcines,
chlortetracyclines, oxytetracylcines, demeclocylcines; nitrofurans
represented by nitrofurazone, furazolidone, nitrofurantoin, furium,
nitrovin and nifuroxime; quinolone carboxylic acids represented by
nalidixic acid, piromidic acid, pipemidic acid and oxolinic acid.
The Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othmer,
editors, Volume 2 (1978), which is hereby incorporated by reference
in its entirety.
[0112] Antiviral agents that can be used with DNP include:
interferons .alpha., .beta. and .gamma., amantadine, rimantadine,
arildone, ribaviran, acyclovir, abacavir, vidarabine (ARA-A)
9-1,3-dihydroxy-2-propoxy methylguanine (DHPG), ganciclovir,
enviroxime, foscarnet, ampligen, podophyllotoxin, 2,3-dideoxytidine
(ddC), iododeoxyuridine (IDU), trifluorothymidine (TFT),
dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, protease
inhibitors such as indinavir, saquinavir, ritonavir, nelfinavir,
amprenavir, etc., and specific antiviral antibodies.
[0113] Anti-cancer drugs that can be used with DNP include, but are
not limited to, various cell cycle-specific agents represented by
structural analogs or antimetabolites of metholtrexate,
mercaptopuorine, fluorouracil, cytarabine, thioguanine,
azacitidine; bleomycin peptide antibiotics, such as podophyllin
alkaloids including etoposide (VP-16) and teniposide (VM-26); and
various plant alkaloids such as vincristine, vinblastine, and
paclitaxel. Anti-neoplastic cell cycle-nonspecific agents such as
various alkylating compounds such as busulfan, cyclophosphamide,
mechlorethamine, melphalan, altaretamine, ifosfamide, cisplatin,
dacarbazine, procarbazine, lomustine, carmustine, lomustine,
semustine, chlorambucil, thiotepa and carboplatin. Anticancer
antibiotics and various natural products and miscellaneous agents
that can be used with DNP include: dactinomycin, daunorubicin,
doxorubicin, plicamycin, mitomycin, idarubicin, amsacrine,
asparaginase, quinacrine, retinoic acid derivatives (etretinate),
phenylacetate, suramin, taxotere, tenizolamide, gencytabine,
amonafide, streptozocin, mitoxanthrone, mitotane, fludarabine,
cytarabine, cladribine, paclitaxel (taxol), tamoxifen, and
hydroxyurea, etc.
[0114] DNP can also be administered with various hormones, hormone
agonists and biologic response modifying agents which include, but
are not limited to: flutamide, prednisone, ethinyl estradiol,
diethylstilbestrol, hydroxyprogesterone caproate,
medroxyprogesterone, megestrolacetate, testosterone,
fluoxymesterone and thyroid hormones such as di-, tri- and
tetraiodothyroidine. The aromatase inhibitor, amino glutethimide,
the peptide hormone inhibitor octreotide and gonadotropin-releasing
hormone agonists such as goserilin acetate and leuprolide can also
be used with DNP. Biologic response modifiers such as various
cytokines, interferon alpha-2a, interferon alpha-2b,
interferon-gamma, interferon-beta, interleukin-1, interleukin-2,
interleukin-4, interleukin-10, monoclonal antibodies
(anti-HER-2/neu humanized antibody), tumor necrosis factor,
granulocyte-macrophage colony-stimulating factor,
macrophage-colony-stimulating factor, various prostaglandins,
phenylacetates, retinoic acids, leukotrines, thromboxanes and other
fatty acid derivatives can also be used with DNP.
[0115] The use of this invention should be under the strict
direction of a qualified and specialized treatment team to insure
safety and effectiveness. The treatment team remains with the
patient throughout the procedure to insure that safe and controlled
dosages of an uncoupler are administered by monitoring real time
changes in V0.sub.2, metabolic rate, temperature, respiratory rate,
heart rate, urine output and clinical status of the patient. This
invention is practiced in controlled steps so as to attain a
predetermined V0.sub.2 and plateau of heating time for a particular
disease or condition. For example, in cases were heat dissipation
mechanisms do not have to be blocked, the specialized team will
periodically recheck V0.sub.2, heart rate, blood pressure, CAT
scan, MRI, etc., and other laboratory and clinical parameters to
insure continued safety and efficacy of DNP therapy. It is
preferred that the specialized team undergo a training period in
the use of this invention prior its administration to human
patients.
[0116] The present invention is further illustrated by reference to
the following examples, which illustrate specific elements of the
invention but should not be construed as limiting the scope of the
invention.
Example 1
Method of Using DNP with Glucagon to Treat Parasitic Infections,
Hydatid Disease of the Liver
[0117] History: A 52 year old white Swiss male, European fox
hunting dog trainer, presented with right upper quadrant pain and
vomiting. Past history revealed he had hepatic "cyst" surgery 2
years ago. Preoperatively, he was treated with albendazole. Only
one dose of albendozale was given because of a "near death"
anaphylactic reaction. He denied history of weight loss, pulmonary,
cardiac, neurologic or thermoregulatory problems. There was no
history of alcohol abuse or medication use. The patient was
adamantly opposed to any further surgery or treatment with
albendazole or mebendazole.
[0118] Physical Examination: Weight=90 Kg; height=177.8 cm;
BP=140/80; HR=76 & reg; Resp.=18 min; T=37.0
[0119] An old well healed scar consistent with prior hepatic
surgery was present. Physical exam otherwise was unremarkable.
[0120] Laboratory studies: EKG, chest X-ray, blood panel, including
serum electrolytes, thyroid studies and liver function tests were
within normal limits (WNL). A complete blood count was unremarkable
except for 20% eosinophilia. Ultrasound and nuclear magnetic
resonance revealed 4, 2 to 3 cm. in diameter, cysts in the right
middle lobe of the liver and a solitary 2 cm semi-solid medullary
cyst in the neck of the right humerus. ELISA serology showed a
diagnostic titer for hydatid disease. Review of previous surgical
liver pathology reports revealed a cestode compatible with
Echinococcus multilocularis.
[0121] Clinical assessment and treatment evaluation: The patient
had no historical or physical contraindications to DNP induced
hyperthermia. Conventional therapy of hydatid disease is either
surgical resection or medical therapy with albendazole for 4 weeks.
Hydatid bone cysts are not amenable to surgery and respond poorly
to standard medical therapy. Echinococcus multilocularis
protoscoleces and the germinal membranes of hydatid cysts are known
to be irreversibly destroyed by heating at 41.degree. C. for 15
minutes. Human liver and hepatocytes can withstand artificial
temperatures of 42.degree. C. for as long as 20 hours without
irreversible damage. Acute glucagon treatment is known to
preferentially stimulate hepatocyte mitochondrial V0.sub.2. Rates
of hepatocyte uncoupled V0.sub.2 are also know to be stimulated up
to 100% in less than 6 minutes after the hormonal action of
glucagon. Acute glucagon treatment has been shown to selectively
increase the pH gradient across hepatocyte mitochondrial membranes.
Thus, it can be empirically presumed that any increase in V0.sub.2
from glucagon administration causes increased thermogenesis,
predominantly in the liver.
[0122] Pretreatment protocol: the patient was given 10 mg diazepam
by mouth and dressed into a modified wet suit. The wet suit was cut
lengthwise at the arms and legs. Velcro strappings were attached at
the cuttings for closure, rapid removal or exposure of the limb(s).
After placement of monitoring sensors, he was started on IV fluids
of 5% dextrose, 0.5 normal saline with 7 meq K.sup.+, infused at an
initial rate of 12 cc/kg/hr. Evaporative heat loss from the head
was minimized by a plastic shower cap and towels. A 401AC
temperature probe (YSI Incorporated, Yellow Springs, Ohio) was
inserted 11 cm. into the rectum. The probe was connected to a Model
4600 telethermometer (YSI 4600 Precision Thermometer) and readings
within 0.1.degree. C. were continuously displayed and recorded at
baseline and during treatment on Hewlett-Packard (HP) computer
systems with customized software developed by MR&S (Manalapan,
N.J.). A TEEM 100 Metabolic Analysis System (AeroSport Inc., Ann
Arbor, Mich.), with a modified face mask and oxygen delivery system
(38-40% 0.sub.2 saturation) for patient comfort and increased
accuracy, was attached to the patient. Oxygen consumption
(V0.sub.2), carbon dioxide production (VC0.sub.2), expired air
volume (V.sub.E), heart rate (HR), and Kcal of heat produced were
measured in 20 second intervals and extrapolated to minute or
hourly rates. All patient data was monitored in real time,
continuously displayed at baseline and during treatment and
recorded on HP computer systems with customized software from
MR&S (Manalapan, N.J.).
[0123] Treatment procedure: After baseline recordings of 10
minutes, the required amount of DNP to raise the initial V0.sub.2
to achieve a temperature in the patient of 40.degree. C. was
calculated as described under "DNP dosage required to increase
V0.sub.2". The patient was given an initial dose of 1 mg/kg of DNP,
infused intravenously over a 3 minute period. After the V0.sub.2
stabilized at 40% above baseline, an additional DNP infusion of 3
mg/kg was given. Upon attaining a stable V0.sub.2, 0.5 mg of
glucagon was administered intravenously. After this stabilization
of V0.sub.2, a glucagon drip was variably infused from 0.5 to 5
mg/kg/hour to additionally control V0.sub.2 and selectively augment
heat production in the liver. The treatment procedure was
discontinued after the patient was maintained at a rectal body
temperature of 40.degree. C. for about 1 hour: The wet suit was
opened and head covering removed. After the patient's body
temperature reached 38.degree. C., the Foley catheter was removed
and intravenous fluids were discontinued. Evaporative and radiant
heat loss lowered the body temperature to a normothermic level
within 30 minutes. No immediate or delayed post-treatment toxicity
was encountered. Monitored patient parameters are shown in FIG.
15.
[0124] Treatment outcome: Serial imaging studies revealed hepatic
and bone cyst shrinkage with increased density at 2 and 4 weeks
post treatment. Repeat magnetic resonance imaging at 4 months
showed complete cyst disappearance in the liver and bone.
Example 2
Method of Using DNP to Treat Viral Infections, HIV Disease
[0125] History: A 38 year old white male, past intravenous heroin
addict, was diagnosed approximately 8 years ago with HIV by ELISA
and positive Western blot for HIV p24 and gp41 antigens after
presenting with weight loss and thrush. His history included
repeated treatment for candidiasis, pneumocystis carinii, and
various subcutaneous abscesses. Past medications included
sulfamethoxazole, ketoconazole, fluconazole, zidovudine, didanosine
and various other antibiotics. For the past year and a half he has
been on highly active antiretroviral therapy (HAART) with various
HIV protease inhibitors combined with thymidine, purine or cytosine
nucleoside and nonnucleoside inhibitors. He was unable to tolerate
nelfinavir because of diarrhea. Ritonavir caused intractable
vomiting and abdominal pain. Current medications include indinavir,
zidovudine and lamivudine. Review of the most recent viral load
(VL) and CD4+ lymphocyte counts showed an initial drop in plasma
HIV RNA (copies/ml) from 200,000 to 2,000 over a 12 week period
with the VL rebounding back to 200,000 at week 16. CD4+ lymphocyte
counts have remained between 100 to 200 cells/mm.sup.3.
Approximately 5 months ago he was treated for oral and
endobronchial Kaposi's sarcoma (KS) with liposomal daunorubicin
followed by liposomal doxorubicin. He denied treatment with
vincristine or bleomycin. There is no history of recent diarrhea,
recent weight loss, hemoptysis, shortness of breath on moderate
exertion, or cardiac problems. There has been no illicit drug use
over the past 2 years. The patient stated no combination of HAART
has been able to lower his viral load and multiple side effects
from the drugs are limiting his compliance to take the medications.
There was no history of thermoregulatory problems.
[0126] Physical examination: weight=60 Kg; height=155 cm;
BP=128/72; Resp=20; T=38.2.degree. C.; and, the pulse was 92 &
reg. Exam revealed asthenia and generalized enlargement of lymph
nodes, some 2 to 3 cm in diameter in the axillary and inguinal
regions. There was diffuse oropharyngeal thrush. Beneath the
thrush, the oral cavity also contained several dark red plaque to
nodular like lesions on the hard palate and gingiva. The lesions
did not blanch on compression with the tongue blade. A crusted
strawberry like mass, 1 by 2 cm, was present at the anus. There
were no neurologic deficits or ocular lesions.
[0127] Laboratory studies: EKG, serum electrolytes, renal and liver
function tests were normal. Hematocrit was 35.5%, WBC was 9,900
with 81% neutrophils, 4 bands, 11 lymphocytes and 4 monocytes.
Platelets were 314,000/mm.sup.3. Viral load was 400,000 copies/ml
(Amplicor HIV Monitor test, Roche). A CD4.sup.+ T cell count was
quantified by flow cytometry at 250/mm.sup.3. He was antibody
positive for hepatitis C. Chest radiograph showed some bilateral
apical patchy opacities. Pulmonary function tests showed all
parameters, including forced expired volume, greater than 80% of
predicted. Karnofsky score was greater than 70. Normal and tumor
tissue biopsies, 3 to 6 mm in diameter, from the oral cavity and
anus were obtained. The tissues were equally divided, weighed and
placed in 4.degree. C. Ringers lactate solution. Histologically
confirmed normal and KS tissues were then subjected to
microcalorimetric measurements in a thermal activity monitor
(ThermoMetric, Jarfalla, Sweden). Recorded heat output (.mu.W/min)
was 8.2-8.5 times greater for the KS sarcoma lesions than
nontumorous oral mucosa. tissues. Repeat measurements with biopsies
specimens in 30 uM DNP increased heat production in tumorous
tissues 20.5 times more than nontumorous specimens.
[0128] Clinical assessment and treatment evaluation: HIV and
HIV-infected T cells are known to be more sensitive to killing by
heat than uninfected lymphocytes. Susceptibility to heat killing is
enhanced with increased oxygen free radical production. Acute and
chronically infected cells have decreased levels of manganous
superoxide dismutase (MnSOD) activity. MnSOD is located exclusively
in mitochondria. Mathematical modeling of human HIV production and
CD4+T cell turnover predicts that reducing both free virus and
actively infected cells by a minimum of 40% with 1 hour of
42.degree. C. therapeutic hyperthermia every third day will promote
recovery of the uninfected T-cell population. Human HIV studies
with extracorporeal hyperthermia of 41-42.degree. C. have reported
isolated cases of extended patient survival, elimination of
detectable virus, and improvement of Kaposi's sarcoma lesions. DNP
is known to generate intracellular hyperthermia and oxygen free
radicals from the level of the inner mitochondrial membrane.
Studies on in vitro inactivation of chronically HIV infected HUT-78
cells by various concentrations of DNP are graphically represented
in FIG. 16.
[0129] The patient has been and remains resistant to treatment with
HAART. Opportunistic infections with candida and Kaposi's sarcoma
herpes virus (KSHV, human herpesvirus type 8) causing his thrush
and Kaposi's sarcoma are comorbid conditions indicative of a
worsening prognosis. In spite of having AIDS with candidiasis and
Kaposi's sarcoma, the patient maintains good cardiac and pulmonary
function. There was no history of thermoregulatory problems. It was
discussed and agreed that hyperthermia treatments with core body
temperatures of 41.degree. C. would be administered on a daily or
every other day basis, as tolerated, for a minimum of 3 hours, not
to exceed 5 hours.
[0130] Pretreatment protocol: all medications were stopped 2 weeks
prior to treatment. The patient refused taking diazepam, placement
of a Foley catheter and oxygen face mask. He dressed himself into a
dry cold water immersion suit (Stearns, ISS-590I, Universal Adult)
designed to prevent heat loss and modified for easy placement of
physiologic monitors. Equipment for measurement of heart rate,
temperature, carbon dioxide production and Kcal of heat produced
were conducted as outlined in Example 1. An oral breathing tube was
used to measure V0.sub.2 from room air. Urine output was measured
when the patient voluntarily urinated through a "Texas" catheter
(superficial condom tightly fitted around the head of the penis
with tubing connected to urine collection bag). The patient was
informed that hyperthermia would be administered as tolerated by
his stamina and monitored clinical parameters, not to exceed 5
hours, on a daily or every other day basis, for a total of 5
sessions.
[0131] Treatment procedure: Baseline reading for 5 minutes
established an average V0.sub.2 of 300 cc/min. An initial dose of 2
mg/kg of DNP was administered over a 2 minute period. V0.sub.2
increased and stabilized at 15 minutes at 340-380 cc/min. An
additional 2 mg/kg DNP infusion was given, the V0.sub.2 increased
and stabilized at 610-630 cc/min. Body core temperature increased
to 39.4.degree. C. within 60 minutes. A gradual fall in blood
pressure was noted at 90 minutes to 100/60 mmHg. Norepinephrine
bitartrate (Levophed) was given IV drip at a dose of 1
microgram/min. and adjusted to maintain blood pressure at 130/80.
Approximately 1 minute after initiating the vasopressor, heart rate
increased from 90 to 100 and V0.sub.2 to 0.85 liters/min. Core body
temperature increased within 20 minutes to 41.5.degree. C. V0.sub.2
was maintained at 1.0 liters/min. by lowering or increasing the
dose of norepinephrine. An additional infusion of 1 mg/kg DNP was
given at hour 4 to correct a dropping V0.sub.2. On occasions when
the core temperature increased above 41.6.degree. C., a lower
extremity was exposed for evaporative heat loss. The patient
withstood the procedure without any untoward effects for a period
of 7 hours. The protocol was repeated consecutively for 5 days
without the additional use of vasopressors.
[0132] Treatment outcome: Immediately after the first treatment
oral candidiasis improved by 50%. The oral and anal Kaposi's
lesions exhibited marked erythema with circumferential areas of
blanching. On the second day of treatment the KS erythema
diminished. There was no evidence of oral candidiasis on the
3.sup.rd day of therapy. The anal tumor was crusted and
approximately 60% diminished in size on the 5.sup.th and last day
of therapy. Lymphadenopathy progressively decreased and was
resolved at 2 weeks post-treatment. At 30 days post-treatment,
there was complete regression of both oral and anal KS lesions.
Repeat blood work on days of treatment showed no significant
hematologic, electrolyte, liver or kidney changes from baseline.
Viral load immediately after treatment day 5 showed 50,000 HIV-RNA
copies/ml. HIV RNA was non-detectable at 4, 6 and 12 weeks
post-treatment. CD4+ T cell lymphocyte counts increased to 380-420
cells/mm.sup.3 by week 4 and remained stable at week 6 and 12. FIG.
17 shows monitored patient parameters on treatment day 1. FIG. 17a)
shows changes in surrogate markers immediately after treatment,
weeks 4, 6 and 12.
Example 3
Use of DNP to Treat Bacterial Infections, Lyme Disease
[0133] History: A 33 year old white female with a textbook case of
Lyme borreliosis related being bitten by a tick and developing a
pathognomonic erythema migrans on her right anterior thigh. The
rash resolved within two weeks but 3 months later she developed
verbal memory impairment, migratory arthritis of the knees, ankles
and tibias. Fibromyalgias, tachycardias and a left sided Bell's
palsy ensued. Constitutional symptoms of fatigue, malaise and
severe depression caused her to undergo psychiatric care for 1-1/2
years before she was definitively diagnosed with chronic Borrelia
burgdorferi infection. She was treated with ceftriaxone, 2 g
intravenously every 12 hours for 14 days. Four months after
apparent improvement she developed photophobia, headaches,
pronounced memory loss, depression, dysesthesias and a painful,
swollen left knee joint. Repeat ELISA, Western blot and DNA-PCR
were all positive for B. burgdorferi. Spinal tap showed pleocytosis
with positive antibody and PCR tests for neuroborreliosis. Over the
next year the patient received prolonged ceftriaxone, 2 g per day
intravenously for 3 months, and 3 individual short courses of oral
ciprofloxacin, minocycline, and azithromycin. Symptoms failed to
resolve. Two months after her last regimen of antibiotics a new
annular erythematous eruption, suggestive of erythema migrans,
reoccurred on the right thigh and developed under her left axilla.
Doxycycline was instituted and the rash subsided. The patient
refused further antibiotic therapy because of associated
intractable diarrhea and has made tentative plans to undergo
"malariotherapy" in China.
[0134] Physical examination: weight=60 Kg; height=160 cm;
BP=130/70; HR=86 & reg; resp=18; T=37.3.degree. C. Physical
exam revealed a swollen and tender left knee. A thin, atrophic
hypopigmented area of skin over the right thigh, typical of
acrodermatitis chronica atrophicans was present. Neurologic exam
showed some verbal memory deficit. There were bilateral, lower
distal extremity paresthesias.
[0135] Laboratory studies: EKG demonstrated a first-degree
atrioventricular block (PR internal >0.2 sec), some widening of
the QRS complex and Wenckebach periodicity. There were no dropped
beats. Left knee arthroscopy showed synovial hyperthrophy with
early erosive arthritis. Synovial fluid analysis revealed a WBC of
50,000 cells/ml with 70% neutrophils and a positive DNA-PCR for
Borrelia burgdorferi. Biopsy sections of synovial tissue showed
chronic nonspecific synovitis. Warthin-Starry and silver staining
histology revealed spirochetal organisms consistent with Borrelia
burgdorferi. Lumbar puncture spinal fluid analysis showed
pleocytosis, elevated gamma globulin and positive PCR for B.
burgdorferi. Spinal fluid cultured for 2 months in
Barbour-Stoenner-Kelly medium was reported positive for B.
burgdorferi. Serum electrolytes, kidney, liver function and
hematologic studies were all within normal limits. The patient
underwent a stress EKG, attaining a maximum heart rate of 165 with
no evidence of arrhythmia or S-T segment depression.
[0136] Clinical assessment and treatment evaluation: Lyme disease
is a zoonosis caused by a slow growing pathogenic spirochete,
Borrelia burgdorferi. In various mammalian species, including man,
these organisms are known to invade heart, kidneys bladder, spleen
and brain. Borrelia spirochetes are very resistant to treatment
with antibiotics, especially if there is evidence of central
nervous system or joint involvement. Viable B. burgdoferi have been
isolated from antibiotic treated monolayers of fibroblasts.
Borrelia spirochetes are known to be facultative intracellular
pathogens in fibroblasts by laser scanning confocal microscopy.
Central nervous system tissue, joints, front chamber of the eye and
intracellular location can provide the Lyme spirochete with a
protective environment against antibiotic therapy and Borrelia
burgdorferi have been reliably cultured from patients with chronic
disease, even from those previously aggressively treated. This
patient has confirmed chronic CNS and joint Lyme disease in spite
of extensive antibiotic therapy.
[0137] The Lyme spirochete is irreversibly inactivated by heating
at 40.degree. C. for 3 hours, 41.degree. C. for 2 hours or
41.5.degree. C. for 1 hour. Susceptibility of all strains of
Borrelia burgdorferi to penicillin and ceftriaxone is increased up
to 16-fold by elevation of temperature from 36.degree. C. to
38.degree. C. At 40.degree. C. Borrelia burgdorferi increases
expression of at least 12 heat shock proteins (HSP), most of which
are strongly immunogenic. The patient had no history of
thermoregulatory problems. She was informed that her body
temperature would be raised between 40 to 41.degree. C. for a
period of 3 hours, the actual level and time under hyperthermia
would depend on her monitored clinical parameters.
[0138] Pretreatment protocol: the evening prior treatment the
patient was instructed not to eat and dress in cotton
undergarments. Approximately 4 hours prior to treatment 2 mg
alprazolam was administered by mouth. The patient dressed herself
into a dry cold water immersion suit (Stearns, previously
described) with headgear. Monitoring sensors, including EKG
display, IV fluids and Foley catheter were attached and the suit
was zipped closed. The patient opted for oxygen supplementation.
The modified face mask was connected to the TEEM 100 metabolic
Analysis System for V0.sub.2 measurements. Data was recorded as
previously described.
[0139] Treatment procedure: baseline recordings of 10 minutes
showed a V0.sub.2 of 220 cc/min., 3.7 cc 0.sub.2/kg/min. The
patient was infused with 1 mg/kg DNP over a 2 minute period.
V0.sub.2 increased and stabilized at 250 cc/min, 5.3 cc/kg/min. A
second dose of 2.0 mg/kg was infused over a 2 minute period and the
V0.sub.2 peaked at 400 cc/min, 8.8 cc O.sub.2/kg/min. An additional
dose of 1.0 mg/kg DNP was given 30 minutes after the second dose.
The V0.sub.2 increased and reached a stable plateau at 600 cc/min,
10.8 cc/kg/min. Rectal temperature continued to climb until a range
of 40.2 to 40.6.degree. C. was reached at 70 minutes after the
initial dose. A fall in V0.sub.2 was noted at 90 minutes, a
dopamine drip at 2-3 mcg/kg/min was initiated. V0.sub.2 increased
back to 680-710 cc/min. The temperature remained stable between
40.1.degree. C. and 40.6.degree. C. throughout the 3 hour plateau
treatment period. The patient periodically requested the V0.sub.2
monitoring mask be removed during the hyperthermia treatment
period. She was accommodated with removal of the mask on two
occasions for periods not exceeding 10 minutes. The patient
experienced no problems during the procedure but was noticeably
fatigued by hour 3. The treatment was terminated 4 hours and 10
minutes after the initial dose of DNP. Twenty five minutes after
the patient was removed from the neoprene survival suit, the rectal
core temperature dropped to 38.5.degree. C. Normothermia was
achieved approximately 60 minutes after cessation of therapy and
removal from the survival suit. Approximately 6.5 to 7 hours after
treatment the patient experienced chills, an increase in oral
temperature to 38.7 degrees centigrade and malaise. IV fluids and
the dopamine drip at 2 mcg/kg/min were restarted and the patient
was closely observed. Her symptoms subsided over 3 hours and by the
next day she felt active and hungry. It was surmised she may have
experienced a delayed Jarisch-Herxheimer reaction. The patients
monitored treatment flow chart is FIG. 18.
[0140] Treatment outcome: at two months follow-up the patient
stated her arthralgias, myalgias, malaise, fatigue and memory
deficits have disappeared. Lower extremity dysesthesias were no
longer present. EKG showed resolution of her first degree A-V
block. The patient was informed of her past positive cerebrospinal
fluid positive culture for the Lyme disease spirochete. It was
suggested a repeat spinal tap be performed for B. burgdorferi by
PCR and culture. If positive, the patient agreed she would be
re-treated with both DNP induced hyperthermia and intravenous
ceftriaxone for maximum synergism. Repeat spinal fluid analysis was
normal, i.e., no elevated protein, no detectable Borrelia DNA by
PCR and no pleocytosis. Three months later, spinal fluid culture on
Barbour-Stoenner-Kelly II medium was reported negative.
Example 4
Method of Using DNP with Vasopressors and Chemotherapy to Treat
Neoplasia, Peritoneal Carcinomatosis
[0141] History: A 55 year old female presented with a distended
abdomen due to ascites. Laparotomy revealed peritoneal
dissemination of a malignancy with histological findings of an
undifferentiated adenocarcinoma, origin unknown.
[0142] Physical examination: weight=55 kg; height=154 cm;
BP=140/90; HR=88 & reg; Resp=22; T=37.6.degree. C. The patient
was a well developed and well nourished Muslim female with a
healing midline laparotomy scar. Ballotable ascites was detected in
the abdomen. There was no lymphadenopathy.
[0143] Laboratory studies: laboratory examination of the ascitic
fluid showed high levels of amylase. She had a hemoglobin of 9.2.
High levels of amylase and tumor markers, including CA15-3, CA 125
and CA72-4 were present in the serum. Blood chemistry, liver and
kidney function tests were within normal limits. Chest X-ray and
EKG was normal. MRI and ultrasound of the abdomen showed normal
pancreas, liver and atrophic ovaries, there were widespread nodular
lesions consistent with peritoneal carcinomatosis.
[0144] Clinical assessment and treatment evaluation: the patient
had an inoperable malignancy of unknown origin. Chemotherapy in
such cases is only of marginal survival benefit. Hyperthermia,
combined with chemotherapy has been shown to be synergistic with
increased tumor response and survival benefit. Tumor antigen
markers are known to be increased by the heat shock response and
may further enhance immunologic surveillance. The patient had no
history of thermoregulatory problems but refused to be placed in
wet suit or survival suit because of a "phobia of enclosed tight
garments".
[0145] It was elected to treat the patient with
hyperthermochemotherapy. Treatment consisted of DNP, and
combination chemotherapy with carboplatin, mitomycin, and
doxifluridine. An .alpha.-1 adrenergic receptor agonist was used to
minimize peripheral vascular dilation and heat loss.
[0146] Pretreatment protocol: the patient was transfused with three
units of packed red blood cells. A Foley catheter was inserted on
each day of treatment. She was covered in a water soaked blanket
containing a polyethylene lining. A shower cap with towels was used
to prevent heat loss from the head. Intravenous lines were placed
into both arms with 19 gauge intracaths. EKG, heart rate, rectal
thermistor, and V0.sub.2 monitors were attached. Oxygen
supplemented facemask and equipment was attached and data monitored
as previously described under Example 1.
[0147] Treatment protocol: the patient was given chemotherapy by
mouth. The total doses of carboplatin, and mitomycin were 450 mg
and 24 mg IV respectively on day 1 and last day of week 6.
Doxifluridine, 600 mg, was orally administered every day for 5 days
and repeated the last 5 days of week 6. On the day of DNP infusion,
baseline recordings were established for 10 minutes. Mephenteramine
sulfate, 30 mg, was given by intramuscular injection. Ten minutes
later her heart rate increased to 96 and her V0.sub.2 increased
from 250 to 320 cc/min. V0.sub.2, heart rate and blood pressure
stabilized after 20 minutes and she was given an initial dose of 1
mg/kg DNP. Additional 0.5 mg/kg infusions of DNP were administered
in 3 successive infusions spaced 20 minutes apart. The patients
V0.sub.2 stabilized between 780-820 cc/min. and her core
temperature increased to a maximum of 41.4.degree. C. After a
plateau temperature of 41.5.degree. C..-+.0.5.degree. C. was
reached, her level of V0.sub.2 and temperature was maintained for a
period of 2 hours and 30 minutes with an additional infusion of 0.5
mg/kg DNP given 50 minutes after the last dose. The DNP treatment
protocol was repeated every fourth day for a period of 6 weeks. A
representative monitored flow chart is shown in FIG. 19.
[0148] Treatment outcome: By the combined treatments outlined
above, ascites resolved by the end of the sixth week. Serum levels
of amylase and all tumor markers decreased after the third week of
treatment and were normal at week 6. Repeat magnetic resonance
imaging and echo re-examination of the abdomen showed complete
resolution of peritoneal metastasis. Nine and a half months after
treatment, the patient is alive without any evidence of tumor
reoccurrence.
Example 5
Use of DNP with Thermosensitive Liposomes
[0149] To overcome the toxicity to normal tissues of many
anticancer agents such as doxorubicin and anti-infectious drugs
such as amphotericin B, liposomal formulations have been developed.
Liposomal doxorubicin is known to have reduced cardiotoxicity and
increased antineoplastic efficacy. Thermosensitive liposomes can
further enhance tumor targeting and decrease toxicity by release of
their water soluble drug contents in response to tumor
hyperthermia. Various synthetic and natural lipids such as
dipalmitoyl phosphatidyl choline and distearoyl phosphatidyl
choline or egg phosphatidyl choline and cholesterol can be combined
in different molar ratios with ethanol, or other agents that have a
biphasic effect on gel-to-liquid phase transition of phosphatidyl
choline bilayers, to produce liposomes that melt (undergo
gel-to-liquid crystalline phase transitions) at a predetermined
hyperthermic temperature.
[0150] Thermosensitive liposomes were prepared form phosphatidyl
choline (PC) and cholesterol (Ch) using the ethanol method of
Tamura et al. A combination of PC:Ch in a 8:1 molar ratio in the
presence of 6% (v/v) ethanol resulted in formation of liposomes
having a transition temperature between 40.2 and 40.8.degree. C.
The anticancer drug dacarbazine [5-(3,3'-dimethyl-1-triazino)
imidazole-4-carboxamide] was encapsulated in these heat-sensitive
liposomes at a concentration of 3 mg/ml. The in vivo efficacy of
the thermosensitive, liposome encapsulated dacarbazine was tested
on Swiss albino mice transplanted with a dimethyl
benzo-dithionaphtene derived ascites fibrosarcoma subjected to DNP
induced hyperthermia.
[0151] Male, 10-12-week-old, Swiss albino mice were injected with
3.times.10.sup.6 viable fibrosarcoma cells into the peritoneum.
After 15 days the animals were divided into various treatment and
control groups receiving intraperitoneal injections of free
dacarbazine, DNP alone, DNP+empty liposomes and DNP+liposome
encapsulated dacarbazine. DNP induced hyperthermia was recorded
with neonatal rectal and 22 ga. hypodermic YSI probes. Temperatures
were recorded 30 minutes after a 20 mg/kg intraperitoneal dose of
DNP. DNP was administered every day for a total of 5 doses. In all
cases the hypodermic, intraperitoneal temperatures were 1.degree.
C. higher than the rectal.
[0152] As shown in FIG. 20, survival curves of animals treated with
DNP alone and DNP+drug containing liposomes were significantly
improved in comparison to controls. DNP-hyperthermia treated
animals remained alive at day 100 whereas sham treated animals all
died by 60.
Example 6
Use of DNP to Induce Autologous Heat Shock Proteins as a Form of
Thermal Preconditioning Prior to Arterial Balloon Catheterization
or Ischemic Surgical Injury
[0153] DNP would be given orally at doses to increase the V0.sub.2
from 1.5 to 5 times above normal per day for a period of 2-6 days
or, as an infusion at doses that would increase V0.sub.2 and core
body temperatures no greater than 39.degree. C. for periods of 5 to
6 hours or, intravenous doses of DNP alone, with vasopressors, or
other short acting metabolic stimulators, that would increase V02
to equivalent core temperatures of 40-41.degree. C. for periods of
15-30 minutes. Within 8-48 hours after cessation of DNP, the
patient would have maximum heat shock protein production. Such DNP
induced stress would improve clinical outcome by induction of
cellular heat shock protein synthesis with protection of the
patient's, organs, tissues and cells from subsequent ischemic
surgical or traumatic procedures.
[0154] This method of DNP induced preconditioning could be used to
decrease intimal thickening and restenosis after angioplasty,
improve ischemia/reperfusion injury in organ and tissue
transplantation, and improve surgical outcome of procedures that
require temporary or prolonged occlusion of arterial blood flow.
Examples of such DNP induced autologous thermotolerance used as a
form of preconditioning are depicted in FIG. 21, which shows
limitation of proliferative arterial catheter balloon injury in
Sprague-Dawley rats pretreated with DNP induced hyperthermia; FIG.
22 shows the protective effect of DNP pretreatment before hepatic
ischemic injury cased by Pringle's maneuver; and, FIG. 23 depicts
improved musculocutaneous flap skin survival after induction of
heat shock proteins by DNP.
Example 7
Method of Using DNP to Enhance Proton Emission Tomography (PET) in
the Diagnosis of Malignancy and/or Malignant Transformation
(GLIOMA)
[0155] History: A 24 year old white male with neurofibromatosis
presented with a six month history of left sided loss of body
sensation, emotional changes, sensory seizures, inattention to
conversations and sensations of jamais vu.
[0156] Physical examination: weight=65 kg; height=175 cm;
BP=135/80; HR=86 & reg; Resp=18; T=37.9.degree. C. The patient
was a well developed well nourished white male with left upper and
lower extremity sensory loss, postural instability and loss of
tactile discrimination. There was a frank left handed
astereognosis. Eye examination was normal, without papilledema.
[0157] Laboratory studies: Complete hemogram, blood chemistry and
endocrine examination were normal. EEG was within normal limits.
MRI with gadolinium enhancement showed a decreased signal in the
right temporoparietal region with no evidence of contrast
enhancement. PET examination with
[.sup.18F]fluoro-2-deoxy-D-glucose(FDG) revealed a homogeneous
hypometabolic area (metabolic Grade 1) consistent with a Low grade
glioma in the right temporoparietal region. There were no zones of
high FDG uptake. Differentiation of displaced noninvaded gray
matter from the tumor was not discernible on PET imaging.
[0158] Clinical assessment and diagnostic evaluation: although Low
grade gliomas generally present histological features of benign
tumor, it is known that the presence of zones of high FDG uptake by
PET scan in such gliomas is associated with a higher percentage of
malignant transformation. PET-FDG with evidence of tumor
hypermetabolism is believed to be an early biochemical marker of
cellular malignant transformation and is of prognostic value in
High grade gliomas. Biochemically, high glucose (uptake of FDG)
utilization in the presence of oxygen, known as aerobic glycolysis,
is believed to be the result of a hyperactive hexokinase attached
to tumor mitochondria. Increased FDG uptake therefore, represents
increased hexokinase activity and is associated with increased
aggressiveness in gliomas, menigiomas and other neoplasms. Since
DNP uncouples oxidative phosphorylation, any shortfall in
mitochondrial ATP production must come from increased glycolysis.
As a result, FDG uptake will be proportionately increased in DNP
treated malignant cells over those that are normal in contralateral
brain white and gray matter. Since no abnormal FDG uptake was
detected in the tumor by standard PET methodology and the PET scan
was unable to clearly delineate the borders of the tumor, it was
elected to give the patient a low dose of DNP to enhance FDG uptake
and repeat the PET scan. Hypermetabolic components of the tumor
would thus permit a more focused PET-guided stereotactic
biopsy.
[0159] Pretreatment protocol: three days prior to DNP dosing and
repeat PET-FDG scan, the patient's dosage of phenyloin was
increased from 100-mg three times daily to 200-mg three times a
day. The same positron emission tomogram, a CTI-Siemens 933/08-12
which provides a 6.75-mm adjacent slices and in-plane spatial
resolution (full-width at half maximum) of .about.5 mm, was to be
used. The highest level of non to DNP stimulated FDG uptake in the
tumor area was to be compared and qualitatively graded by two
radiologists. Independently, each investigator was to visually
evaluate the positron emission tomogram and use the following
metabolic grading scale: I, FDG uptake less than contralateral
white matter; II, uptake between the levels in contralateral white
and gray matter; III, FDG uptake equal to or greater than in
contralateral gray matter.
[0160] Diagnostic--treatment protocol: the patient was given a 300
mg capsule of DNP (approximately 4 mg/kg body weight) three hours
prior to undergoing a PET-FDG scan. Forty minutes prior to the
emission scan he was intravenously injected with a bolus of FDG
according to standard methodology. Immediately prior to the
20-minute emission scan the patients VO.sub.2 uptake was 40% above
that at baseline. The patients DNP/VO.sub.2 flow chart is FIG.
24.
[0161] Diagnostic outcome: DNP enhanced PET-FDG scan revealed two
areas of hypermetabolism. One of the areas surpassed the limits of
the lesion on CT images and consequently only one of the targets
(graded as a III on FDG uptake) was selected in the "abnormal
PET-normal CT" area.
[0162] The plane that best displayed the abnormal FDG
hypermetabolic uptake area was selected and a pixel located in the
center of the zone was interactively pointed at on visual
inspection. The coordinates of that DNP induced hypermetabolic
pixel were then calculated and set as a target for biopsy. A
PET-guided stereotactic biopsy was performed under the procedure
described by Levivier et al., i.e., the target from the PET image
was projected onto the corresponding stereotactic computed
tomographic (CT) slice to control the reliability and precision of
target selection and the trajectory. Serial stereotactic biopsies
were performed along the trajectory by the method described by
Kelly et al.
[0163] On pathologic examination, including analysis of nuclear
polymorphism and cell density, 2 foci of anaplasia consistent with
glioblastoma (Grade III astrocytoma) were noted.
[0164] Treatment outcome: based on the DNP enhanced PET-FDG scan
diagnostics outline above, this patient was found to have a
malignant transformation in his otherwise Low grade glioma. This
diagnostic treatment protocol procedure of detecting foci of
hypermetabolism caused him to undergo systematic radiation therapy
with chemotherapy (dibromodulcitol-procarbazine-carmustine) early
in the course of his malignant process. One year after diagnosis
and therapy the patient again underwent PET scanning. DNP
enhancement (repeated as outlined under "Diagnostic" above)
revealed a single hypermetabolic component (metabolic Grade II) in
the tumor area. Repeat PET-guided biopsy revealed the area to be a
zone of radionecrosis. The remaining viable tumor, even with DNP
enhancement, continued to be a metabolic Grade I. The patient
remains alive one and a half years after his diagnosis, albeit with
left-sided hemiparesis.
Example 8
Method of Using DNP to Enhance Detection of Malignant Tumors by
High Resolution Digital Infrared Imaging (Breast Carcinoma)
[0165] History: a 34 year old white female with existing
fibrocystic disease of the breast underwent yearly mammography and
was found to have an equivocal opacity in the right breast, medial
to the aereola. Two past breast biopsies were negative for
malignancy and consistent with fibroadenomatous disease of the
breast. The patient was opposed to another breast biopsy (would be
third), unless there was a definitive indication of a lesion over
that of her known fibrocystic disease of the breasts.
[0166] Physical examination: WT=60 kg; HT=164 cm; BP=120/72; HR=88
& reg; R=18/min; T=37.7 C. The patient was a normal appearing
white female with scattered to diffuse nodularities in both
breasts. A palpable 3.times.2 cm, non-tender, lump was located 3 cm
medial to the right aereola. There was absence of nipple discharge,
retraction, skin dimpling, rash or discoloration of either breast.
There were no palpable axillary lymphadenopathy.
[0167] Laboratory studies: chest x-ray, EKG, blood chemistry, and
hemogram examination was normal. Mammography, Doppler ultrasound,
MRI, and scintinammography failed to indicate or eliminate a
possible occult carcinoma in this young patient with dense,
fibroadenomatous breast disease. A diffuse, non-cystic, opacity on
the right breast was the only definitive finding from these breast
studies.
[0168] Clinical assessment and diagnostic evaluation: this patient
has had two previous open breast biopsies without evidence of
malignancy. Early detection of breast carcinoma is of crucial
importance to survival. False negative results of mammography (and
other complimentary studies) range between 5-30%. The ability of
infrared imaging technology to detect changes related to increased
metabolism (tumor) and angiogenesis has greatly improved from that
of 30 years ago. High resolution digital computerized infrared
equipment can now detect focal increases in tumor temperature from
as little as 0.05.degree. C., and increases in focal breast
temperatures may be as high as 1-2.degree. C. in malignant tumors
versus normal, contralateral breast sites.
[0169] Since it is known that infrared imaging has at least a 19%
rate of false positives and 17% of false negatives, and equivocal
mammography and abnormal infrared imaging is not uncommon in young
women with dense breast tissue and diffuse fibrocystic disease, the
use of DNP to enhance tumor metabolism (infrared imaging) over that
of normal tissue, could be of substantial diagnostic benefit.
Specifically, DNP would greatly enhance tumor metabolism (infrared
imaging), in comparison to non-DNP enhanced infrared imaging and
would greatly increase tumor detection when there is either
insufficient production or detection of metabolic heat or vascular
changes. Further, the heat differential between DNP enhanced and
non-DNP infrared tumor imaging may also decrease the false positive
rate seen with this procedure, especially in benign conditions such
as fibrocystic disease of the breast. Since non-DNP infrared
imaging is capable of detecting as great as 1-3.degree. C. changes
in focal temperature between normal and malignant tissue, DNP
enhancement would increase the temperature difference several fold
and enhance both the sensitivity and precision of currently
available infrared imaging technology. The patient agreed to have
both of her breasts examined non-invasively with infrared imaging,
before and after intravenous DNP administration to ascertain if
there was increased infrared signaling from the worrisome, palpable
lump in her right breast.
[0170] Prediagnostic protocol: the patient was disrobed to the
waist and sat with her hands interlocked over her head for a five
minute equilibration period in a draft free, thermally controlled
room--kept between 18.degree. C. and 20.degree. C. She did not take
any oral medication, alcohol, coffee, and did not smoke, exercise
or use deodorant three hours prior to testing. A baseline of 4
images consisting of an anterior, undersurface and 2 lateral views
of each breast were generated by an integrated infrared imaging
station consisting of a scanning mirror optical system containing a
mercury-cadmium-telleride detector (Bales Scientific, CA). The
infrared system had a spatial resolution of 600 optical lines, a
central computerized software processor providing multi-tasking
capabilities and a high-resolution color monitor capable of
displaying 1024.times.768 resolution points with 110 colors or
shades of gray per image. Images were stored on retrievable laser
discs.
[0171] Diagnostic treatment protocol: after the above baseline
studies were performed, the patient was given an initial
intravenous dose of 1 mg/kg DNP and observed for a period of 20
minutes. An additional 2 mg/kg of DNP was then administered and 30
minutes thereafter, she was taken to the thermally controlled room
for repeat DNP-enhanced infrared imaging. Immediately prior to
transferring the patient to the thermally controlled room, the
patients VO.sub.2 was incrementally increased to 50% above her
VO.sub.2 baseline, see FIG. 25. Repeat infrared images were then
obtained under the exact protocol used for obtaining baseline
studies.
[0172] Diagnostic--treatment outcome: baseline (non-DNP enhanced)
infrared imaging revealed insignificant vascular asymmetry and no
significant temperature changes when the results were reviewed and
compared to the rest of the ipsilateral or contralateral breast
sites.
[0173] DNP enhanced infrared imaging resulted in a bilateral global
breast temperature increase of approximately 0.5.degree. C. An
abnormal, 2.5.degree. C. increase in temperature was noted in the
palpable, right breast lesion discovered by clinical exam. Since no
non-cancer causes for such a dramatic temperature increase could be
identified, i.e. abcess, trauma, or recent surgery, this 5 fold
increase in heat production (above the DNP baseline increase of
0.5.degree. C.) was highly suspect to be caused by an early
malignancy.
[0174] The patient was admitted to the hospital and under general
anesthesia underwent an open breast biopsy. Frozen section (and
later permanent tissue mounts) revealed a well-differentiated
intraductal carcinoma. Progesterone and estrogen receptors, as
determined by immunocytochemical methods, were negative. A simple,
right mastectomy with axillary lymph node dissection was performed.
A total of twelve lymph nodes were identified: there was no
evidence of tumor. The patient refused chemotherapy and
radiotherapy. She was placed on long-term oral tamoxifen (10 mg
twice a day).
Example 9
The Use of Dinitrophenol with Artificial Electron Receptors (or
Other Free Radical Forming Agents) in the Treatment of Hormone And
Chemotherapy Resistant Malignancy (Prostate Cancer)
[0175] History: a 68 year old Mexican male, developed a gradual
increase in low back pain, right hip pain and several episodes of
hematuria over a 10 month period. He was referred to a urologist
and diagnostic work-up revealed a carcinoma of the prostate with
the extension of the tumor into the bladder. Bony metastasis were
present to the right pelvis, fourth and fifth lumbar vertebra,
right femur, left humerus, right sixth and seventh ribs and right
scapula. He refused any form of surgery but underwent radiation
therapy to the pelvis and symptomatic bony lesions. Treatment was
initiated with megestrol acetate (640 mg/day), prednisone (20
mg/day) and leuprolide (7.5 mg/month). After three months of
therapy the patient continued to have progression of his disease
manifested by increasing bone pain, rising prostatic specific
antigen levels (PSA) and increasing serum acid phosphatase.
[0176] Physical examination: WT=72 kg; HT=175 cm; BP=140/86; R=22;
T=37.6 C; HR=88 & reg; Exam revealed mild emaciation with some
scrotal and +1 pitting bilateral lower extremity edema. There were
scattered bilateral, basilar rales on examination of the chest.
[0177] Laboratory studies: EKG demonstrated a right partial bundle
branch block. Chest x-ray showed mild chronic obstructive pulmonary
disease with minimal fibrosis. There was some patchy, interstitial
edema in both lower lung fields. There were no pulmonary
metastasis. Complete blood count showed a mild anemia with a
hemoglobin of 10.5 and a hematocrit of 34%. Liver function tests
were normal. White blood cell count, differential and platelet
count, was within normal limits. PSA level was 58 ng/ml. Serum acid
phosphatase was 2.times. above normal. Blood electrolytes including
calcium were within normal limits. The acid phosphatase, AST, ALT
and bilirubin levels were normal. Radionucleotide bone scan
revealed multiple metastasis in the axial skeleton and ribs. Review
of past prostatic biopsy slides showed a high grade adenocarcinoma
of the prostate with a Gleason Grade of 8. Pulmonary function
studies showed moderate airflow obstruction with mild hypoxemia and
hypercarbia. Stress EKG was not performed because of his severe
exercise intolerance.
[0178] Clinical assessment and treatment evaluation: the patient
has a metastatic, hormone-refractory prostate carcinoma with
clinical progression documented by increasing bone pain and rising
serial PSA values. Under the TNM classification of the American
Joint Cancer Committee for prostate cancer (T=degree of primary
tumor extension; N=regional lymph node involvement; and, M=presence
of distant metastasis), he has the highest stage (T4 N3 M1).
Histologically, the tumor is aggressive by the Gleason Grading
System. Since death due to prostatic carcinoma is almost invariably
a result of failure to control metastatic disease, and since
prostatic cancers are well-known to be sensitive to heat stress,
the present DNP therapy was undertaken as a last resort effort to
stop tumor progression and/or improve the patients quality of
life.
[0179] In view of the patients age, pulmonary problems and poor
performance status (Karnofsky Score of 6) it was decided to treat
the patient with moderate doses of DNP and a free radical cycling
agent, methylene blue (MB), to induce synergistic tumor killing.
The effect of methylene blue on cellular reduction-oxidation status
(redox) is well known. Methylene blue readily traverses cell
membranes and acts as an electron acceptor from the major
coenzymes. Unlike other oxidizing drugs, it cycles futilely,
transferring electrons from endogenous substrates to oxygen.
Depending on the redox status of a cell, MB can act as either an
intracellular electron acceptor or donor. MR directly catalyzes the
reaction of intracellular reductants, NADPH, NADH and GSH (reduced
glutathione) with oxygen causing the production of hydrogen
peroxide, superoxide anions, and the formation of the potent
cytotoxic oxidant species, peroxynitrite. In DNP partially
uncoupled mitochondria, MB further stimulates respiration due to
its dual action of providing reducing equivalents necessary for
beta-oxidation of fats and electron donating/shuttling capacity,
with respect to the mitochondrial respiratory chain. It is an
effective drug, at doses of 1-3 mg/kg, in treating nitrate-induced
methemoglobinemia. MB is also used as an antidote given as a 100 mg
IV bolus for encephalopathy associated with alkylating
chemotherapy.
[0180] Since uncoupling, heat and MB increase the flux of cellular
free radicals and malignant cells possess a high bioreductive
capacity, the synergistic effects of DNP with MB would allow for
maximum tumor killing with minimum to moderate levels of induced
total body hyperthermia. Additional free radical cycling agents
that can be used in lieu of MB include, but are not limited to:
phenazine methosulfate, xenobiotics such as quinones (e.g.,
menadione, semiquinone, naphthoquinone, duroquinone, indigo
carmine), nitrocompounds (e.g., metronidazole, niridazole,
nitrofurazone, flunitrazepam), eminium ions (e.g., methyl viologen,
benzyl viologen, etc.), and others. In this patient, DNP-MB therapy
was to be administered so as not to exceed the baseline VO.sub.2
level by 50-75%.
[0181] Pretreatment protocol: the patient was transfused with 2
units of packed red blood cells 48 hours prior to undergoing
treatment. Intravenous fluids (Lactated Ringer's solution) were
administered at a rate of 100 cc/hour. The patient was dressed in
comfortable cotton clothing and placed in an air-conditioned room.
Equipment for monitoring heart rate and rhythm, temperature and
oxygen consumption was utilized as outlined in Example 1. An oral
breathing tube was used to conduct TEEM VO.sub.2 measurements.
Oxygen supplementation and "crash cart" was available at
bedside.
[0182] Treatment protocol: baseline VO.sub.2 measurements for 8
minutes established an average VO.sub.2 of 250 cc/minute. DNP, at a
dose of 2 mg/kg, was infused intravenously over a 2 minute period.
Repeat VO.sub.2 at 20 minutes was stabilized at 340-360 cc/minute.
An additional 1 mg/kg DNP infusion was administered, and 15 minutes
thereafter the VO.sub.2 increased and stabilized at 420 cc/minute.
Ten minutes thereafter, an infusion of methylene blue, 2 mg/kg
(dissolved in a 0.4% pyrogen-free isotonic saline solution-35 ml)
was administered over 20 minutes. Repeat VO.sub.2 measurement at 20
minute intervals showed it to rise to and stabilize at 450-500
cc/minute.
[0183] By hour 3, VO.sub.2 declined to the 360-380 cc/minute range.
An additional 1 mg/kg dose of DNP was infused over a 2 minute
period. Repeat VO.sub.2 measurements 20 minutes after this infusion
showed an increase in VO.sub.2 back to the 450-500 cc/minute.
Rectal probe temperature increased to a maximum of 1.3.degree. C.
over baseline. Blood pressure and cardiac rates remained within
normal limits. The patient withstood the procedure without any
adverse effects and therapy was terminated 6 hours after the
initial DNP dose. The protocol was repeated every other day for a
total of 15 treatments (30 days). Therapy was discontinued for 2
weeks and the cycle was again repeated for an additional 30 days,
treatment being administered every other day.
[0184] Treatment outcome: there was no evidence of general toxicity
at any time during treatment. The patient noted a decrease in his
low back, hip and other areas of bone pain on the 6.sup.th day
following therapy. By 2 weeks, the patient was off all narcotic
(morphine) analgesics and had a markedly increased appetite. On day
8, repeat PSA levels were increased by approximately 120% to 125
ng/ml. Acid phosphatase remained unchanged. All other blood
chemistries, including CBC, showed no significant alterations.
[0185] At 6 weeks after treatment, repeat PSA values showed a
significant decline to 30 ng/ml with a concomitant fall in serum
acid phosphatase levels. At the final stage, 10 weeks after
initiation of treatment, a prostatic biopsy was performed.
Histologic examination revealed 95% of the tumor to be necrotic
with only scattered or scarred acini containing an occasional
malignant cell. There was a significant increase in stromal cells
above that seen in his initial biopsy. One of the most striking
changes noted by the pathologist was the formation of cyst-like
structures within the epithelial cells. The patient was seen three
months after initiation of therapy, at which time he had gained 8.2
kg of weight, remained pain free and stated that he felt "normal".
FIG. 26 shows monitored treatment parameters. FIG. 27 shows
biochemical, biopsy and clinical responses.
[0186] Oral DNP therapy (250 mg twice a day, daily for 5 days and
recycled after no medication for 2 days) was initiated after his IV
therapy and continued up to 4 months. A repeat prostate biopsy at
the end of month 4 was obtained. Pathologic examination revealed
disintegration of remaining tumor acini along with the formation of
with many epithelial cysts. Occasional residual tumor cells were
fractured and disrupted with markedly reduced cytoplasm. There was
extensive fibrosis with an apparent increase in the number of
stromal cells. Cytoplasm volume was significantly diminished in
both the residual tumor and normal cells. Overall, there were very
few intact acini or viable acinar cells.
Example 10
Method of Using Dinitrophenol with Biologic Response Modifiers (in
the Treatment of Hepatitis C Infection)
[0187] History: a 32 year old Investment Banker was evaluated for
chronic Hepatitis C infection. She gave a past history of
intermittent jaundice, dark urine, mild anorexia, nausea and
vomiting. This episode occurred 10 years ago, approximately 3
months after a transfusion (3 units of packed red blood cells) for
a cesarean section. She was currently asymptomatic, but on routine
health insurance exam she was found to have elevations in her ALT
and AST (alanine and aspartate aminotransferase) levels: 140 IU/L
and 90 IU/L, respectively. She drank 5-8 glasses of wine per week.
Additional laboratory tests identified anti-HCV antibodies with an
HCV-RNA level of 5.times.10.sup.6/ml. The patient refused to
undergo liver biopsy but agreed to treatment with interferon
alpha-2b (3 million units injected subcutaneously 3 times per week)
and ribavirin (500 mg orally--twice a day). After 12 weeks of
treatment she developed lethargy, severe headaches, fever, nausea
and depression. Anemia was detected with a hemoglobin concentration
of 9.2 g/deciliter. As a result, her dosage of interferon was
reduced to 1.5 million units 3 times a week and the dose of
ribavirin was reduced to a total of 600 mg/daily. After 6 months of
treatment her ALT and AST levels became normal and HCV-RNA became
undetectable.
[0188] An additional six months of therapy however, failed to
sustain her clinical improvement and she was found to have a
relapse. Serum HCV-RNA levels rose to 5.2 million copies/ml and
liver enzymes increased to 2.5-3 times that of the normal range.
She was unable to tolerate any additional ribavirin because of
severe anemia. She persistently refused to undergo a percutaneous
liver biopsy.
[0189] Physical examination: WT=48 kg; HT=150 cm; BP=128/82; HR=76
& reg; R=18; T=37.5.degree. C. Physical examination failed to
reveal any signs of chronic liver disease. She was noted to have
several scattered areas of scalp alopecia which she attributed to
her anti-hepatitis C therapy.
[0190] Laboratory studies: EKG and chest x-ray were normal. CBC
revealed a mild anemia with a hemoglobin of 10.2 and a hematocrit
of 34%. WBC, differential and platelet count were within normal
limits. Alkaline phosphatase was within normal limits. Serum AST
and ALT were elevated to 2.5-3 times that of the upper normal
limit. Serum HCV-RNA levels were found to be at 5.8 million
copies/ml. The infecting hepatitis C strain was of genotype 1b.
Antimitochondrial antibody serology was negative (titer less than
1:20). There were no other blood chemistry, hormone, or urine
laboratory abnormalities.
[0191] Clinical assessment and treatment evaluation: the patient
has a chronic Hepatitis C infection with relapse after combination
ribavirin and interferon alpha-2b treatment. This is not uncommon
in that the rate of relapse after an end-of-treatment response to
interferon-ribavirin therapy may exceed 50%. She was unable to
tolerate additional ribavirin therapy because of a related anemia.
Further, interferon dose escalation in non-responders to initial
interferon therapy has only proved successful in a small number of
cases. Despite her refusal to undergo any form of liver biopsy she
agreed to undergo a combination of DNP and interferon therapy for a
period of 12 weeks.
[0192] The liver is known to be one of the "hottest" organs in the
human body. Liver temperatures exceeding 44.degree. C. have been
documented in humans undergoing strenuous exercise. The hepatitis C
virus is an RNA encoded sphere containing several polyproteins
comprising a capsid, 2 envelope proteins, and at least 6 enzymatic
proteins with varied functions. Hepatitis C virus is known to be
heat sensitive and is inactivated by standard blood banking heating
techniques. Case reports of hepatitis C inactivation with the use
of extracorporeal hyperthermia are known. It has been reported that
HIV positive patients treated with extracorporeal hyperthermia,
many of which were also positive for hepatitis C, the hepatitis C
virus was cleared (as determined by serum viral PCR-RNA
analysis).
[0193] Based on the this patients failure to respond to
conventional treatment, anecdotal and case report studies showing
beneficial results with whole body hyperthermia, the patient
underwent a combination of DNP and interferon therapy. She was
informed that she would undergo daily treatments with intravenous
DNP for five days per week and receive interferon alpha at a dose
of 1.5 million units subcutaneously every two days. This treatment
protocol would continue until her hepatitis C-RNA blood viremia was
no longer detectable.
[0194] Pretreatment protocol: each evening prior to treatment the
patient was instructed not to eat after 7 .mu.m and dress in cotton
clothes. Approximately 6 hours prior to intravenous DNP
administration she was to be given 1.5 million units of
subcutaneous interferon-alpha every 3rd day. Repeat blood work,
including CBC and platelet count, AST, ALT, and hepatitis C-RNA
levels would be initially obtained at 48 hours and weekly
thereafter. No efforts were to be made to prevent body heat loss. A
single intravenous line was placed with a 21-gauge interacath.
Heart rate, rectal thermistor, and VO.sub.2 monitoring was
conducted during therapy as outlined.
[0195] Treatment procedure: the patient presented herself for
outpatient treatment and was given a subcutaneous dose of 1.5
million units of interferon-alpha. Approximately 6 hours
thereafter, at 1 .mu.m, a baseline VO.sub.2 recording of 5 minutes
was 160 cc/min. She was infused with 1 mg/kg DNP over a 2 minute
period. At 20 minutes, her VO.sub.2 increased and stabilized at
approximately 210 cc/min. A second dose of 1 mg/kg DNP was infused
and the VO.sub.2 peaked 20 minutes later at 250 cc/min. An
additional dose of 2.0 mg/kg DNP was given 30 minutes following the
second dose. Repeat VO.sub.2 showed a rise and stabilization 20
minutes thereafter at 360 cc/min. The patient's rectal temperature
increased and never exceeded 1.3.degree. C. above her normal
baseline. Two hours after her last dose of DNP, her VO.sub.2
declined to 280 cc/min. An additional 2 mg/kg dose of DNP was
administered. The patients VO.sub.2 increased and stabilized 20
minutes thereafter to a level of 420 cc/min. She was noted to sweat
profusely. Throughout treatment the patient was permitted to drink
fluids ad libitum. She was notably fatigued at hour 5 of therapy.
Monitored parameters and flow chart are shown in FIG. 23. The 5 day
treatment protocol was repeated after a 2 day "DNP rest period".
This regimen was repeated times 3. Subcutaneous interferon-alpha
was administered for a total of 10 weeks. FIG. 28 shows the
patients DNP/interferon treatment flow chart.
[0196] Treatment outcome: by the treatment regimen outlined above,
hepatitis C-RNA viral load decreased by approximately 2 logs after
48 hours. Over the next 5 days the viral load further decreased by
an additional log. HCV-RNA became undetectable and the HCV viral
genome remained cleared from the bloodstream at week 2 and
thereafter. Alanine transaminase (ALT) levels increased 7 fold at
48 hours and remained elevated until week 3, at which time they
returned to levels slightly below that which existed prior to
therapy. CBC, bilirubin, and blood urea nitrogen (BUN) remained
within normal limits. Alkaline phosphatase levels increased 2 fold
at 48 hours but returned to pretreatment levels at day 7.
[0197] The patients HCV viral genome remained cleared from her
bloodstream 18 months after therapy and there was normalization of
her ALT.
Example 11
Method of Using Dinitrophenol Induced Intracellular Hyperthermia to
Increase Immunogenicity of Human Tumors
[0198] DNP would be given as an intravenous solution, or as an oral
preparation, so as to increase oxygen consumption 2.5-5 times above
normal for a period of 2-3 hours. Such treatment would be
administered every other day for a period of 5-10 days. At 8-24
hours after the last day of treatment, the patient would be
administered standard chemotherapy or specific monoclonal antibody
immunotherapy directed against known mutated or inappropriately
expressed oncogenic proteins (e.g., ras, p53, HER/neu, etc.), or
combination anti-oncogenic immunotherapy with chemotherapy or
radiation.
[0199] Heat shock proteins (HSPs) or stress-induced proteins are
constitutively expressed in all living cells and are among the most
abundant proteins found. However, many members of the HSP family
can further be expressed by cellular stress-causing conditions such
as heat, drugs, glucose deprivation, etc. Of importance to the
present method is that the expression of HSPs in tumors is
associated with a heightened immune and/or cytotoxic T-lymphocyte
response. In particular, it is known that members of the HSP70
family (HSPs are generally classified by their molecular weights
e.g., HSP90 kdaltons, HSP27 kdaltons, HSP70 kdaltons, etc.) are
expressed on cell surfaces. Due to the ability of DNP to induce
intracellular hyperthermia, the enhanced expression of human HSPs
in DNP treated tumors could greatly increase their
immunogenicity.
[0200] This method could be used to broaden the antigen-specific
repertoire of many poorly immunogenic tumors by increasing the
expression of HSP-peptide immunogenic determinants on their cell
surfaces. Such consequences would heighten any endogenous specific
anti-tumor immune response. Moreover, DNP-intracellular
heat-inducible immunogenic targets could further increase the
efficacy of exogenously synthesized and administered monoclonal
antibodies. By example, patients with HER-2/neu overexpressing
metastatic breast cancer (25% of breast cancer patients) would be
treated by the DNP method outlined above. This treatment would then
be followed by a standard loading dose and weekly infusions of
anti-HER-2/neu monoclonal antibodies. Clinical benefits would be
evaluated by overall response rates and duration of response.
Example 12
Synthesis and Use of Novel Conjugates and Derivatives of
2,4-Dinitrophenol
[0201] Formation of novel nitrophenol compounds is of importance in
that their alkyl, alkene, fatty acid, aromatic and other
derivatives may significantly enhance their biologic activity
and/or improve the therapeutic index. Many reactions of the benzene
ring of phenols through halogenation, sulfonation, and nitration
are known. Numerous procedures for C-alkylation of phenols through
reduction of benzylic alcohol, aldehydes, benzonitriles and Mannich
bases are published.
[0202] Alkylations or other "R" group additions have also been
performed on various phenolic substrates using Stille or Negishi
coupling reactions. An example of converting a nitrophenol compound
to the desired alkylated (or other "R" group analog) by a 2 step
procedure utilizing the Stille coupling reaction is illustrated in
FIG. 30. As shown in step 1, DNP is first iodinated with
Barluenga's reagent (IPy.sub.2 BF.sub.4) to yield
2,4-dinitro-3,5-diiodophenol. In step 2, the nitroiodophenol is
then converted to the alkylated derivative (in the instant example
an ethylated derivative) via a co-catalytic, palladium-copper
Stille reaction.
[0203] Compound 3 shown in FIG. 30 is an ethylated derivative of
DNP and is designed to increase uncoupling activity by adding
lipophylic alkyl substituents to the benzene ring. Such analogs
with augmented activity may be particularly useful in the treatment
of bulky tumors and malignancies which possess a high fat content,
e.g. liposarcoma, glioblastoma, etc.
[0204] A representative approach (Step 2) to the palladium-copper,
co-catalytic ethylation of a nitroiodophenol is illustrated by the
conversion of 2,4-dinitro-3,5-diiodophenol to
2,4-dinitro-3,5-diethylophenol. Nitroiodophenol (500 mg, 934
.mu.mol) is added to a pressurized reaction to containing
N-ethylpyrrolidinone (1.5 ml). Pd.sub.2 dba.sub.3CHCL.sub.3 (27 mg,
26 .mu.mol) and triphenylphosphine (50 mg, 191 .mu.mol) is added to
the stirring solution and slowly heated to approximately 50.degree.
C. for 10 minutes. Copper iodide (17 mg, 91 .mu.mol) is added to
the stirring solution. The mixture is again heated to 50.degree. C.
for 10 minutes. The solution is cooled to 32.degree. C. and
tetraethyl tin (285 .mu.L, 2.05 mmol) is added to the stirring
solution. The reaction tube is sealed and heated with continuous
stirring at 65.degree. C. for 12-16 hours. Aqueous workup and ethyl
acetate extraction with drying by magnesium sulfate (MgSO.sub.4)
and concentration yields the end product.
Example 13
Synthesis of an Expanded Combinatorial Library of Putative
Uncoupling Agents Capable of Inducing Intracellular
Hyperthermia
[0205] The spectrum of potential classic uncoupling agents that can
induce intracellular hyperthermia can be greatly expanded through a
designed convergent synthetic approach. An almost limitless variety
of uncouplers can be synthesized through a "combinatorialized"
scheme to produce an expanded "library" of uncoupling agents with
related structures. The scheme specifically presented herein
exemplifies the synthesis of 21 potential uncoupling agents, but
can be expanded to 1,000 to 100,000 putative uncoupling agents.
[0206] Five classes of uncouplers are prepared via the convergent
route shown in FIG. 31. The synthetic scheme depicted in FIG. 31 is
designed as a combinatorial approach to allow access to a library
of structurally related putative uncouplers for biological
evaluation. While the given examples noted in FIG. 31 will allow
formation of at least 21 novel uncouplers, a simple variation in
this synthetic scheme will allow the library of uncouplers to be
expanded to include from 1,000 to 100,000 novel uncoupling agents.
After discussing the general synthetic approach in FIG. 31, the
simple synthetic variations designed to expand the library of
uncouplers will be described. Such variations will be apparent to
those skilled in the art of synthetic organic chemistry and
pharmaceutical development.
[0207] Starting from benzaldehyde (FIG. 31, Compound 1),
diiodination at the 3- and 5-positions using Barluenga's reagent
(IPy.sub.2BF.sub.4) affords Compound 2 which is alkylated using a
co-catalytic, palladium-copper Stille reaction to produce a
3,5-disubstituted Compound 3. This 2 step approach is known for
producing a variety of methylated phenols. Use of tetramethyltin
then produces the dimethyl derivatives [Compound 3, where
R=Me(methyl)]; tetrabutyltin produces the dibutyl derivatives
[Compound 3, where R=Bu(butyl)]; and, tetraphenyltin produces the
diphenyl derivatives [Compound 3, where R=Ph(phenyl)]. A
Baeyer-Villiger oxidation of Compound 3, with meta-chlorobenzoic
peracid (mCPBA) followed by alkaline hydrolysis [KOH(potassium
hydroxide)] of the resulting formate affords phenols, Compound 4.
The homogeneous 2,4-dinitro- or 2,4-dicyano-derivatives are
initially accessed from an intermediate Compound 4. Nitrosation of
Compound 4 with nitrofluoromethylsulfonate salt
(NO.sub.2CF.sub.3SO.sub.3) provides the
3,5-disubstituted-2,4-dinitrophenols shown in Compound 5. Three
different uncoupling agents are produced via this synthetic route.
Diiodination of Compound 4 at the 2- and 4-positions produces
Compound 6 which is treated with copper(I) cyanide (CuCN) to give
the 2,6-dicyanate derivative, Compound 7. Three additional
uncouplers are synthesized by this route. The heterogeneous nitro-,
cyano-uncouplers are also accessed from intermediate Compound 3.
The 2-cyano-, 4-nitro-uncouplers are targeted as the steric effects
of the cyano group at the 2-position is less than the corresponding
2-nitro-derivatives. Mono-iodination of Compound 3 through the
thallium intermediate affords the selective 2-iodo-derivative,
Compound 8. Conversion of Compound 8 to phenol, Compound 9, is
accomplished as before through the Baeyer-Villiger oxidation and
hydrolysis of the resulting formate. Selective 4-nitration to
produce Compound 10 is readily accomplished with
nitrotrifluoromethylsulfonate salt followed by cyanation to afford
2-cyano-,4-nitro-uncouplers, Compound 11. Three additional
uncouplers are produced by this route.
[0208] Additional uncouplers, such as the 2,4,6-tricyano compounds,
can also be produced through the same convergent synthesis.
Exhaustive iodination of Compound 4 affords 2,4,6-triiodinated
Compound 12 which is then directly converted to
tricyano-uncouplers, Compound 13, through copper catalyzed
exchange. Three more uncouplers are produced by this modification.
A 2,4-dicyano-uncoupler carrying three variable substituents at the
3-, 5- and 6-positions is also readily produced through this
convergent approach. Initial selective monobromination of the
phenol Compound 4 at the ortho-position affords Compound 14 which
is diiodinated at the 2,4-positions to produce the 2,4-diiodo-,
6-bromo-Compound 15 derivatives. Selective cyano exchange at the
more reactive aryliodide positions affords the dicyano Compound 16
derivatives. A final co-catalytic, palladium-copper Stille reaction
results in the formation of the 3,5,6-trisubstituted,
2,4-dicyano-uncouplers. Use of the same tin reagents previously
described allows the introduction of either methyl, ethyl, propyl,
butyl, etc., or phenyl at the 6-position. In conjunction with the 3
different substituents at the 3- and 5-positions, 9 additional
uncouplers are afforded by this additional expansive route.
[0209] The synthesis of 21 novel uncouplers depicted by the
convergent approach in FIG. 31 can be further modified. To those
skilled in the art, a simple variation in this exemplary synthetic
approach will allow a greatly expanded library of potential
uncouplers to be synthesized. The expanded library can be produced
by introduction of an array of alkyl and aryl substituents at the
3-, 5-, and/or 6-positions while maintaining the 2,4-dinitro-,
2,4-dicyano, 2-cyano-4-nitro-, and/or the 2,4,6-tricyano-phenol
substrate. This simple synthetic variation is accomplished by using
a variety of well known palladium, zinc, or copper-mediated
reactions at the stage of alkyl or aryl group incorporation, i.e.,
FIG. 31, Compound 2 to 3 and Compound 16 to 17 conversions. This
synthesis is a variation on the Stille reaction, the Heck reaction,
the Negishi coupling, Suzuki couplings, Semmelhack reactions and
cuprate reactions. Such a variation can introduce a nearly of
unlimited array of potential substituents onto the phenol core of
the uncoupler. This combinatorial approach can even be further
expanded by variable halogenation (either bromination or
iodination) at the 3- and 5-positions to allow 2 different
substituents to be introduced at these 2 positions in the ensuing
metal-mediated halogen exchange reactions. This "combinatorial
library" approach will allow a broad range of potential uncouplers
to be synthesized and evaluated for potential biological activity,
including safety and effectiveness.
[0210] Activity of the many diverse conjugates and derivatives of
2,4-dinitrophenol (and other uncoupling agents) may be tested by
known in vitro methods for oxygen consumption, e.g., tissue or
cellular suspensions with Clark type oxygen sensors. Toxicity,
mutagenicity and LD50 studies in animals would be performed under
recognized protocols prior to use of any such novel compounds in
human subjects. Upon establishing toxicity and safety criteria, the
various novel conjugates and derivatives can be administered under
dose escalation trials as outlined previously for the clinical use
of dinitrophenol.
[0211] It will be apparent to those skilled in the art that
numerous modifications and variations can be made to the processes
and compositions of this invention. Thus, it is intended that the
present invention cover the modifications and variations of this
invention provided they come within the scope of the appended
claims and their equivalents.
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