U.S. patent application number 12/803166 was filed with the patent office on 2012-06-14 for method for the treatment or prevention of virus infection using polybiguanide-based compounds.
This patent application is currently assigned to Novaflux Biosciences, Inc.. Invention is credited to Mohammed E. Labib, Richard F. Stockel.
Application Number | 20120148530 12/803166 |
Document ID | / |
Family ID | 30116119 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148530 |
Kind Code |
A1 |
Labib; Mohammed E. ; et
al. |
June 14, 2012 |
Method for the treatment or prevention of virus infection using
polybiguanide-based compounds
Abstract
An inexpensive, easily available, and convenient method of
treating or preventing a virus infection is provided. The present
invention relates to a method for the treatment or prevention of
virus infections using polybiguanide-based compounds administering
a therapeutically effective amount of a compound or a
pharmaceutically acceptable salt thereof The invention relies on
the unique biochemical reaction in which polybiguanide-based
compounds interfere with the spread of virus within or between
organisms. The compositions and formulations described in the
present invention are effective means to reduce the infectivity of
the human immunodeficiency virus type 1 (HIV-1), and human herpes
simplex viruses, and also to kill the causative organisms of many
other sexually transmitted diseases (STDs).
Inventors: |
Labib; Mohammed E.;
(Princeton, NJ) ; Stockel; Richard F.;
(Bridgewater, NJ) |
Assignee: |
Novaflux Biosciences, Inc.
Princeton
NJ
|
Family ID: |
30116119 |
Appl. No.: |
12/803166 |
Filed: |
June 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10435756 |
Aug 27, 2003 |
7772284 |
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12803166 |
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Current U.S.
Class: |
424/85.4 ;
435/366; 435/372.3; 435/375; 514/120; 514/263.38; 514/275; 514/311;
514/365; 514/367; 514/370; 514/383; 514/395; 514/396; 514/43;
514/44R; 514/526; 514/554; 514/635; 514/86 |
Current CPC
Class: |
A61K 31/155 20130101;
Y02A 50/387 20180101; A61P 31/22 20180101; A61P 31/04 20180101;
Y02A 50/385 20180101; A61P 31/18 20180101; Y02A 50/30 20180101;
C08G 73/00 20130101; A61K 31/785 20130101; A61K 31/155 20130101;
A61K 2300/00 20130101; A61K 31/785 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/85.4 ;
514/635; 514/526; 435/375; 435/366; 435/372.3; 514/370; 514/395;
514/367; 514/275; 514/383; 514/365; 514/311; 514/396; 514/554;
514/263.38; 514/120; 514/86; 514/44.R; 514/43 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 31/275 20060101 A61K031/275; A61P 31/18 20060101
A61P031/18; A61P 31/22 20060101 A61P031/22; C12N 5/071 20100101
C12N005/071; C12N 5/0783 20100101 C12N005/0783; A61K 31/426
20060101 A61K031/426; A61K 31/4184 20060101 A61K031/4184; A61K
31/428 20060101 A61K031/428; A61K 31/505 20060101 A61K031/505; A61K
31/4196 20060101 A61K031/4196; A61K 31/427 20060101 A61K031/427;
A61K 31/47 20060101 A61K031/47; A61K 31/4164 20060101 A61K031/4164;
A61K 31/205 20060101 A61K031/205; A61K 31/522 20060101 A61K031/522;
A61K 31/662 20060101 A61K031/662; A61K 31/675 20060101 A61K031/675;
A61K 31/7125 20060101 A61K031/7125; A61K 31/7056 20060101
A61K031/7056; A61K 31/155 20060101 A61K031/155 |
Claims
1-103. (canceled)
104. A pharmaceutical composition comprising an active ingredient
consisting solely of polyethylene-hexamethylene biguanide (PEHMB)
or a pharmaceutically acceptable salt thereof in a pharmaceutically
acceptable carrier, wherein the PEHMB or salt thereof provides a
reduction in the incidence of in-vitro HIV infection of P4R5 cells
and/or human CD4(+)T-lymphocyte cells which express CD4 receptor
and CXCR4 co-receptor when the PEHMB or salt thereof is tested with
the carrier in an in-vitro viral binding/entry assay.
105. The composition according to claim 104 wherein the
polyethylenehexamethylene biguanide has one or both an amino and a
monocyanoguanidine end group.
106. The composition according to claim 104 wherein the
polyethylenehexamethylene biguanide includes end groups selected
from the group consisting of n-octylamine, n-laurylamine,
2-aminothiazole, 2-aminobenzimidazole, 2-aminobenzothiazole,
2-amino-5-chloropyrimidine, 3-amino-1,2,4-triazole, 2-(4-thiazoyl)
benzimidazole, p-cholorobenzylamine, 2,4-dichloroaniline,
8-aminoquinoline, Imidizole, Premuline, 2-aminopyrimidine,
L-tryptophan, 2-guanidinobenzimidazole and mixtures thereof.
107. The composition according to claim 104 wherein the
pharmaceutically acceptable salt of PEHMB is a lactate or arginate
salt.
108. (canceled)
109. The composition of claim 104 wherein the PEHMB or salt thereof
further provides a Therapeutic Index, TI, in the range from 160 to
1000 when the PEHMB or salt thereof is tested with the carrier in
assays of in-vitro cytotoxicity and in-vitro HIV-1 antiviral
activity using P4R5 cells or T-lymphocyte cells.
110. The composition of claim 104 wherein the PEHMB or salt thereof
further provides a reduction in availability of CXCR4 receptors on
the surface of cells expressing the CXCR4 receptor when tested
in-vitro with the carrier by Flouorescence Activated Cell Sorting
(FACS) .
111. The composition of claim 104 wherein the reduction in the
incidence of in-vitro HIV infection of P4R5 cells and/or human
CD4(+)T-lymphocyte persists for at least 4 hours after the
polyethylenehexamethylene biguanide or salt thereof is removed from
a medium in which the cells reside.
112. A method of treating cells that express a CXCR4 receptor to
reduce the availability of the CXCR4 receptor on the cell surface,
said method comprising exposing the cells to
polyethylenehexamethylene biguanide (PEHMB) at a plasma
concentration of 2-50 moles per liter in the medium in which the
cells reside.
113. The method according to claim 112 wherein the
polyethylenehexamethylene biguanide has one or both an amino and a
monocyanoguanidine end group.
114. The method according to claim 112 wherein the
polyethylenehexamethylene biguanide includes end groups selected
from the group consisting of n-octylamine, n-laurylamine,
2-aminothiazole, 2-aminobenzimidazole, 2-aminobenzothiazole,
2-amino-5-chloropyrimidine, 3-amino-1,2,4-triazole, 2-(4-thiazoyl)
benzimidazole, p-cholorobenzylamine, 2,4-dichloroaniline,
8-aminoquinoline, Imidizole, Premuline, 2-aminopyrimidine,
L-tryptophan, 2-guanidinobenzimidazole and mixtures thereof.
115. The method according to claim 112 wherein the cells expressing
the CXCR4 receptor are human cells.
116. The method according to claim 112 wherein the cells expressing
the CXCR4 receptor are human T-lymphocyte cells.
117. The method according to claim 112 wherein the treatment
further comprises exposing the cells to .beta.-cyclodextrin
(BCD).
118. The method of claim 112 wherein the effective amount of PEHMB
is chosen so as to provides a reduction in availability of the
CXCR4 receptor on the surface of cells when measured in-vitro by
fluorescence activated cell sorting (FACS).
Description
[0001] This is a Continuation of co-pending U.S. patent application
Ser. No. 10/435,756 filed Aug. 27, 2003, incorporated herein in its
entirety, by reference.
[0002] Field of the invention: The present invention relates to
polybiguanide-based compounds and a method for the treatment or
prevention of viral infection using polybiguanide-based
compounds.
PRIOR ART
[0003] Valluri et al. 1997 Cornea 16:556-559
[0004] Stephenson J, 2000 J. Am. Med. Assoc. 287:949
[0005] Stafford, et al. 1998 J. AIDS Hum Retroviruses
17:327-331
[0006] Bratt and Hathway, 1976 Macromolecular Chemistry
177:2591-2605
[0007] U.K. 702,268 (Rose and Swain 11359)
[0008] U.K. 1,152,243 (Dickinson et al. 51469)
[0009] U.K. 1,167,249 (Ambler et al. Oct. 15, 1969)
[0010] U.K. 1,432,345 (Drain et al. Apr. 14, 19 76)
[0011] U.K. 1,531,717 (Buckly et al. Nov. 8, 1978)
[0012] U.S. Pat. No. 4,403,078 (McCoy et al. Sep. 6, 1983)
[0013] U.S. Pat. No. 4,558,159 (McCoy et al. Dec. 10, 1985)
[0014] U.S. Pat. No. 4,891,423 (Stockel Jan. 2, 1990)
[0015] U.S. pat. No. 5,741,886 (Stockel et al. Jul. 21, 1998)
[0016] Patent Application Publication 2003/0032768 A1 (Stockel
2/13/03).
[0017] U.S. Pat. No. 5,013,544 (Chantler and Elstein May 7,
1991)
[0018] Application Published under the PCT, International
Publication Number WO 02/17916 A1 (Shetty Mar. 7, 2002)
[0019] Application Published under the PCT, International
Publication Number WO 00/03682 A2 (Races Jan. 27, 200)
BACKGROUND OF THE INVENTION:
[0020] The name polybiguanide (PBG) is used to describe a diverse
group of polymers containing repeating biguanide groups and may be
linear or branched. In addition the biguanide groups may part of
the main chain or incorporated as side-groups or end-groups in the
compound. PBGs are easily and inexpensively synthesized in large
quantities and the reaction yields stable products with several
potential reactive sites for further modifications. PBGs are made
by the condensation polymerization of a biscyanoguanidine and a
diamino compound. Typically, the final PBG product has amino and
monocyanoguanidine end groups at opposite ends of a cationic
polymer backbone (see FIG. 1). These end groups can be subsequently
reacted with either monofunctional amino- or cyanoguanidine-
containing moieties, respectively (the transferred terminal
moieties are termed end-caps). Also, attaching endcaps to the
polybiguanide chain can be made with other reactive groups as is
known in organic chemistry. In FIG. 1, "X" is the hydrocarbon
segment between the biguanide group introduced from the
biscyanoguanidine monomer and "Y" is the hydrocarbon segment
introduced from the diamino co-monomer. The PBG polymer (FIG. 1)
lends itself to modifications and/or additions, namely: (i) size
and nature of backbone segment "X," (ii) size and nature of
backbone "Y," (iii) terminal amino end-cap, (iv) terminal
cyanoguanidine end-cap, and (v) anions. The synthesized product
possesses a distribution of molecular weights that are readily
separated by HPLC, or other means, to distinct fractions with
definite molecular formulae. PBGs are fully ionized at physiologic
pH. Other synthesis routs may be used to produce the polybiguanide
compounds by one skilled the art of organic and polymer
chemistry.
[0021] Previous polybiguanides have been described in the prior
art, therein there are no modified end-groups, mono end cap
modification and di end cap modification (to the best of our
knowledge). Representative prior art patents comprise UK patents
702,268; 1,152,243; 1,167,249; 1,432,345; and U.S. Pat. Nos.
4,403,078; 4,558,159; 4,891,423; 5,741,886; and patent application
number 2003/0032768 A1.
[0022] Advantages of PBGs as HIV-1 Microbicides
[0023] PBGs are already known for their wide-spectrum
anti-bacterial activity and safety, e.g. as a contact lens
disinfectant for over thirty years (Woodcock P. M. Biguanides as
Industrial Biocides. In: K. R. Payne (ed), Critical Reports on
Applied Chemistry: Industrial Biocides, vol. 23 John Wiley and
Sons, New York). Because of their special biological functions,
these well-established low cellular toxicity compounds have
potential as STD microbicides as well as conventional antiviral
agents. The following features represent important technical and
economic advantages of PBGs that have been noted to date: (i) high
activity against a wide range of organisms even in the presence of
organic matter, (ii) low mammalian toxicity (Bratt and Hathway 1976
Macromolecular Chemistry 177:2591-2605; Jangaard et al. 1968
Diabetes 17:96-104; Czyzyk et al. 1968 Diabetes 17:492-498; ICI
Bulletin Cosmocil CQ-an antimicrobial agent for use in cosmetics
and pharmaceuticals. ICI Americas, Inc.), (iii) absence of odor,
(iv) easy handling and application, (v) chemical stability and
non-volatility, (vi) no surface activity; PBGs are not surfactants,
i.e. they do not lower the surface tension of water or dissolve
cellular membranes like surfactants, (vii) inexpensive, (viii) easy
to prepare, and (ix) greater than 96% non-metabolized (Bratt and
Hathway 1976 Macromolecular Chemistry 177:2591-2605; Jangaard et
al. 1968 Diabetes 17:96-104; Czyzyk et al. 1968 Diabetes
17:492-498). The starting chemicals needed to make PBGs are
commercially available in large quantities and their large-scale
production is straightforward. Because of the exorbitant expense of
antiviral therapy in the developing world, the concept of low cost
antiviral agents to prevent the transmission or to combat existing
infections has emerged as one of the most paramount of needs in the
world today. Effective antiviral agents having different
mechanism(s) of action and low cost or low cost microbicides would
be a highly desirable addition to existing therapies, especially
where female control of STD would help dramatically decrease
transmission.
[0024] Safety of Biguanides and PBGs:
[0025] Extensive toxicological studies, covering different
exposures to tissue targets and pathways, have demonstrated the
safety of PBGs (Bratt and Hathway 1976 Macromolecular Chemistry
177:2591-2605; Jangaard et al. 1968 Diabetes 17:96-104; Czyzyk et
al. 1968 Diabetes 17:492-498). Notably, chlorhexidine gluconate
(CHG), a bis-biguanide, has been used as a general disinfectant for
over thirty years with a high level of safety (Rabe and Hillier
2000 Sex Transm Dis 27:74-78; Shubair et al. 1992 Gynecol Obstet
Invest 34:229-233; Stray-Pederson et al. 1999 Int. J. Antimicrob
Agents 12:245-251). Many reports support the safety of CHG in
gynecology and obstetrics as a vaginal douch or as a pre-delivery
vaginal wash (Shubair et al. 1992 Gynecol Obstet Invest 34:229-233;
Stray-Pederson et al. 1999 Int. J. Antimicrob Agents 12:245-251).
For example, Rabe and Hillier report the use of 0.25% chlorhexidine
gel is safe when used vaginally against chlamydia (Rabe and Hillier
2000 Sex Transm Dis 27:74-78; Patton et al. 1998 Sexually
Transmitted Diseases 25:421-426). With vaginal use, CHG did not
disturb flora with respect to Lactobacilli species (Shubair et al.
1992 Gynecol Obstet Invest 34:229-233). Polymeric PBGs have shown
less corneal toxicity, compared to CHG, especially in contact lens
applications (Woodcock P. M. Biguanides as Industrial Biocides. In:
K.R. Payne (ed), Critical Reports on Applied Chemistry: Industrial
Biocides, vol. 23 John Wiley and Sons, New York). Further,
biguanide-based drugs have excellent safety profiles as an
anti-malaria drugs (Proguanil) (Leggat and Haydon 2002 J. Travel
Med. 9:156-159; Chaulet et al. 2002 Arzeimittelforschung
52:407-412; Croft and Herxheimer 2002 Clin Infect Dis.
discussion:1278-1279) and for treating type 2 diabetes (Metformin)
(Stepensky et al. 2002 Drug Metab. Dispos 30:861-868; Zuhri-Yafi et
al. 2002 J. Pediatri Endocrinol Metab 15 Suppl 1:541-546; Wulffele
et al. 2002 Br J. Clin Pharmacol 53:549P-550P; Melikian et al. 2002
Clin Ther 24:460-467) with a daily dose of 2.5 gm. In addition
Cazzanig et al. (2002 Wounds 14:169-176) and Davis et al. (2002
Wounds 14:252-256), report that polyhexamethylene biguanide can
form a barrier to prevent Pseudomonas wound invasion, while Ansorg
et al. (Chemotherapy 48:129-133) have tested the biguanide poly
hexanide against Stahylococcus aureus in the nasal mucosa. Welk et
al. (J. Clin. Periodontology 29:392-399) used PHMB, which has been
used as an antiseptic for many years, as a mouth rinse at 0.12% and
found it significantly more effective in inhibiting plaque than
placebo. Therefore, the positively charged biguanide class of
compounds have consistently demonstrated safety profiles that
allowed for their use in human studies up to and including
regulatory approvals.
[0026] Virus Inactivation mechanisms of PBGs: PBGs in general are
multi-action compounds that could interfere with virus infection by
interaction at the cellular or viral membrane. Their accepted
biological function, as antibacterial agents, is attributed to
their interaction with cell membranes, specifically anionic
phospholipids and possibly proteins (Woodcock P. M. Biguanides as
Industrial Biocides. In: K. R. Payne (ed), Critical Reports on
Applied Chemistry: Industrial Biocides, vol. 23 John Wiley and
Sons, New York ; Gilbert et al. 1990 J. Appl Bacteriol 69:585-592;
Broxton et al. 1984 J. Applied Bacteriol 57:115-124; Broxton et al.
1983 J. Appl Bacteriol 54:345-353; Gilbert et al. 1990 J. Appl
Bacteriol 69:593-598; Broxton et al. 1984 Microbios 41:15-22).
[0027] FIG. 2 depicts the multi-level functions of PBGs as
potential antiviral microbicides. First, due to their cationic
nature, PBGs could retard the movement of virions by binding to
their negatively charged surfaces before they reach the cell
surfaces. Second, PBGs could inhibit cell-free and cell-associated
virus by interacting with viral envelope lipids or negatively
charged viral proteins or with low affinity cell surface receptors
in a non-specific fashion. Third, PBGs could effect cross-linking
of sialic acid groups of mucin and increase its viscosity and
ability to function as a physical barrier to prevent infective
agents from reaching the epithelium. Fourth, PBGs could bind to
acidic phospholipids causing changes in lipid and protein
distribution in the cell and viral membranes with the result of
inhibiting viral infectivity, possibly due to dislocation or
conformational changes in viral receptors or co-receptors or due to
inhibiting the fusion step. Fifth, PBGs could bind specifically to
high affinity virus receptors on the cell surface and therefore
inhibit virus attachment, and/or fusion.
[0028] To further illustrate this last point we will present data
in the examples section of this patent application that shows PBG
compounds that have specific interactions with the HIV-1
co-receptors CXCR4 and CCR5. These data taken together with the
recent reports of positively charged peptides binding to CXCR4 and
inhibiting T cell line tropic strains of HIV (De Clercq, E. 2002
New Anti-HIV Agents and Targets. Medicinal Research Reviews
22:531-565), suggests that a similar mechanism of action is part of
the polybiguanide spectrum of antiviral activity. The chemical
nature of PBGs with variation in the length of the backbone linkers
(X and Y in FIG. 1A) may allow for the formation of a defined
three-dimensional structure that together with the positive charge
characteristics of the PBG class of molecules could lead to a
defined, specific mechanism of action such as that observed for the
positively charged peptides (De Clercq, E. 2002 New Anti-HIV Agents
and Targets. Medicinal Research Reviews 22:531-565). In addition,
B. V. Shetty has disclosed a series of guanidine or biguanide
compounds with antiviral and antimicrobial activity (Shetty
Application Published under the PCT, International Publication
Number WO 02/17916 A1, Mar. 7, 2002). It is apparent from the work
of Shetty and others that positively charged compounds can be
developed as antiviral agents with specific molecular targets.
[0029] Persistence--importance of PBGs binding ability: Due to
their cationic nature, selected PBGs are expected to strongly
interact with both free virus and cell surfaces due to the strong
electrostatic interaction between PBGs and anionic phospholipid
groups. We predict that these strong electrostatic interactions
will ensure that dilution or washing do not readily reverse PBG
binding in the vaginal environment. To minimize the effect of the
cationic charge of PBGs on the overall cellular sensitivity,
several strategies have been identified, including: modulating the
charge density by inserting special moieties in the backbone chain,
altering the pKa of the biguanide group (e.g., by substituting it
with amidine, pKa=9.5 or guanidine, pKa=13.0), tailoring the chain
length or end-caps and selecting their chemistry and optimizing the
anion conjugated with the PBG cation. The ability to design PBGs
with vastly different physical characteristics led to the
identification of PEHMB (PBG in which X=2 carbon atoms and Y=6
carbon atoms in FIG. 1A) which is more potent and less toxic than
all others in this class of compounds tested to date. The reason
for this may lie in the nature of the three dimensional structure
of PEHMB which we believe imparts on the molecule a degree of
specificity in its mechanism of action. Our preliminary data
indicates that of all the PBGs tested at least PEHMB interacts with
cellular receptors in a specific fashion therefore, we can
theorize, this specificity imparts on PEHMB a superior antiviral
profile and reduced cellular toxicity with respect to other members
of this class of molecule.
[0030] The present invention relates to compositions and methods
for inhibiting the transmission of enveloped viruses such as
alphavirus, herpes viruses (e.g. HSV-1 to HSV-8, cytomegalovirus,
varicella zoster, Epstein Bar Virus, etc.), rhabdoviruses,
orthomyxoviruses (e.g. influenza), retroviruses (e.g. human
immunodeficiency virus type 1, HIV-1), flaviviridae (e.g. Hepatitis
C, West Nile, Dengue, and yellow fever viruses), and Pox viruses
(e.g. smallpox, and vaccinia viruses).
[0031] Human immunodeficiency virus type 1 (HIV-1), a member of the
retrovirus family, is the causative agent in the development of
acquired immune deficiency syndrome (AIDS). This condition is a
catastrophic, fatal disease that presently infects millions of
people worldwide. Major efforts are being made to develop novel
antiviral agents with unique mechanisms of action to be used in
drug therapy and on methods of preventing the transmission of
HIV-1, methods of curing the AIDS disease state once contracted,
and methods of ameliorating the symptoms of AIDS.
[0032] Despite almost 20 years of AIDS/HIV-1 prevention efforts and
research, the sexually transmitted HIV-1 epidemic continues to be a
major health problem throughout the world and is accelerating in
many areas. To date the HIV epidemic has infected over 42 million
people predominantly through sexual intercourse at the end of 2002.
Of these there has been 3.1 cumulative deaths from the disease
worldwide (from the Joint United Nations Program on HIV/AIDS and
the World Health Organization's AIDS Epidemic Update Report,
December 2002).
[0033] Virtually all the compounds that are currently used or are
the subject of advanced clinical trials for the treatment of HIV
infections belong to one of the following classes:
[0034] 1) Nucleoside analogue inhibitors of reverse transcriptase
functions.
[0035] 2) Non-nucleoside analogue inhibitors of reverse
transcriptase functions
[0036] 3) HIV-1 Protease inhibitors.
[0037] 4) Virus fusion inhibitors (the 36 amino acid fusion
inhibitor T20 has been approved for sale by the FDA)
[0038] The HIV-1 replication cycle can be interrupted at many
different points. As indicated by the approved medications the
viral reverse transcriptase and protease enzymes are good molecular
targets as is the entire process by which the virus fuses to and
injects itself into host cells. Thus the recently approved drug T20
is the first in a novel class of anti-HIV-1 agents. However, in
addition to the drugs already approved for treatment of HIV-1
infection, work continues on the discovery and development of
additional treatment modalities because of the virus's propensity
to mutant and thus renders ineffective the existing therapies.
[0039] At present combination therapy comprising at least three
anti-HIV drugs has become the standard treatment for HIV infected
patients. Virtually all drugs that have been licensed for clinical
use for the treatment of HIV infection fall into one of the four
categories listed above, comprising three molecular targets.
However one problem with current therapy is the cost associated
with the need to use multiple drugs used in combination. Estimates
of $15000 to $20000 U.S. per year per person are close
approximations. This cost makes it virtually impossible for many
people to afford combination therapy, especially in developing
nations where the need is greatest. Another problem with existing
therapeutic regimens, as stated above, is the ability of the virus
to develop resistance to the individual medications and many times
to develop resistance to the combination therapy. This works
against the population in two ways. First, the individual infected
will eventually run out of treatment options and second, if the
infected individual passes along a virus already resistant to many
existing therapeutic agents, the newly infected individual will
have a more limited treatment option than the first. Therefore, the
need for new, improved and hopefully inexpensive medications is
evident.
[0040] Most importantly in the search for new medications to combat
the spread of the HIV-1 virus is the search for chemotherapeutic
interventions that work by novel mechanism(s) of action. Several
potential areas for intervention that are under consideration or
have active programs in include 1) blocking the viral envelope
glycoprotein gp120, 2) additional mechanisms beyond gp120 to block
virus entry such as blocking the virus receptor CD4 or co-receptors
CXCR4 or CCR5, 3) viral assembly and disassembly through targeting
the zinc finder domain of the viral nucleo capsid protein 7 (NCp7)
and 4) by interfering with the functions of the viral integrase
protein, and by interruption of virus specific transcription
processes.
[0041] Vaginal contraceptive products have been available for many
years and usually contain nonoxynol-9 or other detergent/surfactant
as the active ingredient that are toxic to cell membranes. However
frequent use of N-9 causes irritation and inflammation of the
vagina (M. K. Stafford et al. J. AIDS human retrovirology, 1998
17:327-331). N-9 is also toxic to vaginal and cervical cells
increasing the permeability of vaginal tissue, and can inactivate
lactobacilli. Lactobacilli produce lactic acid and hydrogen
peroxide that serve to maintain the acidic pH of the vagina
(.about.pH 3.5 to 5.0). At this pH, a number of sexually
transmitted disease (STD) causing organisms like HIV, and
spermatozoa are inactivated. Disturbance of the vaginal microbial
flora can lead to vaginal infections, which in turn increase the
chance of HIV/STD transmission. The most recent data (Stephenson,
J. Am. Assoc. 2000, page 284-949) in which a topical formulated N-9
product strongly suggest that the compound may even enhance HIV
transmission.
[0042] Therefore it is extremely important to identify and evaluate
new contraceptive antimicrobial agents, microbicides, which can be
used vaginally in effective doses without inactivating lactobacilli
or causing overt vaginal irritation or other toxicity.
[0043] A successful microbicide should (i) be effective against
infection caused by cell-free and cell-associated virus, (ii)
adsorbs tightly with its molecular target(s), i.e., its adsorption
should not be reversed by dilution or washing, (iii) permanently
"inactivate" the virus, (iv) inactivate free virus and infected
cells faster than their rate of transport through the mucus layer,
(v) have persistent activity for more than one episode of coitus,
(vi) be safe to host cells and tissues--causing no irritation or
lesions, (vii) be easy to formulate, (viii) remain stable in the
formulated state, (ix) not activate mucosal immunity, (x) retard
transport in mucus and entire vaginal and rectal mucosa, and (xi)
be inexpensive for worldwide application.
[0044] Current HIV-1 microbicide candidates fall into two
categories--either surfactants or polyanionic compounds (Pauwels
and De Clercq 1996 J. AIDS Hum Retroviruses 11:211-221;
Recommendations for the development of vaginal microbicides. 1996
International Working Group on Vaginal Microbicies AIDS 10:1-6).
However, these proposed agents may not satisfy all of the necessary
criteria for a successful microbicide as mentioned above. In
addition, most of the compounds under current investigations as
microbicides are non-specific and emerged from either excipients or
related compounds used in conventional topical formulations --
almost none of the compounds used have definite chemical formulae,
and many are based on natural or synthetic water-soluble polymers.
For example, despite the effectiveness of N-9 with respect to HIV-1
inactivation in vitro, its failure to effectively prevent HIV-1
infection in vivo has been attributed to its high irritation
profile and indiscriminate disruption of epithelial cells (Feldblum
et al. 1986 N.C. Med J. 47:569-572; Alexander, 1990 WHO Global
Programme on AIDS Fertil Steril. 54:1-18; Niruthisard et al. 1991
Sex Transm Dis 18:176-179; Roddy et al. 1993 J STD AIDS 4:165-170;
Kreiss et al. JAMA 1992 268:477-482). In order to satisfy the
diverse criteria stated above, the target molecule needs to be
custom tailored to provide several functions at the same time. The
ability to manipulate by synthetic means the molecular structure of
the current classes of agents under investigation as potential
microbicides (such as N-9 or C31G surfactants, or sulfated
polysaccharides) is limited, or in some cases even impossible. In
contrast polybiguanide-based molecules provide a wealth of
possibilities with respect to targeted synthetic manipulation.
These compounds are safe, inexpensive, and highly effective
anti-HIV-1 microbicides that can be synthesized with or without
spermicidal activity.
[0045] Herpes viruses are another class of virus that like HIV-1
develop resistance to existing therapy, and can cause problems from
a STD (especially Herpes simplex virus type 2, HSV2) as well as a
chronic infection point of view. For example human cytomegalovirus
(HCMV) is a serious, life threatening opportunistic pathogen in
immuno compromised individuals such as AIDS patients (Macher et al.
1983 NEJM 309:1454;Tyms et al. 1989 J Anitmicrob Chemother
23:89-105) or organ transplant recipients (Meyers, J. D., 1991 Am
J. Med. 81:27-38). Over the past decade there has been a tremendous
effort dedicated to improving the available treatmes for herpes
viruses. At the present time acyclovir is still the most prescribed
drug for HSV1 and HSV2, while for HCMV ganciclovir, foscarnet,
cidofovir, and fomivirsen are the only drugs currently available
(Bedard et al. 2000 Antimicrobial Agents and Chemotherapy
44:929-937). None of the current treatments for herpesviruses are
effective at preventing the sexual transmission of the viruses
therefore there is still an urgent need for new drugs that have
unique mechanisms of action and modes of therapeutic
intervention.
SUMMARY OF THE INVENTION:
[0046] The present invention provides compounds of Formula I: And
pharmaceutically acceptable salts or formulations thereof,
wherein:
[0047] It can be deduced from this generalized formula that there
are five different parts of the macromolecule where modifications
can be performed: [0048] (a) "Z.sup.-" is an anion where said anion
is a halide, carboxylate, hydroxy carboxylate, amino carboxylate,
organophosphate, organophosphonate, organosulfonate, or
organosulfate [0049] (b) "A" is an amino end group while "B" is a
cyanoguanidine group which can be reacted with the corresponding
monoanimo (reacts with the cyanoguanidino end group), or
monocyanoguanidine (reacts with cyanoguanidine end group). The
monoamino or monocyanoguanidino end group modifiers can be
aliphatic, cycloaliphatic, heterocyclic, aryl, alkaryl, aralkyl,
and oxyalkylene radicals. [0050] (c) X and Y can be the same or
different organic radical bridging groups. Suitable examples of the
organic radicals represented by X and Y include C2 to C140,
aliphatic, cycloaliphatic heterocyclic, aryl, alkaryl, aralkyl, and
oxyalkylene radicals. X and/or Y can also be polyalkylene radical
optionally interrupted by oxygen, nitrogen, or sulfur atoms, or by
saturated or unsaturated cyclic nuclei. [0051] (d) The number of
repeat units for the core biguanide (n) can be 1 to 100.
##STR00001##
[0052] The present invention provides a method for treating or
preventing a viral infection in a host comprising administering a
therapeutically effective amount of a compound having the Formula
I.
[0053] In another aspect, there is provided a pharmaceutical
formulation comprising the compounds of the invention in
combination with a pharmaceutically acceptable carrier or
excipient.
[0054] Still another aspect, there is provided a method for
treating or preventing a viral infection in a host comprising
administering to the subject a combination comprising at least one
compound according to Formula I and at least one further
therapeutic agent.
[0055] In another aspect of the invention is the use of a compound
according to Formula I, for the preparation of a medicament for
treating or preventing viral infections in the host.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1. PBG structures and synthesis strategies. A schematic
depicting: (A) general structure of a PBG backbone with cationic
biguanide groups (rectangles) separated by methylenic linker or
spacers (lines); (B) formula of PHMB chloride (linkers are
hexamethylene) and (C) optimized PEHMB backbone (alternating di-
and hexamethylene linkers). R1 and R2 represent two end-caps
although the end caps can be the same or different. The counter
anion is not shown in B or C.
[0057] FIG. 2. PBGs have many potential sites of action. Potential
anti-viral activities of PBGs include:
[0058] (i) retardation of virion and infected cell movement before
reaching the epithelium,
[0059] (ii) inactivation of the virus before it reaches the cell
surfaces,
[0060] (iii) electrostatic cross-linking of mucus with PBGs to form
a viscous barrier over on the surface of the epithelium, and
[0061] (iv) interference with the viral binding and entry events by
interacting with viral receptors/co-receptors (or inhibiting
fusion).
[0062] FIG. 3. Effect of end cap moiety on cellular toxicity and
antiviral profile of PEHMB (A) P4R5 cell viability was assessed by
MTT assay after 2 hr exposures to the indicated compounds.
[0063] (B) Inhibition of viral binding was assessed after
incubation of P4R5 cells with HIV-1IIIB and the
[0064] indicated compounds for 2 hr at 37.degree. C.
[0065] FIG. 4. A combination of PEHMB and BCD is able to inhibit
HIV-1 binding and entry. (A) P4R5 cell viability was assessed by
MTT assay after 2 hr exposures to the indicated compounds or
compound combinations. The b-BCD combination contained 0.1% PEHMB
combined with the indicated concentrations of BCD; PEHMB contained
0.3% BCD with varied amounts of PEHMB. (B) Inhibition of viral
binding was assessed after incubation of P4R5 cells with HIV-1IIIB
and the PEHMB (0.1%), BCD (5 mM or approximately 0.6%), or PEHMB
(0.1%)
[0066] FIG. 5. PEHMB protection from HIV-1 infection persists for
at least 4 hr after the compound has been removed from the media.
(A) P4R5 target cells were preincubated with PEHMB (0.1%) or
dextran sulfate (DS; 0.1%) for 2 hr at 37.degree. C., washed three
times with PBS, and challenged with HIV-1IIIB (1:250, ABI) 0, 0.5,
1, 2, 3, or 4 hr after the cells were washed. Control wells were
infected for 2 hr in the absence of compound, or with the
simultaneous addition of PEHMB or DS. (B) The reduction in
infectivity as a function of time.
[0067] FIG. 6 HIV-1 co-receptor CXCR4 availability on the cell
surface is reduced in the presence of PEHMB and a combination of
PEHMB and BCD. P4-R5 cells were incubated in the absence or
presence of PEHMB (0.1%), BCD (0.3%), or a mixture of both
compounds (at the afore mentioned concentrations) for 2 hr at
37.degree. C. and subsequently analyzed by FACS analysis using an
antibody to CXCR4. (A) untreated; (B) PEHMB-treated; (C)
BCD-treated; (D) PEHMB/BCD treated.
[0068] FIG. 7 HIV-1 co-receptor CXCR4 availability on the cell
surface is reduced in the presence of PEHMB and a combination of
PEHMB and BCD. PM-1 cells were incubated in the absence or presence
of PEHMB (0.1%), BCD (0.3%), or a mixture of both compounds (at the
afore mentioned concentrations) for 2 hr at 37.degree. C. and
subsequently analyzed by FACS analysis using an antibody to CXCR4.
(A) untreated; (B) PEHMB-treated; (C) BCD-treated; (D) PEHMB/BCD
treated.
[0069] FIG. 8. Detection of CCR5 is increased by exposure of PM1
cells to PEHMB. PM-1 T cells wee incubated in the presence or
absence of PEHMB (0.1% for 2 hr at 37.degree. C.) and then analysed
by FACS analysis using an anti-CCR5 antibody (R&D Systems
Fab183F) either immediately (0 hr) or 2 hr post compound removal
from culture media. The inset depicts the 0 hr analyses using
untreated or treated cells an IgG2B isotype antibody
DETAILED DESCRIPTION OF THE INVENTION:
[0070] In one embodiment of the invention the viral infection is
selected from the group consisting of retrovirus infections.
[0071] In one embodiment, the retrovirus infection is selected from
the group consisting of human immunodeficiency virus types 1 and
2.
[0072] In another embodiment, the retrovirus infection is human
immunodeficiency virus type 1 (HIV-1).
[0073] In one embodiment of the invention the viral infection is
selected from the group consisting of herpes virus infections.
[0074] In another embodiment, the herpes virus is selected from the
group consisting of herpes simplex virus type 1 and herpes simplex
virus type 2.
[0075] In another embodiment, the herpes virus is herpes simplex
virus type 2 (HSV2).
[0076] In one embodiment, the compounds and methods of the present
invention comprise those wherein the following embodiments are
present, either independently or in combination:
[0077] In one aspect of the present invention, X in Formula I is an
organic bridging group such as an aliphatic group containing 2 to
140 carbon atoms.
[0078] In another aspect X is C.sub.2 to C.sub.140 cycloaliphatic,
heterocyclic, aryl, alkaryl, aralkyl, or oxyalkylene radicals.
[0079] In another aspect X can be a polyalkylene radical optionally
interrupted by oxygen, nitrogen, or sulfur atoms, or by saturated
or unsaturated cyclic nuclei.
[0080] In one aspect of the present invention, Y in Formula I is
the same or different organic bridging group as X, such as an
aliphatic group containing 2 to 140 carbon atoms.
[0081] In another aspect Y in Formula I is the same or different as
X, and consists of C2 to C140 cycloaliphatic, heterocyclic, aryl,
alkaryl, aralkyl, or oxyalkylene radicals.
[0082] In another aspect Y in Formula I can be a polyalkylene
radical optionally interrupted by oxygen, nitrogen, or sulfur
atoms, or by saturated or unsaturated cyclic nuclei, and can be the
same or different as X.
[0083] In another aspect the number of biguanide repeat units (n in
Formula I) can be one to one hundred.
[0084] In another aspect of the present invention the anion used to
form the PBG salt (Z in Formula I) could be drawn from any one or a
combination of the following groups that includes halides,
carboxylic acids, hydroxy carboxylic acids, amino acids, sulfonic
acids, phosphonoic acids or phosphates.
[0085] In a further embodiment, the present invention relates to a
method for the treatment or prevention of virus infections using
polybiguanide-based compounds administering a therapeutically
effective amount of a compound having the Formula I or a
pharmaceuticaly acceptable salt thereof:
##STR00002##
[0086] wherein A, B, n, X, Y, and Z.sup.- are as defined above.
[0087] In a further embodiment, the present invention relates to a
method for the treatment or prevention of virus infections using
polybiguanide-based compounds administering a therapeutically
effective amount of a compound having the Formula I or a
pharmaceutically acceptable salt thereof, wherein the virus is
selected from the group of retroviruses.
[0088] In a further embodiment, the present invention relates to a
method for the treatment or prevention of virus infections using
polybiguanide-based compounds administering a therapeutically
effective amount of a compound having the Formula I or a
pharmaceutically acceptable salt thereof, wherein the virus is the
human immunodeficiency virus type 1 (HIV-1).
[0089] In a further embodiment, the present invention relates to a
method for the treatment or prevention of virus infections using
polybiguanide-based compounds administering a therapeutically
effective amount of a compound having the Formula I or a
pharmaceutically acceptable salt thereof, wherein the virus is
selected from the group of herpesviruses.
[0090] In a further embodiment, the present invention relates to a
method for the treatment or prevention of virus infections using
polybiguanide-based compounds administering a therapeutically
effective amount of a compound having the Formula I or a
pharmaceuticaly acceptable salt thereof, wherein the virus is
herpes simplex virus type 2 (HSV2).
[0091] There is also provided pharmaceutically acceptable salts of
the compounds of Formula of the present invention. By the term
pharmaceutically acceptable salts of the compounds of Formula I are
meant those derived from pharmaceutically acceptable inorganic and
organic acids. Examples of suitable acids include halides,
carboxylic acids, hydroxy carboxylic acids, amino acids, sulfonic
acids, phosphonoic acids or phosphates.
[0092] As used in this application, the term "alkyl" represents an
unsubstituted or substituted (by a halogen, nitro, CONH.sub.2,
COOH, O--C.sub.1-6, alkyl, O--C.sub.2-6, alkynyl, hydroxyl, amino,
or COOQ, wherein Q is C.sub.1-6, alkyl; C.sub.2-6, alkenyl;
C.sub.2-6alkynyl) straight chain, branched chain or cyclic
hydrocarbon moiety (e.g. isopropyl, ethyl, fluorhexyl, or
cyclopropyl). The term alkyl is also meant to include alkyls in
which one or more hydrogen atoms are replaced by an halogen, more
preferably, the halogen is fluoro.
[0093] The terms "alkenyl" and "alkynyl" represent an alkyl
containing at least one unsaturated group (e.g. allyl).
[0094] The term hydroxy protecting group" is well known in the
field of organic chemistry. Such protection groups may be found in
T. Greene, Protective Groups in Organic Synthesis, (John Wiley and
Sons, 1981). Examples of hydroxy protecting groups include but are
not limited to acetyl-2-thioethyl ester, pivaloyloxymethyl ester
and isopropyloxycarbonyloxymethyl ester.
[0095] The term "aryl" represents an unsaturated carbocyclic
moiety, optionally mono- or di-substituted with OH, SH, amino,
halogen, or C.sub.1-6 alkyl.
[0096] The term "heteroaryl" represents an aryl wherein at least
one carbon ring atom is substituted by an heteroatom (e.g. N, 0, or
S).
[0097] The term "aminoalkyl" represents an alkyl which is
covalently bonded to the adjacent atom through a nitrogen atom.
[0098] The term "thioalkyl" represents an alkyl which is covalently
bonded to the adjacent atom through a sulfur atom.
[0099] The term "alkoxy" represents an alkyl which is covalently
bonded to the adjacent atom through an oxygen atom.
[0100] Halogens are chosen from F, Cl, I, and Br.
[0101] The term "host" represents any mammals including humans.
[0102] In one embodiment, the host is human.
[0103] The compounds of the present invention can be prepared by
methods well know in the art. Previously polybiguanides have been
described in the prior art wherein there are no modified
end-groups, mono- or di- end cap modification (to the best of our
knowledge). Representative prior art patents comprise UK patents
702,268; 1,152,243; 1,167,249; 1,432,345; 1,531,717; U.S. Pat. Nos.
4,403,078; 4,558,159; 4,891,423; 5,741,886; and patent application
publication 2003/0032768 A1.
[0104] According to one embodiment, it will be appreciated that the
amount of a compound of Formula I of the present invention required
for use in treatment will vary not only with the particular
compound selected but also with the route of administration, the
nature of the condition for which treatment is required and the age
and condition of the patient and will be ultimately at the
discretion of the attendant physician or veterinarian. In general
however a suitable dose will range from about 0.01 to about 750
mg/kg of body weight per day, preferably in the range of 0.5 to 60
mg/kg/day, most preferably in the range of 1 to 20 mg/kg/day for
systemic administration, or for topical applications a suitable
dose will range from about 0.001 to 5% wt/vol, preferably in the
range of 0.1 to 1% wt/vol of formulated material.
[0105] The desired dose according to one embodiment is conveniently
presented in a single dose or as a divided dose administered at
appropriate intervals, for example as two, three, four or more
doses per day.
[0106] In another embodiment, the compound is conveniently
administered in unit dosage from; for example containing 10 to 1500
mg, conveniently 20 to 1000 mg, most conveniently 50 to 700 mg of
active ingredient per unit dosage form.
[0107] According to another embodiment of the present invention,
the active ingredient is administered to achieve peak plasma
concentrations of the active compound of from about 1 to about 75
uM, preferably about 2 to 50 uM, most preferably 3 to 30 uM. This
may be achieved, for example, by the intravenous infection of a 0.1
to 5% solution of the active ingredient, optionally in saline, or
orally administered as a bolus containing about 1 to about 500 mg
of the active ingredient. Desirable blood levels may be maintained
by a continuous infusion to provide about 0.01 to about 5.0
mg/kg/hour or by intermittent infusions containing about 0.4 to
about 15 mg/kg of the active ingredient.
[0108] While it is possible that for use in therapy a compound of
Formula I of the present invention may be administered as the raw
chemical, it is preferable according to one embodiment of the
invention, to present the active ingredient as a pharmaceutical
formulation. The embodiment of the invention thus further provides
a pharmaceutical formulation comprising a compound of Formula I or
a pharmaceutically acceptable salt thereof together with one or
more pharmaceutically acceptable carriers thereof and, optionally,
other therapeutic and/or prophylactic ingredients. The carrier(s)
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0109] According to one embodiment of the present invention,
pharmaceutical formulations include but are not limited to those
suitable for oral, rectal, nasal, topical, (including buccal and
sub-lingual), transdermal, vaginal or parenteral (including
intramuscular, sub-cutaneous and intravenous) administration or in
a form suitable for administration by inhalation or insufflation.
The formulations may, where appropriate, be conveniently presented
in discrete dosage units and may be prepared by any of the methods
well known in the art of pharmacy. All methods according to this
embodiment include the steps of bringing into association the
active compound with liquid carriers or finely divided solid
carriers or both and then, if necessary, shaping the product into
the desired formulation.
[0110] According to another embodiment, pharmaceutical formulations
suitable for oral administration are conveniently presented as
discrete units such as capsules, cachets or tablets each containing
a predetermined amount of the active ingredient, as a powder or
granules. In another embodiment, the formulation is presented as a
solution, a suspension or as an emulsion. In still another
embodiment, the active ingredient is presented as a bolus,
electuary or paste. Tablets and capsules for oral administration
may contain conventional excipients such as binding agents,
fillers, lubricants, disintegrants, or wetting agents. The tablets
may be coated according to methods well know in the art. Oral
liquid preparations may be in the form of, for example aqueous or
oily suspensions, solutions, emulsions, syrups or elixirs, or may
be presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may contain
conventional additives such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives.
[0111] The compounds in Formula I according to an embodiment of the
present invention are formulated for parenteral administration
(e.g. by injection, for example bolus injection or continuous
infusion) and may be presented in unit dose form in ampoules,
pre-filled syringes, small volume infusion or in multi-dose
containers with an added preservative. The compositions may take
such forms as suspensions, solutions, emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively, the active
ingredient may be in powder form, obtained by aseptic isolation of
sterile solid or by lyophilisation from solution, for constitution
with a suitable vehicle, e.g. sterile, pyrogen-free water, before
use.
[0112] For topical administration to the epidermis (mucosal or
cutaneous), the compounds of Formula I, according to one embodiment
of the present invention, are formulated as ointments, creams or
lotions, or as transdermal patch. Such transdermal patches may
contain penetration enhancers such as linalool, carvacrol, thymol,
citral, menthol, and t-anethole. Ointments and creams may, for
example, be formulated with an aqueous or oily base with the
addition of suitable thickening and/or gelling agents. Lotions may
be formulated with an aqueous or oily base and will in general also
contain one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents.
[0113] Pharmaceutical formulations suitable for topical
administration in the mouth include lozenges comprising active
ingredient in a flavored base, usually sucrose and acacia or
tragacanth; pastilles comprising the active ingredient in an inert
base such as gelatin and glycerin or sucrose and acacia; and
mouthwashes comprising the active ingredient in a suitable liquid
carrier.
[0114] In another embodiment of the present invention a
pharmaceutical formulation suitable for rectal administration
consists of the active ingredient and a carrier wherein the carrier
is a solid. In another embodiment, they are presented as unit dose
suppositories. Suitable carriers include cocoa butter and other
materials commonly used in the art, and the suppositories may be
conveniently formed by admixture of the active compound with the
softened or melted carrier(s) followed by chilling and shaping in
moulds.
[0115] According to one embodiment, the formulations suitable for
vaginal administration are presented as pessaries, tampons, creams,
gels, pastes, foams, or sprays containing in addition to the active
ingredient such carriers as are known in the art to be
appropriate.
[0116] For intra-nasal administration the compounds, in one
embodiment of the invention, are used as a liquid spray or
dispersible powder or in the form of drops. Drops may be formulated
with an aqueous or non-aqueous base also comprising one or more
dispersing agent, solubilising agent, or suspending agent. Liquid
sprays are conveniently delivered from pressurized packs.
[0117] For administration by inhalation the compounds, according to
one embodiment of the invention are conveniently delivered from an
insufflator, nebulizer or pressurized pack or other convenient
means of delivering an aerosol spray. In another embodiment,
pressurized packs comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
another embodiment, the dosage unit in the pressurized aerosol is
determined by providing a valve to deliver a metered amount.
Alternatively, in another embodiment, for administration by
inhalation or insufflation, the compounds of Formula I according to
the present invention are in the form of a dry powder composition,
for example a powder mix of the compound and a suitable powder base
such as lactose or starch. In another embodiment, the powder
composition is presented in unit dosage form in, for example,
capsules or cartridges or e.g. gelatin or blister packs from which
the powder may be administered with the aid of an inhalator or
insufflator.
[0118] In one embodiment, the above-described formulations are
adapted to give sustained release of the active ingredient.
[0119] The compounds of the invention may also be used in
combination with other antiviral agents.
[0120] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chose
from a list that includes but is not limited to antiviral protease
inhibitors, polymerase inhibitors, virus/cell fusion inhibitors,
integrase inhibitors, virus/cell binding inhibitors, helicase
inhibitors, and/or virus binding inhibitors.
[0121] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst agents approved for use in humans by government
regulatory agencies.
[0122] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst approved HIV-1 reverse transcriptase inhibitors, HIV-1
protease inhibitors, HIV-1 fusion inhibitors.
[0123] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst HIV-1 reverse transcriptase inhibitors, HIV-1 protease
inhibitors, HIV-1 fusion inhibitors, HIV-1 binding inhibitors.
[0124] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst herpes virus DNA polymerase inhibitors, herpes virus
protease inhibitors, herpes virus fusion inhibitors, herpes virus
binding inhibitors, and ribonucleotide reductase.
[0125] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst HSV DNA polymerase inhibitors, HSV protease
inhibitors, HSV fusion inhibitors, HSV binding inhibitors, and
ribonucleotide reductase inhibitors.
[0126] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from amongst acyclovir, ganciclovir, foscarnet, cidofovir, and
fomivirsen.
[0127] In one embodiment, the compounds of the invention may be
employed together with at least on other antiviral agent chosen
from Interferon and Ribavirin.
[0128] In one embodiment, the compounds of the invention may be
employed together with at least on other antiviral agent chosen
from Interferon-a and Ribavirin.
[0129] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation and
thus pharmaceutical formulations comprising a combination as
defined above together with a pharmaceutically acceptable carrier
therefore comprise a further aspect of the invention.
[0130] The individual compounds of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical formulations.
[0131] When the compound of Formula I or a pharmaceutically
acceptable salt or modification thereof is used in combination with
a second therapeutic agent active against the same virus the dose
of each compound may either be the same as or differ from that when
the compound is used alone. Appropriate doses will be readily
appreciated by those skilled in the art.
[0132] The following examples are provided to illustrate various
embodiments of the present invention and shall not be considered as
limiting in scope.
EXAMPLES
Example 1
Synthesis of Biguanide Compounds
[0133] The PBGs of this invention are readily prepared by reacting
biscyanoguanides with diamino compounds in the presence of
sufficient protic acids to form the polymer carried out in the neat
or by using suitable solvent. The end group modifications (mono or
di types) can be accomplished either by a post reaction after the
initial polymer is formed, or simultaneously during the formation
of the polybiguanide. All these syntheses are described in British
patents numbered 1,167,249; and 1,531,717; and U.S. Pat. Nos.
4,891,423; 5,741,886; and patent application publication
2003/0032768 A1. The synthesis of mono end-capped PBGs is readily
described in U.S. Pat. No. 5,741,886 while that of di-end capped
PBGs is described in US 2003/0032768 A1.
[0134] The present invention provides compounds of formula (I):
##STR00003##
[0135] The first line of the equation describes the synthesis of
the biscyanoguanidine reactant, while the second shows the reaction
of the diamino compound with the biscyanoguanidine compound to form
the polybiguanide product as shown as Formula I. The compound in
Formula I is referred to as end capped polybiguanide (PBG).
[0136] It can be deduced from this generalized formula that there
are five different parts of the macromolecule where modifications
can be performed: [0137] (e) "Z.sup.-" is an anion where said anion
is a halide, carboxylate, hydroxy carboxylate, amino carboxylate,
organophosphate, organophosphonate, organosulfonate, or
organosulfate. [0138] (f) "A" is an amino end group while "B" is a
cyanoguanidine group which can be reacted with the corresponding
monoanimo (reacts with the cyanoguanidino end group), or
monocyanoguanidine (reacts with cyanoguanidine end group). The
monoamino or monocyanoguanidino end group modifiers can be
aliphatic, cycloaliphatic, heterocyclic, aryl, alkaryl, aralkyl,
and oxyalkylene radicals. [0139] (g) X and Y can be the same or
different organic radical bridging groups. Suitable examples of the
organic radicals represented by X and Y include C2 to C140,
aliphatic, cycloaliphatic heterocyclic, aryl, alkaryl, aralkyl, and
oxyalkylene radicals. X and/or Y can also be polyalkylene radical
optionally interrupted by oxygen, nitrogen, or sulfur atoms, or by
saturated or unsaturated cyclic nuclei. [0140] (h) The number of
repeat units for the core biguanide (n) can be 1 to 100.
[0141] The compositions and method of preparation of the
polybiguanides described in the sited prior art patents are thereby
incorporated in the body of this invention. During our studies
involving PBGs as microbicides we found that the counter ion
(anion) can play an important part in the overall efficacy of the
positively charged PBG. Fortunately it is quite easy to carry out
the exchange of anions either by using an anion exchange resin, or
by using the corresponding conjugate acid of the anion in the
original synthesis when reacting the diamino reactant with the
dicyanoguanidine in the presence of the desired conjugate acid,
either neat or in a suitable solvent provided it has a proton which
is sufficiently acidic with a pKa of about 5.0 or less. Further the
anion replacement of compounds represented by Formula I can be
exchanged by precipitating the free base by adding an alkali
hydroxide and then neutralizing the resulting free base with the
corresponding acid that carries the desired anion.
[0142] To illustrate the versatility of end-capping Table 1 lists
amines, which can also represent cyanoguanides as modifiers.
TABLE-US-00001 TABLE 1 Common End cap modifications for
polybiguanide compounds. Monoamine Abbreviation Formula
n-octylamine OA NC.sub.8H.sub.17NH.sub.2 n-laurylamine LA
nC.sub.12H.sub.25NH.sub.2 2-aminothiazole 2-AT ##STR00004##
2-aminobenzimidazole 2-ABI ##STR00005## 2-aminobenzothiazole 2-ABT
##STR00006## 2-amino-5-chloro pyrimidine 3-amino-1,2,4-triazole
##STR00007## 2-(4-thiazoyl) benzimidazole p-chlorobenzylamine
2,4-dichloroaniline 8-aminoquinoline Imidazole Primuline
2-aminopyrimidine L-tryptophan 2-guanidinobenzimidazole 2-GBI
##STR00008## Suylfonamine MNPA 1000 * ##STR00009## Jeffamine M-2005
** *Surfonamine MNPA and **Jeffamine M-2005 are surface agent
monamines having multiple polyoxyethylene and/or polyoxypropylene
groups available from Huntsman Chemical Company
[0143] It is obvious to one skilled in synthetic organic chemistry
that Table 1 represents only a partial list, and that many more
examples are possible provided that no other reactive
functionalities are present which would compete with the primary
desired reaction of forming a biguanide moiety. It is also possible
for one skilled in the art to find one or more active compounds in
this class by performing the above synthesis or similar methods
using combinatorial synthesis or equivalent schemes by altering X,
Y, or the associated anion Z. This type of experimentation is
deemed obvious by adopting the systematic scientific method by one
skilled in the art
Example 2
Cytotoxicity Analysis of Polybiguanide Compounds
[0144] A. Monomeric Biguanides and bis-Biguanides.
[0145] HeLa cells (ATCC designation CCL-2) were maintained in
Dulbecco's Modified Eagle's medium (DMEM). P4-CCR5 (P4R5 cells)
(AIDS Reagent Program #3580) were cultured in DMEM with 0.1 ug/ml
puromycin as described by Charneau et al. (1994 J. Mol. Biol.
241:651-652). Sup-T1 human T lymphocytes (ATCC designation
CRL-1942) were cultured in RPMI 1640. All three cell types were
cultured in media supplemented with 10% fetal bovine serum (FBS),
L-glutamate (0.3 mg/ml), antibiotics (penicillin, streptomycin, and
kanamycin at 0.04 mg/ml each), and 0.05% sodium bicarbonate. Cells
of the Vk2/E6E7 human vaginal karatinocyte cell line were cultured
as described by Fichorova et al. (1997 Biol. Reprod.
57:847-855).
[0146] As part of our initial efforts to design and test PBG-based
compounds optimized for cytotoxicity and anti-HIV-1 activity,
experiments were performed with small molecules that contain the
same biguanide group found in the higher molecular weight
polybiguanides (PBGs). The experiments described below were
conducted to determine the activity and cytotoxicity of short chain
compounds having one or two biguanide groups. To do this HeLa cells
were incubated for 2 hours in the presence of the indicated
compound after which time the cells were assessed for viability
using the tetrazolium dye MTT as described by Rando et al. (1995 J.
Biol. Chem. 270:1754-1760). The cytotoxic concentration (CC50,
concentration of compound needed to reduce cell viability after a
two hour exposure by 50%) and the inhibitory concentration (IC50,
concentration needed to reduce cell-free virus infectivity by 50%)
are shown in Table 2. The efficacy determinations were made using
P4R5 cells, which allow for the detection of bacterial
.beta.-galactosidase activity upon release of viral tat protein
into the parent HeLa cell. In this assay bacterial enzyme levels
are monitored colormetrically 48 hours post virus infection using a
Tropix Galactro-Star machine using methods similar to those
reported by Ojwang et al. (1995 Antimicrobial Agents and
Chemotherapy 39:2426-2435).
TABLE-US-00002 TABLE 2 Cytotoxicity and efficacy of monomeric
biguanides # of CC50 IC50 Chemical name Biguanides (%) (%)
1,1-dimethyl biguanide 1 NT NA (metformin) 1-phenyl biguanide 1
0.150 0.522 xyleneylene bis-biguanide 2 0.385 NA hexamethylene
bis-biguanide 2 NT NA 1-phenethyl biguanide 1 0.538 ND (phenformin)
nonoxynol-9 (N-9) 0 0.007 0.005 Table 2. Monomeric biguanide and
bis-biguanide molecules have little or no activity against
cell-free HIV-1. Each entry lists the compound's chemical name, the
CC50 (the concentration at which HeLa cells were reduced in
viability by 50% during 2 hr exposure to the compound) and the IC50
(the concentration at which cell-free infectivity was reduced by
50% following a 2 hr exposure of cells to the compounds in the
presence of virus). NT = not toxic; NA = No activity; ND = Not
done.
[0147] B. Toxicity of Polybiguanides.
[0148] To build on the information provided by the monomeric
biguanide data a series of polybiguanide molecules were synthesized
with varied linker length as described in Example 1. In these
molecules the methylene spacer arms between the biguanide
functionalities (X and Y in Forumla I) have been changed. In this
respect the nomenclature used for this series of variants is as
follows: PHMB is polyhexamethylene biguanide and has six methylene
groups at both the X and Y positions; hence, it is a 6-6 PBG. PHMB
is FDA approved and EPA registered. In this experiment P4R5 cells
(modified HeLa cells that contain HIV-1 coreceptors were incubated
for 2 hours in the presence of the indicated compound after which
time the cells were assessed for viability using the tetrazolium
dye MTT as described by Rando et al. (1995 J. Biol. Chem.
270:1754-1760). The cytotoxic concentration (CC50, concentration of
compound needed to reduce cell viability after a two-hour exposure
by 50%) and the inhibitory concentration (IC50, concentration
needed to reduce cell-free virus infectivity by 50%) are shown in
Table 3.
TABLE-US-00003 TABLE 3 Cytotoxicity of PBGs with variations in
linker length. Chemical name (X, Y)PBG Abreviation CC50 (%) IC50 (
% ) 2-2 PBG PEB >0.2 >0.2 2-4 PBG 2-6 PBG PEHMB 0.8 0.004 2-8
PBG 0.1 2-10 PBG 0.01 2-12 0.01 3-6 0.15 0.003 4-4 PTMB 0.2 0.0025
4-6 0.05 0.002 6-6 PHMB 0.005 0.0015 nonoxynol-9 N-9 0.007
0.005
[0149] Compounds were synthesized with varied linker lengths
(between the biguanide groups in the chain) to alter
hydrophobicity,charge density, and chain flexibility of the
backbone--smaller number of methylene linkers will limit bending
and rotation of the PBG chain. For the first set of experiments, a
series of compounds was synthesized with one linker shorter than
the other (4-6, 3-6, and 2-6 PBG). While all of these compounds
were considerably less cytotoxic than N-9 (0.003% CC50) and PHMB
(0.005% CC50), there was a clear correlation between linker length
and cytotoxicity (Table 3). Compared to PHMB, which was the most
cytotoxic PBG compound, the other compounds were all less cytotoxic
(6-6>4-6>3-6>2-6 in order of decreasing cytotoxicity). In
vitro analyses demonstrated that a 2-2 PBG (a PBG with very limited
chain flexibility) is non-cytotoxic up to the highest concentration
tested (.about.0.2%). The 2-2 PBG was also not active against
anti-HIV-1 in this assay system up to the highest concentration
tested (.about.0.2%). The least cytotoxic compound in this series,
the 2-6 molecule known as PEHMB (0.799% CC50, or higher based on
the nature of associated anion), had no detrimental effect on cell
viability at concentrations as high as 0.316%. Having identified
the 2-6 PBG compound as the least cytotoxic in the above series,
subsequent experiments were performed using compounds with 2-Y
backbones, where Y was increased to 8, 10, or 12 methylene groups.
These experiments demonstrated that the 2-8 PBG compound was less
cytotoxic than either the 2-10 or 2-12, again indicating a
relationship between linker length and cytotoxicity (Table 3).
Cumulatively, these studies identified the 2-6 molecule (PEHMB) as
the optimal compound synthesized to date with respect to in vitro
cytotoxicity.
[0150] C. Toxicity of polybiguanides with modified counter
anions.
[0151] See Example 3B.
[0152] D. Toxicity of Polybiguanides with Modified End Caps.
[0153] See Example 3C.
[0154] E. Toxicity of Polybiguanides in Primary Vaginal
Keratinocytes.
[0155] As described above the effect of PEHMB and related compounds
on P4R5 cell viability after a two-hour exposure to the compound
was compared to the effects of control compounds such as N-9. In
those experiments (Table 3) the PBG compounds were usually
substantially less toxic than N-9. To further evaluate the effect
of this class of compound on various types of cells (in particular
PEHMB P4-R5 cells, ME-180 cells (ATCC HTB-33; cervical epidermoid
carcinoma), VK2/E6E7 human vaginal cell line (Fichorova et al. 1997
Biol. Reprod. 57:847-855), and human primary vaginal karatinocytes
(isolated from tissues obtained from vaginal reconstructive
surgery)) the compounds were incubated with the different cells for
various lengths of time before assessment of cell viability. The
results from these experiments are presented in Table 4.
[0156] The pairing of different anions with PBGs may result in a
combination that (i) has superior antiviral activity to (for
example) PEI-IMB that uses chloride as the counter ion, (ii)
imparts or introduces an additional mechanism of anti-HIV-1
activity that differs from, and complements, the activity of the
PBG, or (iii) further decreases the already low cytotoxicity of the
PBG. Our studies demonstrated that combining PHMB with a lactate
anion (PHMB-L) resulted in a compound that was somewhat less
cytotoxic than the chloride salt of PHMB. The same strategy was
tested using PEHMB. In Table 4, PEHMB-L indicates PEHMB with a
lactate counter anion.
[0157] The attachment of specific chemical moieties on one or both
ends of PBGs also has the potential to (i) further reduce the low
cytotoxicity of PBGs, (ii) augment the anti-HIV-1 activity of PBGs,
and (iii) broaden the activity of PBGs by introducing additional
moieties with mechanisms that will contribute anti-viral activity
(possibly against additional pathogens than the PBG target). A
derivative of PEHMB was synthesized with a single 1-aminoadamantane
moiety on the cyanoguanidine end of the molecule (PEHMB-A).
TABLE-US-00004 TABLE 4 Exposure of multiple cell types for varying
periods of time to PEHMB and its derivatives. CC50 % 10 minute 2 hr
6 hr Cell type/compound exposure exposure exposure P4-R5 cells
PEHMB 0.9 0.8 0.7 PEHMB-L 0.9 0.7 0.65 PEHMB-A 0.9 0.7 0.4 N-9 0.2
0.008 0.005 Dextran Sulfate ~2.0 >1.0 >1.0 ME-180 cells PEHMB
2.0 0.6 0.7 PEHMB-L 1.0 0.6 >1.0 PEHMB-A 1.5 0.6 0.7 N-9 0.20
0.008 0.007 Dextran Sulfate ~2.0 2.0 >1.0 Vk2/E6E7 cells PEHMB
1.5 0.9 0.7 PEHMB-L 1.0 0.9 0.7 PEHMB-A 1.0 0.3 0.1 N-9 0.03 0.006
0.002 Dextran Sulfate ~2.0 >1.0 >1.0 Primary vaginal
keratinocytes PEHMB 0.2 N-9 0.007 Dextran Sulfate 0.7
Example 3
Efficacy Analysis of Polybiguanide Compounds
[0158] Antiviral assays include a viral binding/entry assay in
which reporter cells such as P4R5 are incubated with virus in the
presence of compound for two hours at which time the drug is washed
off and the cells incubated for 48 hrs before measuring the
intracellular production of .beta.-galactosidase (Ojwang et al.
1995 Antimicrobial Agents and Chemotherapy 39:2426-2435). In
cell-associated virus inhibition (CAI) assays, HIV-1111B infected
SUP T1 cells are pelleted to remove cell free virus and incubated
with each compound for ten minutes at 37.degree. C. before a 1:10
dilution in media and incubation with P4R5 indicator cells for 48
hours before measuring .beta.-galactosidase activity. In cell-free
virus assays HIV-1IIIB or BaL were incubated with each compound for
10 minutes at 37.degree. C. before a 1:100 dilution in RPM! 1640,
and incubated for 2 hours with P4R5 cells, and subsequent assays
for viral infectivity were performed 48 hours later using the
.beta.-galactosidase method.
[0159] A. Anti-HIV-1 Efficacy of Polybiguanides.
[0160] Select compounds were assessed for their ability to inhibit
events necessary for HIV-1 binding and entry. Four compounds (2-6
PBG, 3-6 PBG, 4-6 PBG, and PHMB) were able to inhibit HIV-1 IIIB
binding and entry with activity equal to or greater than the
control compound N-9 (0.003% IC50) but not dextran sulfate (data
not shown). In these studies we compared the PBG class of compounds
against representatives from the surfactant class of microbicide
(N-9) and the polyanion class of compounds, dextran sulfate. All
four PBG-based compounds had similar activities (0.0015% to 0.004%
IC50). However, when combined with the cytotoxicity results, the
2-6 PBG compound (PEHMB) had the highest in vitro therapeutic index
(200) compared to 3-6 PBG (50), 4-6 PBG (80), and N-9 (1.4). Based
on numerous assays of in vitro cytotoxicity and antiviral activity,
we have calculated therapeutic indices for PEHMB that range from
160 to 1000.
TABLE-US-00005 TABLE 5 Efficacy against HIV-1 of PBGs with
variations in linker length. Compound CC50 (%) IC50 (%) TI 2-2 PBG
(PEB) >0.2 >0.2 ND 2-4 PBG 2-6 PBG (PEHMB) 0.8 0.004 200 2-8
PBG 0.1 2-10 PBG 0.01 2-12 0.01 3-6 0.15 0.003 50 4-4 (PTMB) 0.2
0.0025 80 4-6 0.05 0.002 25 6-6 (PHMB) 0.005 0.0015 3 nonoxynol-9
0.007 0.005 1.4
[0161] B. Anti-HIV-1 Efficacy and Cytotoxicity of Polybiguanides
with Modified Counter Anions.
[0162] The pairing of different anions with PBGs may result in a
combination that (i) has superior antiviral activity to (for
example) PEHMB that uses chloride as the counter ion, (ii) imparts
or introduces an additional mechanism of anti-HIV-1 activity that
differs from, and complements, the activity of the PBG, or (iii)
further decreases the already low cytotoxicity of the PBG. Previous
studies demonstrated that combining PHMB with a lactate anion
(PHMB-L) resulted in a compound that was somewhat less cytotoxic
than the chloride salt of PHMB. The same strategy was tested using
PEHMB. In an in vitro assay of cytotoxicity, PEHMB-L was
non-cytotoxic at concentrations up to and including 0.1% (Table 4),
as was PEHMB. In an assay of viral inhibition, PEHMB-L (0.054%
IC50) was slightly less effective than PEHMB (0.009% IC50) (Table
6). These results indicate that (i) anion changes can affect the
efficacy of the parental PBG molecular cation, and (ii) the
activity of PEHMB can be supplemented or complemented by the choice
of anion. The data presented in Table 6 show the results obtained
after a two-hour exposure of P4R5 cells to the indicated compound
in the presence of HIV-1IIIB.
TABLE-US-00006 TABLE 6 Efficacy against HIV-1 of PBGs with
variations in counter ion. X-Y Compound (ion) CC50 (%) IC50 (%) TI
P4R5 cell data (2 hr exposure) 6-6 PHMB (chloride) 0.004 0.002 2
6-6 PHMB-L (lactate) 0.008 0.003 2.6 2-6 PEHMB (chloride)) 0.8
0.004 200 2-6 PEHMB (lactate) 0.5 0.05 10 Nonoxynol-9 0.005 0.005
1.4 Vk2/E6E7 cell data (2 hr exposure) 6-6 PHMB (chloride) 0.002
6-6 PHMB-L (lactate) 0.005
[0163] C. Anti-HIV-1 Efficacy and Cytotoxicity of Polybiguanides
with Modified End Caps.
[0164] The attachment of specific chemical moieties on one or both
ends of PBGs also has the potential to (i) further reduce the low
cytotoxicity of PBGs, (ii) augment the anti-HIV-1 activity of PBGs,
and (iii) broaden the activity of PBGs by introducing additional
moieties with mechanisms that will contribute anti-viral activity
(possibly against additional pathogens than the PBG target). A
derivative of PEHMB was synthesized with a single 1-aminoadamantane
moiety on the cyanoguanidine end of the molecule (PEHMB-A). This
end-cap was hypothesized to add antiviral activity to PEHMB since
"amantadine" is known to have antiviral activity against other
viruses (Ahmad et al. 2002, Dig. Dis Sci 47:1655-1656; Englund, et
al. 2002, Semin Pediatr Infect Dis. 13:120-128; Stilianakis et al.
2002 Lancet 359:1862-1863). In an MTT assay to evaluate
cytotoxicity, the end-capped PEHMB molecule was non-cytotoxic at
concentrations at or below 0.316% (0.6% CC50), and had a level of
cytotoxicity similar to that of PEHMB (FIG. 3).
[0165] These data clearly show that end cap modifications can be
designed and synthesized so that minimal changes in compound
toxicity occur. It is likely therefore that modifications that
enhance antiviral activity can also be achieved.
[0166] In this regard we have also modified the ends of PBGs using
long chain hydrocarbon moieties resulting in compounds that
enhanced the antiviral activity of the parent compound in cell free
HIV-1 inhibition assays. Specifically we attached MNPA-1000 (Table
1) to the cyanoguanidinium end of PHMB. When PHMB (6-6 PBG)
containing the MNPA-1000 end cap was tested in the cell-free virus
anti-HIV-1 assays we observed an improved virus killing on the
order of 10 to 100-fold. These results open up an avenue for
modifications to improve the antiviral potency of PBGs. End group
modifications are also critical to the overall three-dimensional
shape of polymers such as PBGs, therefore the addition of
hydrocarbon end caps may help stabilize these molecules in solution
while also helping the PBG molecule interact with the hydrophobic
portions of viral membranes. These and other end-cap modifications
designed to augment the efficacy of PEHMB-based compounds will be
explored in Specific Aim 1.
[0167] D. Anti-HIV-1 Efficacy of Polybiguanides in Combination with
other Compounds.
[0168] The paradigm for effective HIV-1 therapy (for systemic
infections) is the use of combination drug regimens. Combination
therapy has proven effective at reducing virema, delaying the onset
of AIDS, and retarding the emergence of drug-resistant virus. At
this time the most effective microbicide regimen has not been
established. It may be that in order to block sexual transmission
of HIV-1 several drugs, having different mechanisms of action will
need to be applied in the same formulation. Therefore, our strategy
for augmenting or broadening the spectrum of PEHMB activity is to
combine PEHMB with other compounds that have different mechanisms
of action against HIV-1. As an example, we have investigated the
use of PEHMB combined with .beta.-cyclodextrin (BCD), a compound
that has the ability to sequester cholesterol from plasma membrane
lipid rafts and down-modulate presentation of the HIV-1 co-receptor
CXCR4 from the cell surface and consequently inhibit infection of
HIV-1 susceptible cells (Liao et al. 2001 AIDS Res Hum Retroviruses
17:1009-1019). In vitro cytotoxicity experiments demonstrated that
combinations of PEHMB and BCD, in which the concentration of one
component was varied while the other was kept constant, were
non-cytotoxic at the concentrations tested, as was the case for
PEHMB and BCD tested alone (FIG. 4A). In an assay of viral binding
inhibition using HIV-1 strain IIIB, PEHMB (0.1%) was very
effective, reducing infectivity to 3% of mock-treated cells. While
BCD (0.3%) also had considerable activity (27% remaining
infectivity), the combination of PEHMB and BCD was equally or more
effective (1% remaining infectivity) than PEHMB alone.
[0169] E. Persistent Anti-HIV-1 Efficacy of Polybiguanides.
[0170] An underlying general mechanism of the activity of PBG
compounds involves electrostatic interactions with negatively
charged phospholipids and with membrane-bound proteins including a
direct interaction with CXCR4. We have noted that the activity of
PEHMB is greater in assays of viral binding inhibition compared to
experiments in which activity against cell-free virus was measured.
These results suggested that PEHMB may interact more efficiently
with the cellular plasma membrane compared to the viral envelope,
and that this interaction may specifically disrupt events necessary
for viral binding and entry, serving to protect the cells from
infection. Because the interaction between PBGs and cellular
membranes involves interactions with phospholipids and protein
components, we hypothesized that PEHMB may provide protection from
infection even after the compound has been removed from the media
in which the cells are maintained. In a modified viral binding
inhibition assay (VBI) assay, HIV-1-susceptible P4R5 cells were
maintained for 1 or 2 hr in the presence of PEHMB (0.1% and 0.01%)
and then washed extensively before challenge with HIV-1 IIIB.
Despite the absence of PEHMB in the media during exposure to HIV-1,
infection of PEHMB pre-treated cells (2 hr at 0.1%) was reduced to
52% compared to control cells infected without PEHMB pre-treatment
(data not shown). In contrast, dextran sulfate (Dextralip 50, 0.1%
or 0.01%) had no residual antiviral activity after removal of the
compound from the culture media (data not shown).
[0171] Having demonstrated the persistence of antiviral activity
immediately after removal of PEHMB from the media, subsequent
experimentation was performed to determine the duration of the
protection in the absence of compound (FIG. 5). HIV-1-susceptible
target cells were incubated with PEHMB (0.1%) for 2 hr, washed
extensively with PBS, and subsequently challenged with HIV-1 IIIB
at 0, 0.5, 1, 2, 3, and 4 hours after washing. Introduction of
virus immediately after the cells were washed resulted in a level
of infection that was 37% of the infection achieved in the absence
of any compound (FIG. 5A). At 30 min and 1 hr after washing the
cells, the protection provided by PEHMB resulted in infections that
were 45% and 55% of control levels, respectively. Even after a
period of 4 hr between washing and virus challenge, residual
protection provided by PEHMB pre-treatment reduced the infection to
69% of the control infection. Although the reduction in infectivity
decreased over the 4 hr period, the reduction in infectivity after
4 hr was still considerable (FIG. 5B). These results indicate that
PEHMB or the effect of PEHMB on cells is retained by the cells even
after they have been washed.
[0172] F. Effect of Polybiguanides on HIV-1 Coreceptors CXCR4 and
CCR5
[0173] To explore the hypothesis that PEHMB may affect the
presentation of cell surface proteins involved in viral binding and
entry events (specifically the HIV-1 co-receptors CXCR4 and CCR5),
fluorescence activated cell sorting (FACS) analysis techniques were
used as described by Liao et al. (2001 AIDS Res Hum Retroviruses
17:1009-1019) to document the presence of CXCR4 on the surface of
target cells and to demonstrate the effect of PEHMB, BCD, and
combinations of both compounds. After a 2 hr exposure to PEHMB
(0.1%), P4-R5 cell presentation of CXCR4 was reduced from 98% to
29% (FIG. 6). Addition of BCD to PEHMB did result in an additional
reduction in CXCR4 presentation (down to 6%), suggesting at least
an additive, or possibly synergistic, effect was produced by the
combination.
[0174] A similar experiment (FIG. 7) was performed using the PM-1 T
lymphocyte cell line (Lusso et al. 1995 J. Virol 69:3712-3720).
Incubation of PEHMB (0.1%, FIG. 7B) or BCD (0.3%, FIG. 7C) resulted
in reductions of CXCR4 presentation from 45% to 5% and 22%,
respectively, while the combination of PEHMB and BCD reduced
expression even further (3%) (FIG. 7D). However, reductions in CD3
were not seen in response to compound exposure, indicating
specificity of action with respect to PEHMB's impact on CXCR4 (data
not shown). These results suggest that T cells may be more
sensitive to the actions of both PEHMB and BCD, since (i) both
compounds caused greater fold reductions in PM-1 CXCR4 and (ii) a
reduction in CXCR4 was noted in PM-1 cells at a BCD concentration
that had no apparent effect in P4-R5 cells.
[0175] Additional FACS analyses have been performed to examine the
impact of PEHMB on the cell surface expression of the HIV-1
receptor CD4 and the coreceptor CCR5 in the PM-1 T cell line. These
studies have indicated that PEHMB, under the conditions examined,
only minimally reduced the cell surface expression levels of CD4
and CD3 (data not shown) but had a pronounced effect on CCR5 (FIG.
8). In this figure it is easy to see that treatment of cells with
PEHMB dramatically increased the availability of epitopes
recognized by the monoclonal antibodies used in this study
suggesting a large change in the conformation of CCR5 has occurred.
Therefore it is quite likely that the anti-HIV-1 effects observed
for PBGs in general and PEHMB in particular is due at least in part
to a disruption of virus binding to cells at the level of the viral
co-receptors CXCR4 and CCR5.
[0176] G. Effect of PBGs on Herpes Simplex Virus Infections.
[0177] Herpes simplex virus plaque reduction assays were performed
as described by Fennewald et al. (1995 Antiviral Research
26:37-54). This assay was a variation on the cytopathic effect
assay described by Ehrlich et al. (1965 Ann N.Y. Acad. Sci
130:5-16). Basically cells such as Vero cells are seeded onto a
96-well culture plate at approximately 1 x 10.sup.4 cells/well in
0.1 ml of minimal essential medium with Earle salts supplemented
with 10% heat inactivated fetal bovine serum (FBS) and pennstrep
(100 U/ml penicillin G, 100 ug/ml streptomycin) and incubated at
37.degree. C. in a 5% CO.sub.2 atmosphere overnight. The medium was
then removed and 50 ul of medium containing 30-50 plaque forming
units (PFU) of HSV1 or HSV2, diluted in test medium and various
concentrations of test compound are added to the wells. Test medium
consists of MEM supplemented with 2% FBS and pennstrep. The virus
was allowed to adsorb to the cells, in the presence of test
compound, for 10 min at 37.degree. C. The test medium is then
removed and the cells are rinsed 3 times with fresh medium. A final
100 ul of test medium is added to the cells and the plates are
returned to 37.degree. C. Cytopathic effects are scored 40-48 hr
post infection when control wells (no drug) showed maximum
cytopathic effect.
[0178] In these experiments PEHMB was added to cells in the
presence of HSV2 as described above for ten minutes, washed off,
and the cells incubated for an additional 40-48 hrs. At this time
control wells that received no drug treatment had over 300 plaques
per well. Wells treated with 1% PEHMB for the indicated amount of
time had less than 200 plaques per well while wells treated with 2%
PEHMB had no visible plaques.
[0179] These results demonstrate that PBGs in general and PEHMB in
particular are potent inhibitors of herpes simplex viruses.
* * * * *