U.S. patent application number 12/943744 was filed with the patent office on 2012-05-10 for physico-chemical-managed killing of penicillin-resistant static and growing gram-positive and gram-negative vegetative bacteria.
Invention is credited to Francis G. Defalco, Alex F. Farris, III, Robert Chaffee Richmond, Harry F. Schramm, JR..
Application Number | 20120114762 12/943744 |
Document ID | / |
Family ID | 46019849 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120114762 |
Kind Code |
A1 |
Richmond; Robert Chaffee ;
et al. |
May 10, 2012 |
Physico-Chemical-Managed Killing of Penicillin-Resistant Static and
Growing Gram-Positive and Gram-Negative Vegetative Bacteria
Abstract
Systems and methods for the use of compounds from the Hofmeister
series coupled with specific pH and temperature to provide rapid
physico-chemical-managed killing of penicillin-resistant static and
growing Gram-positive and Gram-negative vegetative bacteria. The
systems and methods represent the more general physico-chemical
enhancement of susceptibility for a wide range of pathological
macromolecular targets to clinical management by establishing the
reactivity of those targets to topically applied drugs or
anti-toxins.
Inventors: |
Richmond; Robert Chaffee;
(Huntsville, AL) ; Schramm, JR.; Harry F.;
(Winchester, TN) ; Defalco; Francis G.; (Houston,
TX) ; Farris, III; Alex F.; (Birmingham, AL) |
Family ID: |
46019849 |
Appl. No.: |
12/943744 |
Filed: |
November 10, 2010 |
Current U.S.
Class: |
424/601 |
Current CPC
Class: |
A61K 47/02 20130101;
A61P 31/04 20180101; A61K 31/43 20130101; A61K 33/42 20130101; A61K
33/02 20130101; A61K 33/04 20130101; A61K 9/0014 20130101; A61K
9/0019 20130101; A61K 33/02 20130101; A61K 9/08 20130101; A61K
31/43 20130101; A61K 9/0073 20130101; A61K 31/19 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/19 20130101;
A61K 33/04 20130101; A61K 33/42 20130101 |
Class at
Publication: |
424/601 |
International
Class: |
A61K 33/42 20060101
A61K033/42; A61P 31/04 20060101 A61P031/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] The invention described herein was made in part by an
employee of the United States Government and may be manufactured
and used by and for the Government of the United States for
governmental purposes without the payment of any royalties thereon
of therefor.
Claims
1. A pharmaceutical compound comprising: a potassium cation; an
ammonium cation; a phosphate anion; a sulfate anion; an acetate
anion; and a penicillin; wherein said potassium cation, said
ammonium cation, said phosphate anion, said sulfate anion, said
acetate anion, and said penicillin are in a solution having a pH
ranging from 3.5 to 10; wherein said solution has a temperature
ranging from 0.degree. C. to 43.degree. C.; wherein each of said
potassium cation, said ammonium cation, said phosphate anion, said
sulfate anion, and said acetate anion has a molar concentration
ranging from 0.01 M to 4.0 M and are of a concentration capable of
inducing alteration of in situ target proteins to establish
sensitivity of bacteria to otherwise ineffective penicillins.
2. The pharmaceutical compound of claim 1 wherein said potassium
cation has a molar concentration of 2.6 M.
3. The pharmaceutical compound of claim 1 wherein said ammonium
cation has a molar concentration of 3.7 M.
4. The pharmaceutical compound of claim 1 wherein said phosphate
anion has a molar concentration of 3.1 M.
5. The pharmaceutical compound of claim 1 wherein said sulfate
anion has a molar concentration of 0.4 M.
6. The pharmaceutical compound of claim 1 wherein said acetate
anion has a molar concentration of 0.2 M.
7. The pharmaceutical compound of claim 1 which further includes
free ammonia.
8. The pharmaceutical compound of claim 1 wherein said solution is
at 22.degree. C.
9. The pharmaceutical compound of claim 1 wherein said solution is
at 37.degree. C.
10. The pharmaceutical compound solution of claim 1 wherein said
bacteria are Gram-positive.
11. The pharmaceutical compound solution of claim 10 wherein said
bacteria are methicillin-resistant Staphylococcus aureus
(MRSA).
12. The pharmaceutical compound solution of claim 1 wherein said
bacteria are Gram-negative.
13. The pharmaceutical compound solution of claim 12 wherein said
bacteria are Pseudomonas aeruginosa.
14. The pharmaceutical compound solution of claim 1 wherein said
penicillin is semi-synthetic penicillin.
15. The pharmaceutical compound solution of claim 14 wherein said
penicillin is cloxacillin.
16. The pharmaceutical compound solution of claim 1 wherein said
penicillin concentration is between 512 .mu.g/ml and 4096
.mu.g/ml.
17. A method of making a pharmaceutical compound comprising the
steps of: providing a potassium cation, an ammonium cation, a
phosphate anion, a sulfate anion, an acetate anion, a penicillin;
and ammonium hydroxide; placing said potassium cation, said
ammonium cation, said phosphate anion, said sulfate anion, and said
acetate anion into an aqueous solution in a proportional
concentration capable of inducing alteration of in situ target
proteins to establish sensitivity of bacteria to otherwise
ineffective penicillins; adding said penicillin to said solution;
and using said ammonium hydroxide to bring said solution to a
desired pH.
18. The method of claim 17 which further comprises the step of
bringing said solution to a desired temperature.
19. The method of claim 18 wherein said desired temperature is
22.degree. C.
20. The method of claim 18 wherein the desired temperature is
37.degree. C.
21. The method of claim 17 wherein said compound is adapted for
administration by topical application.
22. The method of claim 17 wherein said compound is adapted for
administration by oral ingestion.
23. The method of claim 17 wherein said compound is adapted for
administration by inhalation.
24. The method of claim 17 wherein said compound is adapted for
administration by instillation.
25. The method of claim 17 wherein said bacteria are in a
stationary growth phase.
26. The method of claim 17 wherein said bacteria are in a
logarithmic growth phase.
27. The method of claim 17 wherein said bacteria are
methicillin-resistant Staphylococcus aureus (MRSA).
28. The method of claim 17 wherein said bacteria are Pseudomonas
aeruginosa.
29. The method of claim 17 wherein said penicillin is
semi-synthetic penicillin.
30. The method of claim 29 wherein the penicillin is
cloxacillin.
31. The method of claim 17 wherein said penicillin concentration is
between 512 .mu.g/ml and 4096 .mu.g/ml.
32. The pharmaceutical compound of claim 17 wherein said potassium
cation has a molar concentration of 2.6 M.
33. The pharmaceutical compound of claim 17 wherein said ammonium
cation has a molar concentration of 3.7 M.
34. The pharmaceutical compound of claim 17 wherein said phosphate
anion has a molar concentration of 3.1 M.
35. The pharmaceutical compound of claim 17 wherein said sulfate
anion has a molar concentration of 0.4 M.
36. The pharmaceutical compound of claim 17 wherein said acetate
anion has a molar concentration of 0.2 M.
Description
FIELD OF INVENTION
[0002] The present invention relates to the field of pharmaceutical
compounds and more particularly to physico-chemical alteration of
macromolecular targets and target-accessibility to a drug or
antitoxin resulting from inclusion of components of the Hofmeister
series.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1 illustrates the non-substantial killing effect of a
pH 5.5 PBS solution having 1024 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase methicillin-resistant Staphylococcus
aureus (MRSA) culture over a 20 minute period.
[0004] FIG. 2 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 512 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase MRSA culture over a 20 minute period.
[0005] FIG. 3 illustrates the non-substantial killing effect of a
pH 5.5 SS having 1024 .mu.g/ml cloxacillin at 22.degree. C. on a
logarithmic-phase MRSA culture over a 20 minute period.
[0006] FIG. 4 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 22.degree. C. on a logarithmic-phase
MRSA culture over a 60 minute period.
[0007] FIG. 5 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0008] FIG. 6 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0009] FIG. 7 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0010] FIG. 8 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
MRSA over a 60 minute period.
[0011] FIG. 9 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
MRSA culture over a 24 hour period.
[0012] FIG. 10 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
MRSA culture over a 20 minute period.
[0013] FIG. 11 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
MRSA culture over a 20 minute period.
[0014] FIG. 12 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
MRSA culture over a 20 minute period.
[0015] FIG. 13 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
MRSA culture over a 20 minute period.
[0016] FIG. 14 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 35.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0017] FIG. 15 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 35.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0018] FIG. 16 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 35.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0019] FIG. 17 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 35.degree. C. on a stationary-phase
MRSA culture over a 60 minute period.
[0020] FIG. 18 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution at 22.degree. C. and 35.degree. C. on a
logarithmic-phase Pseudomonas aeruginosa culture over a 60 minute
period.
[0021] FIG. 19 illustrates the non-substantial killing effect of a
pH 7.4 SS at 22.degree. C. and 35.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 60 minute period.
[0022] FIG. 20 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution at 22.degree. C. and 35.degree. C. on a
stationary-phase Pseudomonas aeruginosa culture over a 60 minute
period.
[0023] FIG. 21 illustrates the non-substantial killing effect of a
pH 7.4 SS at 22.degree. C. and 35.degree. C. on a stationary-phase
Pseudomonas aeruginosa culture over a 60 minute period.
[0024] FIG. 22 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 512 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0025] FIG. 23 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 1024 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0026] FIG. 24 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 2048 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0027] FIG. 25 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 4096 .mu.g/ml cloxacillin at 22.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0028] FIG. 26 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 22.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0029] FIG. 27 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 22.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0030] FIG. 28 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 22.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 10 minute period.
[0031] FIG. 29 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 22.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 10 minute period.
[0032] FIG. 30 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 512 .mu.g/ml cloxacillin at 35.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0033] FIG. 31 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 1024 .mu.g/ml cloxacillin at 35.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0034] FIG. 32 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 2048 .mu.g/ml cloxacillin at 35.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0035] FIG. 33 illustrates the non-substantial killing effect of a
pH 7.4 PBS solution having 4096 .mu.g/ml cloxacillin at 35.degree.
C. on a logarithmic-phase Pseudomonas aeruginosa culture over a 20
minute period.
[0036] FIG. 34 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0037] FIG. 35 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0038] FIG. 36 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0039] FIG. 37 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 35.degree. C. on a logarithmic-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0040] FIG. 38 illustrates the killing effect of a pH 7.4 SS having
512 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0041] FIG. 39 illustrates the killing effect of a pH 7.4 SS having
1024 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0042] FIG. 40 illustrates the killing effect of a pH 7.4 SS having
2048 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
Pseudomonas aeruginosa culture over a 20 minute period.
[0043] FIG. 41 illustrates the killing effect of a pH 7.4 SS having
4096 .mu.g/ml cloxacillin at 22.degree. C. on a stationary-phase
Pseudomonas aeruginosa culture over a 20 minute period.
GLOSSARY
[0044] As used herein, the term "humectant" refers to a substance
that absorbs water, helps another substance retain moisture, and/or
disrupts or affects the water activity of macromolecules.
Humectants include compounds in the Hofmeister series, including,
but not limited to chaotropes, kosmotropes, and astringents or
styptics, such as alum, Burow's solution (i.e., aluminum acetate),
and silver nitrate, which at concentrations of approximately 10 mM
or less also acts as an anti-toxin and antiseptic.
[0045] As used herein, the term "penicillin" refers to any of a
group of broad-spectrum antibiotic drugs of the central formula
R--C.sub.9H.sub.11N.sub.2O.sub.4S, obtained from penicillium molds
or produced synthetically, and which are most active against
Gram-positive bacteria principally due to a beta-lactam ring
reacting with a -serine-X--X-lysine-amino acid motif in the
bacteria's transpeptidases, where X is any amino acid. Penicillins
are used in the treatment of various bacterial infections and
diseases. Penicillins include, but are not limited to, methicillin,
cloxacillin, amoxicillin, ampicillin, carbenicillin, dicloxacillin,
oxacillin, and therapeutic equivalents.
[0046] As used herein, the term "salt" refers to a chemical
compound derived from an acid by replacing a hydrogen, wholly or
partly, with a metal or an electropositive radical. This includes
ionic products of Bronsted-Lowry acid-base reactions and ionic
products of Lewis acids in water, i.e., conjugate bases, where both
these forms of salts are found within the Hofmeister series.
[0047] As used herein, "SS" is an abbreviation for a salt solution
for denaturing, i.e., altering the structure of, macromolecules,
and which is comprised of compounds within the Hofmeister
series.
[0048] As used herein, "PBS" is an abbreviation for non-denaturing
phosphate buffered saline, a buffer solution commonly used to
suspend and wash cells.
BACKGROUND
[0049] Both Gram-positive and Gram-negative pathogenic bacteria are
causing significant health problems around the world due to these
bacteria developing, or innately presenting, biochemical mechanisms
that thwart medical management by various types of antibiotics.
Effective use of penicillins, one major class of antibiotics, is
particularly being threatened. For examples, Gram-positive
methicillin-resistant Staphylococcus aureus (MRSA) has become
resistant to control by penicillins, and Pseudomonas aeruginosa, an
opportunistic member of Gram-negative bacteria, is innately beyond
control of penicillins.
[0050] One area of concern is hospital-acquired or nosocomial
parenteral antibiotic-resistant bacterial infections from topical
colonized bacteria or suppurating infections. These types of
bacteria frequently escape sterilization efforts prior to invasive
procedures allowing them to enter the body and establish
infection.
[0051] The acquiring of penicillin resistance by bacteria is
life-threatening and is being addressed by the pharmaceutical
industry through the development of new generations of penicillins.
The pharmaceutical industry largely directs its efforts to creating
new molecular alterations of existing penicillins in order to
circumvent continually evolving resistance that in turn defeats
efficacy of such new penicillins. Each generation of penicillins
successively targets penicillin-resistant mechanisms in the
bacterial coat in a way designed to circumvent biochemical
resistance mechanisms that have evolved within pathogenic bacteria
to resist previous generations of penicillins. It is unlikely that
this cycle of new biochemical specificity for penicillin activity,
followed by evolving resistance to that specificity, will be
therapeutically successful since the percentages of
penicillin-resistant pathogenic variants that defeat antibiotic
management is rapidly increasing.
[0052] Penicillins bind to penicillin-binding proteins (PBPs) in
the bacterial coat, and especially in Gram-positive bacteria those
targets tend to evolve into non-binding or non-accessible motifs
where, for example, one binding motif is said to be a 4-amino acid
sequence -serine-X--X-lysine- that provides covalent acylation of
serine by the beta-lactam ring of penicillins. In Gram-negative
bacteria, resistance to penicillins is additionally complicated by
the presence of transporters in the coat-associated outer membrane
that export the influx of penicillin, and by similarly located
porins that can restrict uptake of penicillin. Therefore, it is
important to resolve both the evolved resistance to binding of
penicillin to amino acid target motifs and the blockage of uptake
of penicillin into cells, which together largely account for
observed antibiotic resistance.
[0053] It is known that covalent binding of penicillins to PBPs of
actively replicating bacterial cells leads to defective coats,
which ultimately cause cell lysis and death. It is known that this
covalent binding is commonly defeated by evolution of structural
alteration in PBPs during development of penicillin resistance.
[0054] In addition, penicillin transport mechanisms also require
proteins of specific structure to perform the function of
penicillin efflux. Structural alterations of these proteins by pH,
salt concentration, or dehydration are often reversible. For
example, for at least one strain of MRSA, penicillin resistance is
observed at pH 7.4; however, penicillin sensitivity is returned
when those bacteria are exposed to penicillin at pH 5.6. Conversely
to physico-chemically induced reversible denaturation, covalent
binding of penicillin to PBP targets is not reversible, but rather
immutable whether achieved in growing or static bacterial
cells.
[0055] It is desirable to have a system and method for killing
topical bacteria known to be penicillin-resistant, particularly
MRSA and Pseudomonas aeruginosa.
[0056] It is desirable to have a system and method for reversing
the levels of penicillin-resistant bacterial infections that plague
individuals in both community and hospital settings.
[0057] It is desirable to have a system and method for managing
penicillin-resistance by mechanisms other than biochemical advances
in the structure and/or activity of penicillin.
[0058] It is further desirable to have a system and method for
altering in situ targets and inaccessibility of penicillin in
bacteria by physico-chemical treatments, providing novel paradigms
for effective topical applications of antibiotics and other drugs
and antitoxins.
SUMMARY OF THE INVENTION
[0059] The present invention is embodied as a pharmaceutical
solution at pH 7.4 comprised of high concentrations of phosphate,
sulfate, and acetate anions, potassium and ammonium cations, a
trace of free ammonia, penicillin, and water. In an exemplary
embodiment, the SS is applied within a range of temperatures,
specifically 22.degree. C. and 35.degree. C. The SS is capable of
inducing alteration of bacterial in situ target proteins to create
sensitivity of the bacteria to otherwise ineffective
penicillins.
DETAILED DESCRIPTION OF INVENTION
[0060] For the purpose of promoting an understanding of the present
invention, references are made in the text to exemplary embodiments
of a system and method for the physico-chemical alteration of
penicillin-binding proteins in penicillin-resistant Gram-positive
and Gram-negative bacteria to induce sensitivity to otherwise
ineffective penicillins, only one of which is described herein. It
should be understood that no limitations on the scope of the
invention are intended by describing these exemplary embodiments.
One of ordinary skill in the art will readily appreciate that
alternate but functionally equivalent use of compounds, solvents,
concentrations, pH, and methods may be used to expand biochemical
target reactivity and accessibility to reactive drugs and
anti-toxins. The inclusion of additional elements, such as drugs
and anti-toxins, depending upon the specific biochemical targets
and conditions involved, may be deemed readily apparent and obvious
to one of ordinary skill in the art. Specific elements disclosed
herein are not to be interpreted as limiting, but rather as a basis
for the claims and as a representative basis for teaching one of
ordinary skill in the art to employ the present invention.
[0061] Moreover, the terms "substantially" or "approximately" as
used herein may be applied to modify any quantitative
representation that could permissibly vary without resulting in a
change in the basic function to which it is related.
[0062] The physico-chemical aspect of the SS, less penicillin, is
conceived upon knowledge of water activity relative to biological
macromolecular rearrangements, and the knowledge that temperature
and pH also affect these rearrangements. The specifics of the SS,
less penicillin, are taken from the extensive Hofmeister series for
enhancing reversible denaturation of macromolecules in
Gram-positive bacteria (e.g., methicillin-resistant Staphylococcus
aureus) and Gram-negative bacteria (e.g., Pseudomonas aeruginosa).
The Hofmeister series is comprised of compounds known to effect
water activity and stability of a substantial and varied range of
macromolecules. The physico-chemical conditions of reversible
denaturation rearrange biological macromolecules, thereby altering
target motifs in protein, inhibiting structure-specific protein
activities, altering passive diffusion through structural barriers,
and imposing a temporal static state upon many metabolic processes,
thereby improving outcome from coincident therapeutic treatment of
targets. That is, such a disrupted and static condition in bacteria
can be used as an advantage by including one or more different
drugs or anti-toxins in this denaturing solution; drugs and
anti-toxins can include a range of known biochemical agents, such
as penicillin, antiseptics, or disinfectants selected for
compatibility with normal tissue at the site of topical
application. In this embodiment, cloxacillin, an otherwise
substantially ineffective penicillin, was chosen and proved to be
highly efficient in directly killing bacteria normally resistant to
penicillin.
[0063] FIGS. 1 through 41 illustrate the efficiency of a SS
formulation derived from members of the Hofmeister series for
physico-chemically inducing alteration of in situ target proteins
to establish sensitivity of Gram-positive (i.e., MRSA) and
Gram-negative bacteria (i.e., Pseudomonas aeruginosa) to otherwise
ineffective penicillins.
[0064] The SS formulation at pH 7.4, but not pH 5.5, is effective
in reversing penicillin-resistance in a methicillin-resistant
strain of Staphylococcus aureus (MRSA) and in Pseudomonas
aeruginosa, important bacterial pathogens. The SS affects water
activity, which in turn alters the macromolecular structure of the
target. In this manner, penicillin-resistant proteins are
rearranged by physico-chemical conditions such that covalent
binding of penicillins is allowed and the structures of penicillin
transport proteins are altered or thereby inactivated to defeat the
function of penicillin efflux.
[0065] In an exemplary embodiment for treatment of planktonic
bacteria, the SS contains high, at times saturated, concentrations
of phosphate, sulfate, and acetate anions, potassium and ammonium
cations, plus a small concentration of free ammonia, all prepared
in water, and applied at 22.degree. C. or 35.degree. C. For
example, in one embodiment, the molar concentrations of the
phosphate, sulfate, and acetate anions may be 3.1 M, 0.4 M, and 0.2
M, respectively, and the potassium and ammonium cations may be 2.6
M and 3.7 M, respectively. In an exemplary embodiment, ammonium
hydroxide is added to the SS in order to bring the SS to the
desired pH (e.g., pH 7.4), resulting in a small amount of free
ammonia. In various other embodiments, sulfuric acid, acetic acid,
a combination thereof, or another acid is added to lower the pH of
the SS. In various embodiments, each compound may have a molar
concentration ranging from 0.01 M to 4.0 M. In addition, the SS may
include any of the compounds in the Hofmeister series; the specific
formula of the SS may be adjusted for maximal physico-chemical
effectiveness in each specific application.
[0066] In various physico-chemical embodiments, the pH,
concentrations, solvents and/or temperature of the formulations may
vary in order to maximize the effectiveness of macromolecular
rearrangements and the outcome for different pathological
biomolecular targets, all with regard for the tolerance of the
normal tissue involved in topical applications. For example, the
temperature may range from freezing to 43.degree. C., the limiting
temperature for thermal pain sensation. In various embodiments, the
solvent (i.e., water) may be blended with or replaced with a
substance(s) that is known to affect water activity and
macromolecular rearrangements, such as alcohols (e.g., ethanol,
propanol, butanol) or aprotic solvents (e.g., dimethyl acetamide,
dimethyl formamide, dimethyl sulfoxide).
[0067] In an exemplary embodiment, the SS is prepared to an
effective pH of 7.4. In various other embodiments, the SS may be
prepared to a pH ranging from 3.5 to 10. The pH may be varied to
maximize the effectiveness of the macromolecular rearrangements and
the outcome for different pathological biomolecular targets.
[0068] In an exemplary embodiment, the SS formulation, often acting
as a humectant, is dictated by the alteration of water activity
that leads to reversible denaturation of biomolecules, both those
that are targets and those affecting accessibility to targets,
thereby maximizing the effectiveness of the macromolecular
arrangements and the resulting outcome. In various embodiments, the
formulation of SS may be varied by the addition of additives that
affect the water activity of biomolecules. These additives are
taken from the Hofmeister series, and include chaotropes and
kosmotropes, including humectants characterized as astringents or
styptics, such as alum, Burow's solution (i.e., aluminum acetate),
and silver nitrate. These additives are used to effect the
reversible denaturation of biomolecules resulting in effective
presentation of a variety of new biomolecular targets to drugs or
anti-toxins, such as silver nitrate, contained within the topical
solutions.
[0069] FIGS. 1 through 17 illustrate kill curve time course
experiments of stationary and logarithmic-phase MRSA bacterial
suspensions exposed to PBS as well as denaturing SS at pH 5.5 or pH
7.4 in the presence of cloxacillin concentrations spanning from 512
.mu.g/ml to 4096 .mu.g/ml. Experiments were conducted at 22.degree.
C. or 35.degree. C. The completion time for the experiments ranged
from 20 minutes to 24 hours.
[0070] For these experiments, the MRSA strain was grown overnight
in Mueller-Hinton broth (MH) +2% NaCl at 35.degree. C., subcultured
into fresh media, and grown to mid-logarithmic phase. The cells
were centrifuged and resuspended in pH 7.4 MH +2% NaCl to produce
an OD.sub.600 nm known to indicate logarithmic-phase or
stationary-phase of growth. An appropriate volume of cells was
added to 1 ml of buffered SS in a 10 ml Falcon tube to yield a
final concentration of ca. 1.times.10.sup.8 colony forming units
(CFU)/ml, except for the experiments of FIGS. 1-4 where the final
concentration of ca. 1.times.10.sup.6 CFU/ml applies. Survival was
determined for bacteria held for varied exposure times of up to 24
hours in the SS or in PBS at pH 5.6 or pH 7.4, each altered with
cloxacillin concentrations ranging in doublings from 512 .mu.g/ml
to 4096 .mu.g/ml. Survival was measured from samples held at
22.degree. C. and 35.degree. C. After exposure, samples of 50 .mu.l
were then withdrawn and a serial dilution series established. Cells
were then plated on MH +2% NaCl agar plates and incubated at
22.degree. C. or 35.degree. C. for CFU formation, after which
colonies were counted, surviving fractions recorded, and survival
curves constructed. Triplicate experiments were conducted for each
exposure condition. The cells were treated under static conditions
in all cases and treatments were always made on cells held in salt
solutions, never on cells held in growth media. In addition to
logarithmic-phase cells, stationary-phase cells removed from growth
cycle were also treated.
[0071] Cells exposed to SS at pH 7.4 in the presence of cloxacillin
are killed. Killing is more efficient for logarithmic-phase cells
than for stationary-phase cells. Both logarithmic-phase and
stationary-phase cells are killed more effectively by treatment at
35.degree. C. than treatment at 22.degree. C.
[0072] Killing efficiency is evaluated as therapeutically
sufficient according to the 10.sup.-5 level of killing in 5 minutes
specified by the British Standard BS EN 1276:1997 Chemical
Disinfectants and Antiseptics, which is referred to as the gold
standard for efficacy of bactericidal agents. Typically, agents
meeting the British Standard are too toxic for human topical
application. However, this is not a restriction for effective SS
containing cloxacillin used in the illustrated embodiment.
[0073] As developed in FIGS. 1 through 4, substantial killing of
static logarithmic-phase MRSA by cloxacillin is achieved at pH 7.4
at 22.degree. C. for SS only. PBS at pH 5.5 or at pH 7.4 is not
effective, and neither is SS at pH 5.5. Regarding the results shown
in FIG. 4, no survivors were seen on any of the treated plates;
therefore, the surviving fraction for all time points is less than
the 2.times.10.sup.-4 indicated as a conservative maximum allowed
for the dilution factor plated. Experiments were also conducted on
MRSA logarithmic-phase cells with SS alone at 22.degree. C. No
killing effect was seen at pH 5.6 or pH 7.4 for SS alone.
[0074] As FIGS. 5 through 9 illustrate, the efficiency of killing
of stationary-phase MRSA cells at 22.degree. C. does not provide
the 10.sup.-5 level of killing in 5 minutes specified by the
British Standard. However, at the highest concentration of
cloxacillin, that is, 4096 .mu.g/ml, the 10.sup.-5 level of killing
is achieved in 3 hours, as detailed in FIG. 9.
[0075] As shown in FIGS. 10 through 13, killing is enhanced by
exposure of logarithmic-phase MRSA to cloxacillin at 35.degree.
C.
[0076] As FIGS. 14 through 17 illustrate, the enhanced killing
extends to stationary cells exposed at 35.degree. C. as well. The
efficiency of killing of both logarithmic-phase and
stationary-phase MRSA cells at 35.degree. C. approaches or exceeds
the 10.sup.-5 level of killing in 5 minutes specified by the
British Standard.
[0077] The level of killing is concentration dependent and is based
upon the conditions of the treatment, such as species and initial
sensitivity/resistance, whether the cells are in a stationary or
logarithmic growth phase, temperature, and specific Hofmeister
series compounds used.
[0078] FIGS. 18 through 41 illustrate kill curve time-course
experiments of stationary-phase and logarithmic-phase Pseudomonas
aeruginosa bacterial suspensions exposed to PBS, to SS, to PBS in
the presence of cloxacillin concentrations spanning from 512
.mu.g/ml to 4096 .mu.g/ml, or to SS in the presence of cloxacillin
concentrations spanning from 512 .mu.g/ml to 4096 .mu.g/ml.
Experiments were conducted at 22.degree. C. or 35.degree. C. over
the course of 10, 20, or 60 minutes.
[0079] Pseudomonas aeruginosa cells were prepared as described
above for MRSA with a few exceptions. The Pseudomonas aeruginosa
cells were first grown in trypticase soy broth and then frozen in
aliquots, which were later rescued into MH broth +2% NaCl for use
as in the MRSA experiments. An appropriate volume of cells was
added to 1 ml of comparative salt solutions in a 10 ml Falcon tube
to yield a final concentration of ca. 1.times.10.sup.8 colony
forming units (CFU)/ml. Survival was determined for bacteria held
for varied exposure times of up to 60 minutes in PBS, in SS, in PBS
altered with cloxacillin concentrations ranging in doublings from
512 .mu.g/ml to 4096 .mu.g/ml, or in SS containing cloxacillin
concentrations ranging in doublings from 512 .mu.g/ml to 4096
.mu.g/ml. The salt solutions were compared at a pH of 5.6 or 7.4.
Survival was measured from samples held at 22.degree. C. or
35.degree. C. After exposure, samples of 50 .mu.l were then
withdrawn and a serial dilution series established. Cells were then
plated on MH +2% NaCl agar plates and incubated at 22.degree. C. or
35.degree. C. for CFU formation, after which colonies were counted,
surviving fractions recorded, and survival curves constructed.
Triplicate experiments were conducted for each exposure condition.
The cells were treated under static conditions in all cases and
treatments were always made on cells held in salt solutions, never
on cells held in growth media. In addition to logarithmic-phase
cells, stationary-phase cells removed from growth cycle were also
treated.
[0080] FIGS. 18 through 21 show the results of the control
experiments. For the control experiments, logarithmic-phase
Pseudomonas aeruginosa cells were treated in PBS (FIG. 18) or SS
(FIG. 19), and stationary-phase Pseudomonas aeruginosa were treated
in PBS (FIG. 20) or SS (FIG. 21). All experiments were conducted at
both 22.degree. C. and 35.degree. C. for 60 minutes. No substantial
killing was observed for any of these controls.
[0081] FIGS. 22 through 25 illustrate the non-substantial killing
logarithmic-phase Pseudomonas aeruginosa using a PBS solution with
cloxacillin concentrations ranging from 512 .mu.g/ml to 4096
.mu.g/ml at pH 7.4. The experiments were conducted at 22.degree. C.
for 20 minutes.
[0082] FIGS. 26 through 29 illustrate the efficiency of killing
logarithmic-phase Pseudomonas aeruginosa using SS with cloxacillin
concentrations ranging from 512 .mu.g/ml to 4096 .mu.g/ml at pH
7.4. For cloxacillin concentrations of 2048 .mu.g/ml and 4096
.mu.g/ml, the experiments were conducted at 22.degree. C. for 20
minutes. For cloxacillin concentrations of 512 .mu.g/ml and 1024
.mu.g/ml, the experiments were conducted at 22.degree. C. for 10
minutes. At all concentrations of cloxacillin, the SS, but not the
PBS solution, was substantially efficient in killing Pseudomonas
aeruginosa.
[0083] FIGS. 30 through 33 illustrate the non-substantial killing
of logarithmic-phase Pseudomonas aeruginosa using a PBS solution
with cloxacillin concentrations ranging from 512 .mu.g/ml to 4096
.mu.g/ml at pH 7.4. The experiments were conducted at 35.degree. C.
for 20 minutes.
[0084] FIGS. 34 through 37 illustrate the substantial killing of
logarithmic-phase Pseudomonas aeruginosa using the SS with
cloxacillin concentrations ranging from 512 .mu.g/ml to 4096
.mu.g/ml at pH 7.4. The experiments were conducted at 35.degree. C.
for 20 minutes.
[0085] FIGS. 38 through 41 illustrate the substantial killing of
stationary-phase Pseudomonas aeruginosa using SS with cloxacillin
concentrations ranging from 512 .mu.g/ml to 4096 .mu.g/ml at pH
7.4. The experiments were conducted at 22.degree. C. for 20
minutes.
[0086] Killing is more efficient for cells exposed to higher
concentrations of cloxacillin. In the embodiments shown, the
highest concentration of cloxacillin tested was 4096 .mu.g/ml;
however, it is not necessarily the maximum effective or tolerated
concentration. The concentration of cloxacillin or other drug may
be varied depending on the contact effect required for topical
applications in any given situation. That is, the preferred
concentration should be determined for each specific
application.
[0087] The use of various concentrations of compounds from the
Hofmeister series can affect macromolecular hydration and protein
denaturation, which may expose novel penicillin-binding amino acid
motifs of PBPs and other non-specific proteins. In addition, high
salt concentrations inactivate efflux transporters and porins. As a
result, both penicillin-resistant Gram-positive and Gram-negative
pathogenic bacteria may be created as penicillin-sensitive by
physico-chemical denaturation induced by exposure to the embodied
SS and related salt solutions when containing penicillin. It is
expected that under these specific conditions, universal creation
of protein target sensitivity to penicillin results from such
physico-chemical treatment, that is, a field effect of covalent
binding of proteins to penicillin is established during the
physico-chemical treatment by SS in the presence of penicillin.
This field effect, whereby any affected bacterial proteins may be
modified to enhance penicillin-binding, promotes direct killing and
allows, for the first time, the potential application of penicillin
to control static cells, including stationary-phase cells, as well
as actively growing cultures of bacteria. Penicillin dissolves well
in the SS described and may be used for treatment in the form of a
topical application.
[0088] Cloxacillin at relatively high concentration in the SS
substantially kills static logarithmic-phase and static
stationary-phase penicillin-resistant MRSA and Pseudomonas
aeruginosa cells rapidly upon exposure at room temperature, and the
degree of killing is advanced using variables of pH and
temperature. This substantial killing of static
penicillin-resistant bacteria at room temperature is notable in
that the killing effectiveness is established in both
logarithmic-phase and stationary-phase Gram-positive and
Gram-negative bacteria.
[0089] The embodiment of discovery and application described for
physico-chemically induced alteration of in situ target proteins
producing creation of sensitivity to otherwise ineffective
penicillin is of great value in regard to both concepts and
applications for continuing efforts to remedy clinical
penicillin-resistance, and by extension a spectrum of related
drug-resistance.
[0090] The fact that penicillin-resistant static bacteria, both
Gram-positive and Gram-negative, are killed directly using a
topically innocuous solution immediately applies to topical
treatments such as those involved in pre-surgical fields so that
contaminating penicillin-resistant bacteria will be purged prior to
surgical procedures, thus eliminating subsequent parenteral
infections that currently plague clinical invasive procedures.
Topical applications include, but are not limited to skin
colonization, suppurating infections, infections of the eye, nares,
throat and mouth, wounds, burns, encysted infections, cellulitis,
fulminating fasciitis, and also bacterial infection upon internal
structures, such as the vagina, urinary tract and bladder,
peritoneal cavity, large intestine, lung and trachea, stomach,
etc., all of which are accessible to instillation procedures.
[0091] In addition, bacterial infections are a complicating factor
in space travel. Injuries and radiation burns do not heal as well
in space, resulting in nagging injuries which may affect an
astronaut's performance. The present invention provides a new
ability to fight infections in space.
[0092] Bacterial infections are also a major factor for the
Department of Defense, especially during war times. Often, injuries
that occur in the field become infected with the subject bacteria
and are barely treatable. Use of the present invention provides an
improved method for wound and burn healing. Likewise, the
Department of Homeland Security is concerned about infections that
result from radiation burns occurring at dirty bomb sites. The use
of the present invention would provide a new and simple way of
dealing with that issue.
[0093] The present invention operates in physico-chemical mode,
which exposes normally inaccessible target motifs for covalent
binding of penicillin, thus demonstrating for the first time a
mechanism of action for penicillin that is outside the dogma of
biochemical action of penicillin upon only transpepidases in
actively growing bacterial cells.
[0094] The present embodiment claims rearrangement of
macromolecules using compounds from the Hofmeister series, thereby
yielding new target motifs accessible by drugs and/or anti-toxins
carried in the SS, which may be applied topically to manage
resistant or problematic organisms and/or macromolecules. The scope
of this discovery includes topical applications for treatment of
bacteria; viruses; molds and yeasts; topical parasites, such as
ring-worm, ticks, and cutaneous and mucocutaneous Leishmania; and
pathological macromolecules, such as bacterial exotoxins and
endotoxins, snake and spider venoms, super-antigens, and prions.
"Topical application" includes application not only to skin, but
also to cystic and suppurating infections, wounds, burns, nares,
throat, mouth, as well as all instillations such as to the urinary
tract and bladder, vagina, peritoneal cavity, large intestine,
stomach, lung, and trachea. The mechanism of denaturation and
rearrangement of macromolecules, and not only proteins, contributes
to the disruption of pathologic processes by compounds of the
Hofmeister series, opening new accessibility to molecular target
motifs for effective management by drugs, such as penicillins, and
anti-toxins, such as silver ions, carried in specifically optimized
SS. Combinations of drugs and anti-toxins may be applied in this
regard. For example, in topical applications of the SS to
fulminating fasciitis, a condition requiring rapid and multiple
reversals of currently resistant pathologic processes, penicillin
may be included to kill bacteria and silver ions may be included to
inactivate super-antigens.
[0095] The present embodiment claims management of water activity
and structure associated with macromolecules in bacterial pathogens
by use of compounds of the Hofmeister series, and examines the
substantial killing effects due to new target motifs that react
with penicillin in planktonic bacteria during exposures to the SS.
Topical clinical applications of the SS carrying drugs and/or
anti-toxins will, however, encounter sessile, as well as planktonic
bacteria, the principal additional barrier to accessibility by
drugs and/or anti-toxins then being the biofilm of polysaccharides,
mucopeptides, etc. secreted by and covering the sessile bacteria.
It is expected that dehydration and denaturation by humectant-like
and other compounds from the Hofmeister series in the SS will
permeabilize this highly hydrated biofilm to drugs and/or
anti-toxins, as well as expose new target motifs and inactivate
outer membrane-associated accessibility barriers as shown in the
embodiment herein.
* * * * *