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WO1993018172A1 - Composes servant a moduler le developpement d'agents infectieux - Google Patents

Composes servant a moduler le developpement d'agents infectieux Download PDF

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Publication number
WO1993018172A1
WO1993018172A1 PCT/US1992/005847 US9205847W WO9318172A1 WO 1993018172 A1 WO1993018172 A1 WO 1993018172A1 US 9205847 W US9205847 W US 9205847W WO 9318172 A1 WO9318172 A1 WO 9318172A1
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Prior art keywords
growth
catecholamine
organisms
gram
vectors
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Mark Lyte
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MANKATO STATE UNIVERSITY
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MANKATO STATE UNIVERSITY
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to a method of modulating the proliferation of microorganisms or other infectious vectors and, more particularly, to a method of introducing a neurochemical to augment, repress or otherwise affect the growth of Gram-reactive organisms.
  • the process involves the introduction (or application) of effective amounts of a group of neurochemicals known as the catecholamines.
  • Each microorganism tested has been shown to have specific requirements for one or more of the subject catecholamines.
  • suppression or enhancement of growth may be effected.
  • growth regulation can be effected in vivo and/or in vitro.
  • the characterization of the receptor through which the catecholamine binds to the cell or cellular components as novel enables further control of cell growth by the application of either receptor agonists or antagonists specific for this novel receptor.
  • Sepsis is the generally febrile pathologic state resulting from the presence of microorganisms or their poisonous products in the blood stream particularly in humans or other mammals. It occurs when spreading infectious agents are not successfully arrested within the lymph nodes, and thus directly invade venous channels. Although it is not uncommon to have periodic invasions of bacteria into the blood stream, called bacteremia, these outbursts are normally handled quickly and effectively by macrophages circulating in the blood. However, in some circumstances, such large numbers of bacteria may be involved in the invasion that the macrophages are overwhelmed and under-effective, resulting in the symptoms of fever, chills, general malaise and lethargy known as septicemia.
  • antimicrobial agents typically in conjunction with the use of vasoactive drugs such as norepinephrine and dopamine.
  • vasoactive drugs such as norepinephrine and dopamine.
  • antimicrobial agents are typically designed to affect bacterial wall structure or bacterial metabolic processes. Such agents may be particularly targeted at a specific metabolic process or cellular receptor.
  • conventional antimicrobial therapy is unsuccessful in a large number of cases. In particular, it has been documented that up to 60% of all patients diagnosed with sepsis eventually succumb to the condition. Thus, despite the great strides forward, there remains great need for more specific and more effective antimicrobial agents or treatment protocols.
  • vasoactive drugs in septic conditions has been the restoration of normal hemodynamics.
  • a typical clinical picture involving sepsis is the patient who fails to respond to traditional treatment or who undergoes intensive antibiotic therapy and apparent resolution of sepsis, with improvement in condition two to three days post admission to hospital.
  • nearly 40% of patients relapse on the third to fourth day of treatment, with death ensuing rapidly thereafter.
  • catecholamines such as norepinephrine and dopamine
  • Catecholamines in humans are a class of hormones that evoke a response by activation of adenyl cyclase. These compounds are targeted to specific hormone receptors in mammals and produce varied responses, depending upon the nature of the target tissue. The majority of catecholamines evoke their characteristic response by influencing the activity of pre-existing cellular enzyme systems. Thus, the response evoked may be almost instantaneous, such as in the case of neurotransmitters including norepinephrine and epinephrine.
  • Norepinephrine, epinephrine and dopamine are the characteristic hormones of the mammalian sympathetic nervous system. All are a ine derivatives of the catechol nucleus (dihydroxybenzene) . These compounds have clearly identified peripheral effects.
  • Classic feedback inhibition processes control the production of these compounds, in which the rate limiting step in the pathway is hydroxylation of the amino acid precursor tyrosine to form dihydroxyphenylalanine (dopa) .
  • synthesis of catecholamines is a unique feature of sympathetic nervous tissue.
  • hormone producing tumors may release catecholamines directly into the circulation and, thus, manifest peripheral effects of these compounds as a result of plasma concentration instead of local tissue concentration.
  • Other states, such as stress are also classic activators of the production of catecholamines concomitant with the presumed suppression of the immune system. Otherwise, most manifest a non- circulatory effect.
  • bacteria lack a nervous system and, thus, have no apparent need for neurotransmitters such as norepinephrine, epinephrine and dopamine
  • neurotransmitters such as norepinephrine, epinephrine and dopamine
  • Such a class-dependent response presents great opportunity for novel approaches in drug design.
  • the identification of the receptors by which bacteria may use these neurochemicals leads to the design of receptor antagonists which may be as potent in the control of bacterial growth as current antimicrobial therapy, including the application of antibiotics.
  • Gram- negative bacteria having a growth-enhancing response to neurochemicals are open to treatment by any inhibitor which intercedes at any point in the catecholamine biosynthetic pathway, such as onoamine oxidase inhibitors, in order to interrupt specific steps in the conversion pathways of these catecholamines.
  • Another object of the present invention is to provide a new and improved method for the treatment of living patients suffering the effects of a microbial, parasitic or viral infection.
  • a further object of the present invention is to provide a new and improved method for the suppression of bacterial replication within living patients.
  • Another object of the present invention is to provide a new method for specifically binding the novel receptor identified herein.
  • Yet another object of the present invention is to provide a new method for application in the field of the design of drugs and therapeutics, by which recognition of this novel receptor identified herein is useful in suppressing the proliferation of infectious agents.
  • the modulation of bacterial growth is effected in vitro by the addition of one or more catecholamines to buffered basal culture medium containing the subject bacterial cells and control or catecholamine solution in an amount ranging from about 10 "4 to 10 "10 M concentration. Modulation of growth rate can be detected by a number of standard methodologies. For example, scintillation counts of 3 H-thymidine, optical density readings or plate counts may be performed.
  • known antagonists which specifically block receptor binding of catecholamines, such as norepinephrine and epinephrine, may be added to bacterial cultures in the presence or absence of a preselected concentration of norepinephrine.
  • Known agonists are also tested.
  • a method of treatment of infections has been devised in which treatment protocol is based not upon whether the causative agent is infectious, but upon the nature of the cell wall in microorganisms (or other binding characteristics in viruses or vectors) . It has been discovered that growth is enhanced in Gram-negative microbes when in the presence of certain catecholamines. Thus, the accepted practice of administering catecholamines in such patients should be suspended. However, growth of Gram-positive bacteria is suppressed in these conditions, which contraindicates suspension. Because the affinity of these microorganisms for certain catecholamines is receptor-mediated, as demonstrated herein, the method of treatment of infections caused by these organisms is directed at manipulation of the receptor. Furthermore, the receptor involved is not blocked by known ⁇ ,, ⁇ . or ⁇ adrenergic agents.
  • FIG. 1 is a block diagram of a preferred embodiment of the method of the present invention for in vitro applications
  • Figure 2 is a block diagram of an alternative embodiment of the present invention for in vivo applications
  • Figure 3 is a plot illustrating differing rates of 3 H- thymidine incorporation into newly synthesized DNA when E. coli is cultured in various concentrations of norepinephrine, epinephrine and dopamine at an initial inoculum of 15 CFU per well;
  • Figure 4 is a plot similar to Figure 3 illustrating differing rates of 3 H-thymidine incorporation into newly synthesized DNA when E. coli is cultured in various concentrations of norepinephrine, epinephrine and dopamine at an initial inoculum of 1500 CFU per well;
  • Figure 5 is a plot illustrating the differing rates of 3 H-thymidine incorporation into newly synthesized DNA when
  • Y. enterocolitica is cultured in various concentrations of norepinephrine, epinephrine and dopamine at an initial inoculum of 10,000 CFU per well;
  • Figure 6 is a graph illustrating the change in optical density with various concentrations of norepinephrine in a culture of E. coli with an initial inoculum of 25 CFU per well;
  • Figure 7 is a graph illustrating pour plate counts for cultures of P. aerucrinosa cultured in various concentrations of norepinephrine
  • Figure 8 is a plot illustrating differing rates of 3 H- thymidine incorporation into newly synthesized DNA when S. aureus is cultured in various concentrations of norepinephrine, epinephrine and dopamine at an initial inoculum of 1600 CFU per well
  • Figure 9 is a plot similar to Figure 8 illustrating the suppression of growth of E. coli in the presence of the anti-adrenergic compound chlorpromazine with an initial inoculum of 2200 CFU per well;
  • Figure 10 is a plot similar to Figures 8 and 9 illustrating the lack of an effect of adrenergic receptor agonists octopamine and ephedrine on the growth of E. coli with an initial inoculum of 25 CFU per well;
  • Figure 11 is a plot similar to Figure 10 illustrating the growth enhancing effect of high concentrations of the j8-receptor agonist isoproterenol on the growth of E. coli at an initial inoculum of 25 CFU per well;
  • Figure 12 is a plot illustrating the lack of an effect of the ⁇ -adrenergic receptor antagonist benextramine tetrachloride on growth of E. coli with an initial inoculum of 30 CFU per well;
  • Figure 13 is a plot illustrating the lack of a growth enhancing effect of high concentrations of the 0-adrenergic receptor antagonist alprenolol on the growth of E. coli at an initial inoculum of 25 CFU per well;
  • Figure 14 is a plot similar to Figure 10 illustrating a lack of decrease in growth of S. aureus in the presence of the dual a- and ⁇ -adrenergic receptor agonist ephedrine at an initial inoculum of 27 CFU per well;
  • Figure 15 is a graph illustrating the increase in glucose production at increasing concentrations of norepinephrine in cultures of E. coli;
  • Figure 16 is a plot illustrating the increase in ⁇ - galactosidase activity with increasing concentrations of norepinephrine in cultures of E. coli:
  • Figure 17 is a graph illustrating a 3 H-norepinephrine competition/displacement curve
  • Figure 18 is a saturation curve for 3 H-norepinephrine
  • Figure 19A is a competition/displacement curve illustrating the non-displacement with non-specific binding which occurs with the addition of 3 H-prazosin
  • Figure 19B is a competition/displacement curve illustrating the non-displacement with non-specific binding which occurs with the addition of 3 H-rauwolscine;
  • Figure 19C is a competition/displacement curve illustrating the non-displacement with non-specific binding which occurs with the addition of 3 H-dihydroalprenolol.
  • Figure 20 is a tissue dependency graph demonstrating saturation in which all of the novel receptors are saturated with catecholamines at high concentrations of bacteria. DESCRIPTION OF THE PREFERRED EMBODIMENT
  • the modulation of microbial growth may be effected in vitro by the addition of one or more catecholamines in an amount ranging from about 10 "4 to 10 "10 M concentration to culture medium containing the subject microbial cells.
  • the culture medium should preferably be a relatively simple medium so as to facilitate the inclusion of the catecholamines in a known concentration.
  • a basal medium consisting of various constituents such as dextrose (0.5 g/1, 2.78 mM) , ammonium nitrate (0.5 g/1, 6.25 mM) , potassium phosphate (0.25 g/1, 1.84 mM) , potassium chloride (0.25 g/1, 3.35 mM) and magnesium sulfate (0.25 g/1, 1.01 mM) adjusted to a final pH of 7.5.
  • hepes buffer may be added at a final concentration of 10 mM to provide additional buffering capacity.
  • This medium may then be further supplemented with 30% V/V of bovine serum, although equivalent results may be obtained with serum from other sources such as pig, mouse and man.
  • Suitable antioxidants such as ascorbic acid may also be added to prevent oxidation of catecholamines.
  • such compounds should be used judiciously, since the antibacterial effects of antioxidants are well recognized. It is recognized that individual cells may require different basal medium preparations to insure optimum growth in catecholamine supplemented medium.
  • the desired catecholamine is then dissolved in an appropriate amount of chilled boiled water.
  • the catecholamine solution is then added to the basal medium in a final volume-to-volume addition of, for example, 20%.
  • the subject microbial cells are prepared in serum supplemented basal medium at varying concentrations of 15 to 15,000 CFU per ml and as depicted at Block 3, 100 ⁇ l of suspensions may be added into the wells of standard tissue culture plates.
  • 100 ⁇ l of catecholamine or control solution is added to the wells containing, for example, bacteria and the plates are incubated for varying lengths of time at an appropriate temperature in a humidified incubator, as at Block 4. This process may be altered as necessary for application to a variety of non-microbial vectors as well.
  • Modulation of growth rate can be detected by a number of standard methodologies.
  • the cultures can be further supplemented at time of culture initiation by the addition of 50 ⁇ l of a 20 ⁇ Ci solution of 3 H-thymidine in PBS per 200 ⁇ l of culture.
  • Culture contents can then be harvested onto filter mats using standard plate harvesters which separate unbound radioactive material from radioactive material that has been incorporated into the cell's DNA.
  • the filter mats with bound material are then counted in a scintillation counter.
  • the raw counts resulting from incorporation of the 3 H-thymidine into newly synthesized DNA is used as measure of cell proliferation.
  • contents of plate wells may be gently resuspended and the optical density readings taken in a standard microplate reader at an optical density of 550 nm or 630 nm.
  • Increases in optical density readings in catecholamine supplemented wells as compared to control wells indicates greater mass of cells due to increased proliferation.
  • plate counts may also be performed by standard pour-plate methodology in which the contents of the plate well are gently resuspended and an aliquot removed and serially diluted samples are plated. After 24-48 hours, the number of colonies are enumerated with an increase in colonies relative to control plates indicating enhanced cell proliferation.
  • FIG. 5 depicts a method for in vivo treatment of infection using a suggested method of the present invention. As denoted at Block 1, the causative nature of the infection must first be assessed.
  • the appropriate therapy is commenced, as at Block 2.
  • This therapy would include either application of antimicrobial or antiparasitic agents, or pertinent anti-viral treatment.
  • a decision must be made as to whether or not to introduce systemic catecholamines to the patient, as designated at Block 3. If the decision is reached that catecholamines should be applied, metabolic and cardiac functions should be monitored as denoted at Block 4. As described further hereinafter, it is also appropriate at this time to screen for any increase in the concentration of microbes, whether Gram-negative or Gram- positive.
  • the therapy commenced at Block 2 should be continued (Block 5) with intermittent assessment and monitoring, as denoted at Block 4.
  • Block 6 therapy should be discontinued. If, however, the decision is made that due to the nature of the causative organism, catecholamines should not be applied, subsequent steps denoted at Blocks 7 and 8 include monitoring of metabolic and cardiac functions as well as a continuation of appropriate anti-microbial or anti-viral therapy.
  • Block 9 to identify the agent causing the infection. It is to be emphasized that these steps (Block 1 and Block 9) are undertaken simultaneously, due to the customary time delay in arriving at a proper identification and the deleterious impact which would ensue if therapy were delayed. Once the agent is identified, it may be necessary to alter or adjust therapy accordingly, as at Block 10.
  • catecholamines specifically norepinephrine and epinephrine
  • receptors include the a- and 3-receptors and their constituent subtypes.
  • known antagonists which specifically block receptor binding of catecholamines, i.e., norepinephrine and epinephrine, are added to bacterial cultures in the presence or absence of a preselected concentration of norepinephrine.
  • the / 5-adrenergic receptor antagonist alprenolol hydrochloride (HC1) was dissolved in chilled boiled water in an amount ranging from 5 x 10 "4 to 5 x 10 "12 M.
  • Norepinephrine was also dissolved in chilled boiled water in an amount of 5 x 10 "5 M.
  • alprenolol HC1 and norepinephrine were added alone or in combination.
  • the subject bacterial cells were prepared in serum supplemented basal medium at varying concentrations of 15 to 15,000 CFU per ml and 100 ⁇ l of suspensions were added into 96 well tissue culture plates.
  • Figure 3 depicts a positive correlation between increase in concentration of a Gram-negative bacterium and increased 3 H-thymidine incorporation (DPM) into newly synthesized DNA in the presence of the catecholamines norepinephrine, epinephrine and dopamine. Data presented are for a 10-hour culture of E. coli with an initial inoculum of 15 colony-forming units (CFU) per well. Of these catecholamines, it can be seen that norepinephrine is the most potent enhancer of growth at low initial concentration of Gram-negative bacterium. To show that the effect depicted in Figure 3 was not dependent upon the initial concentration of bacterium, experiments were repeated at a variety of initial concentrations. For the Gram-negative bacterium, E.
  • Figure 4 depicts a 10-hour culture of E. coli with an initial inoculum of 1500 CFU per well.
  • DPM 3 H-thymidine incorporation
  • Figure 5 depicts a 36 hour culture of Yersinia enterocolitica with an initial inoculum of 10,000 CFU per well. The data represented herein were selected because they presented typical values for incorporation of 3 H-thymidine into newly synthesized DNA at high concentration of catecholamine. The enhancing effect of catecholamines is representative of all tested Gram- negative bacteria at high initial concentrations. No Gram- negative organism was found which did not demonstrate a selective preference for one or more of the catecholamines tested.
  • Table l depicts data representative of the contents of Figures 3 through 5 and further includes data on the effect of administration of catecholamine metabolites. These data confirm the impression presented in Figures 3 through 5 that Y_;_ enterocolitica is considerably more selective in substrate usage than E. coli.
  • the ubiquitous nature of Ij . coli is borne out by the fact that it is even capable of fueling growth by utilization of the metabolites 4-hydroxy- 3-methoxyphenylglycol piperazine salt (MHPG) and normetanephrine (NOR) .
  • MHPG 4-hydroxy- 3-methoxyphenylglycol piperazine salt
  • NOR normetanephrine
  • Data represent mean DPM values with initial concentrations of 15 and 1500 CFU for E. coli and 80 CFU for Y. enterocolitica. The mean DPM values were obtained from quadruplicate cultures and the standard error was 10% or lower.
  • Figure 6 depicts spectrometric data for a 20 hour culture of E. coli with an initial inoculum of 25 CFU per well measured at 630 nanometers. A separate blanking plate containing all media and chemicals, but lacking a bacterial inoculum, was measured to provide a correction factor for all concentrations of norepinephrine and control. The same enhancement of growth of E. coli was detected with this method as was shown using scintillgraphic data.
  • Figure 7 further illustrates that the growth-enhancing capacity of norepinephrine in Gram-negative bacteria is not method dependent.
  • standard plate counts for a 20 hour culture of Pseudomonas aerucrinosa increasing concentrations of norepinephrine correlated to an increased number of mean CFU for increasing concentrations of norepinephrine as compared to a control medium lacking norepinephrine.
  • Figure 8 demonstrates that these compounds have essentially the reverse effect in Gram-positive bacteria. This trend was consistent for all Gram-positive bacteria tested and exemplified by the plot in Figure 8.
  • a 20 hour culture of Staphyloccus aureus representing an initial inoculum of 1600 CFU per well manifested a decline in 3 H-thymidine uptake into new DNA. As concentration of catecholamines increased, suppression of growth increased.
  • Figure 9 depicts the effect of the anti-adrenergic compound chlorpromazine on the synthesis of new DNA in EL. coli.
  • Data presented represent an initial inoculum of 2200 CFU per well and a culture period of 11 hours.
  • the growth suppressive effect on E. coli is what would be expected if the phenomena demonstrated herein were due to a receptor- mediated mechanism.
  • Figure 10 delineates the lack of an effect of selected adrenergic receptor agonists upon this process.
  • the ⁇ -receptor agonist octopamine, and the dual ⁇ - and 0-receptor agonist ephedrine were found to have no effect on Gram-negative growth.
  • This particular graph presents a 10 hour culture of E. coli having an initial inoculum of 25 CFU per well.
  • Figure 11 depicts the effect of active and inactive enantiomers of the /3-receptor agonist isoproterenol. Although the exact mechanism of this effect has not yet been elucidated, it appears that the modality of the effect of catecholamines in these microorganisms is receptor- modulated. Thus, it would be anticipated that isoproterenol would have an effect similar to the data of Figure 10, i.e. no effect. Conversely, at very high concentration there is, in fact, an extremely significant effect. For this reason, both the active (-) and the inactive (+) enantiomers were tested. The data presented are for a 10-hour culture of E. coli with an initial inoculum of 25 CFU per well.
  • Figure 12 demonstrates the lack of effect of the a- adrenergic receptor antagonist benextramine on the growth of a 20 hour culture of E. coli with an initial inoculum of 30 CFU per well.
  • the bar graph designated "A” the effect of norepinephrine on E. coli growth is compared to control medium (CON) .
  • CON control medium
  • norepinephrine enhanced growth continues as anticipated irregardless of benextramine concentration. Note that the final concentration of norepinephrine is the same (5xl0 "5 M) .
  • Figure 13 depicts the effect of the / 5-adrenergic receptor antagonist alprenolol on a 20 hour culture of E coli with an initial inoculum of 30 CFU per well. Again, norepinephrine stimulates growth in comparison to control and the presence of alprenolol has no inhibiting effect on norepinephrine enhanced growth, as depicted in graph "B".
  • Graph A demonstrates norepinephrine-induced growth in the absence of alprenolol.
  • Figure 14 demonstrates the effect of a dual ⁇ - and ⁇ - receptor agonist, ephedrine, on a 20 hour culture of Staphyloccus aureus with an initial inoculum of 27 CFU per well.
  • the growth of this Gram-positive bacterium is not decreased in the presence of this agonist as it is in the presence of norepinephrine.
  • the ⁇ -receptor agonist, octopamine was tested and found to produce some inhibition of growth. This is somewhat analogous to the events in Figure 11 involving active and inactive enantiomers of isoproterenol.
  • Table 2 demonstrates the effect of norepinephrine and various ⁇ - and j8-receptor agonists on the growth of Y. enterocolitica.
  • Mean DPM values were obtained for quadruplicate cultures of the indicated compounds, The standard error of the mean did not exceed 10%. Concentrations for all compounds are 10 "4 M. Abbreviations are: 0, control (no compound added); NE, (-)- norepinephrine; OCT, (-)-octopamine; (-)-isoproterenol; (+)-isoproterenol; and (-)-ephedrine. Values shown were obtained at 36 hours. The number of colony forming units for CON indicates the number of bacteria present in each well at initiation of culture. Once again, the significant growth enhancing effect of norepinephrine is evident in view of insignificant effect by related ⁇ - and /3-agonists.
  • an initial inoculum of 130 CFU per well was treated with (+)-octopamine (an ⁇ - agonist) , (-)-isoproterenol (a / 3-agonist) , -(+)- isoproterenol (an inactive enantiomer) , and (-)-ephedrine (a dual ⁇ -, /3-agonist) .
  • the incubation period was 36 hours.
  • Figure 16 demonstrates expression of / 3-galactosidase activity in a culture of E. coli. Although total expression of /3-galactosidase activity is increased, actual activity per organism is suppressed. This indicates alteration of this operon and activation of genes which allow the protein to be expressed in E. coli is modulated in the presence of norepinephrine. Thus, this provides another example of a significant enhancement in the production of commercially viable products which may be attained by the inclusion of catecholamines, such as norepinephrine, in the appropriate culture medium.
  • catecholamines such as norepinephrine
  • Figures 17-20 demonstrate that the effect of norepinephrine as described herein is receptor mediated, and that the receptor involved is a new type of receptor that is not one of the presently known receptors, e.g. ⁇ .,, ⁇ 2 / & ⁇ ⁇ or &z ⁇
  • ⁇ ., ⁇ 2 / & ⁇ ⁇ or &z ⁇ These figures were obtained using widely accepted methods of neurotransmitter receptor analysis, including methods described in Methods of Neurotransmitter Receptor Analysis. Yamamura H.I., S.J. Enna, and M.J. Kuhar, Eds., New York: Raven Press, 1990. According to these accepted methods, these figures satisfy the accepted criteria for identification of a novel receptor.
  • Figure 17 provides a competition/displacement curve for 3 H-norepinephrine, in which the log of the concentration of cold norepinephrine is plotted on the abscissa and the total amount of bound 3 H-norepinephrine (DPM) is plotted on the ordinate.
  • DPM total amount of bound 3 H-norepinephrine
  • Zero concentration for cold norepinephrine is represented by a box, whereas the presence of cold norepinephrine is indicated by circles.
  • the resultant curve demonstrates that once 3 H-norepinephrine has been bound to its receptor on E. coli. the addition of increasing amounts of cold norepinephrine results in displacement of the 3 H-norepinephrine from the receptor.
  • Figure 18 provides a saturation curve for 3 H- norepinephrine. Bound radioligand is presented on the ordinate and free radioligand is presented on the abscissa, as obtained from a mixture of free radioligand and cold norepinephrine. Thus, nonspecific binding (NSB) , specific binding (SB) , and total binding (TB) are plotted as a function of free radioligand concentration.
  • the curves presented in Figure 18 are analogous to those predicted for any receptor mediated process, wherein nonspecific binding appears as an essentially straight line, and is essentially parallel to the high concentration region of the curve for total binding of radioligand.
  • Figures 19A through 19C provide competition/displacement curves for the binding of E. Coli, grown in commercially available nutrient broth.
  • known antagonists for the known adrenergic receptors are applied to cultures killed by sodium azide, to investigate if the binding of catecholamines is through one of the known ⁇ .,, ⁇ 2 , or /3-adrenergic receptors.
  • the value for zero concentration of cold norepinephrine is represented by a box, whereas the presence of cold norepinephrine is indicated by circles.
  • Figure 19A demonstrates that the addition of 3 H-prazosin, a known ⁇ 1 receptor antagonist, does not result in any binding that can be displaced by the addition of cold norepinephrine. If the receptor was of a known a, subtype, then a competition/displacement curve similar to that shown for Figure 17 would have been obtained.
  • Figure 19B demonstrates that the addition of 3 H-rauwolscine, a known ⁇ 2 receptor antagonist, does not result in any binding that can be displaced by the addition of cold norepinephrine. If the receptor was of a known ⁇ 2 , subtype, then a competition/displacement curve also similar to that shown for Figure 17 would have been obtained.
  • Figure 19C demonstrates that the addition of 3 H-dihydroalprenolol, a known jS, and /3 2 receptor antagonist does not result in any binding that can be displaced by the addition of cold norepinephrine. If the receptor was of a known ⁇ subtype, then a competition/displacement curve similar to that shown for Figure 17 would have been obtained.
  • Figure 20 provides a tissue dependency curve for E. coli.
  • specific binding (SB) to the novel receptor is proportional to tissue (bacterial) concentration at low levels, and counts of 3 H-norepinephrine specifically bound to its receptor on the bacteria plateau at high concentrations of bacteria when high numbers of receptors are present.
  • SB specific binding
  • DPM specific binding
  • Figures 17-20 demonstrate the specificity of this novel receptor for the catecholamines, it does not preclude that differences in receptor number (or density) , as well as receptor affinity, may differ among various infectious agents. Further, my preliminary findings indicate that the density and affinity of the receptor for a given infectious agent may differ as a function of the medium or nutritive environment in which the infectious agent is grown. For example, culture of the gram-negative bacterium Escherichia coli in a serum-containing medium results in a catecholamine receptor density and affinity which is higher than when E. coli is cultured in a nutrient broth of standard microbiological medium not containing serum.
  • a method of affecting the rate of proliferation of living organisms or vectors, such as bacteria may be effected by the introduction of a compound, such as a known agonist or antagonist, which will specifically bind with the novel receptor demonstrated in Figures 17 through 19.
  • a compound such as a known agonist or antagonist, which will specifically bind with the novel receptor demonstrated in Figures 17 through 19.
  • an initial assessment of a need to modify the level of presence of a neurotransmitter chemical, such as norepinephrine is performed. Such an assessment may be made by incubating the organism or vector, then evaluating the need to modify its population level.
  • this agent determines the antimicrobial agent and treatment protocol which will be administered.
  • this agent will be selected upon proper identification of a pathogen, typically by culturing.
  • a pathogen typically by culturing.
  • increased levels of norepinephrine are released by a host mammalian organism.
  • certain gram- negative pathogens have been identified as possessing a novel receptor for circulating catecholamines, and because growth is augmented in the presence of these compounds, it is desirable to block this receptor by administration of a suitable agent, since it is not possible, or if possible, not practical, to block the host organism's release of these compounds. Blocking this novel receptor in the presence of an abundance of such compounds ensures that norepinephrine-enhanced growth will be suppressed.
  • a microbe such as E. coli..
  • This may be accomplished on an industrial scale by including a catecholamine, such as norepinephrine, in the culture broth and insuring that this compound remains available as the population grows.
  • a catecholamine such as norepinephrine
  • populations of E.coli grown in a lactose broth feature a substantially increased yield of glucose when norepinephrine is added to the medium.
  • the method involved in obtaining this result includes the steps of determining the quantity of neurotransmitter substance required to produce a threshold effect on the rate of proliferation of the E. coli.
  • the living organisms or vectors which possess the genetic complement enabling possession of the novel receptor described herein include vertebrates, invertebrates, unicellular animals, multicellular animals, living tissue, unicellular plants, multicellular plants, and phages. Although described in reference to gram negative bacteria, this method has particular applicability to the control of proliferation of all infectious agents, including mycobacteria and viruses.

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Abstract

Procédé permettant de moduler in vivo/ou in vitro le développement bactérien par l'administration de substances neurochimiques. Le procédé implique la reconnaissance d'un nouveau récepteur de ces composés et comprend les étapes consistant à déterminer le besoin d'appliquer une substance chimique neurotransmetteur en fonction de la nature de l'organisme vivant, à déterminer la quantité de substance chimique neurotransmetteur nécessaire pour produire un effet sur la cadence de prolifération, à appliquer la substance chimique neurotransmetteur à l'organisme vivant, à déterminer l'efficacité de cette application dans la réduction ou l'accroissement de la cadence, et à répéter ces étapes de manière intermittente pour contrôler la cadence effective de prolifération, soit accélérée soit réduite. On décrit en outre un procédé dans lequel la substance chimique neurotransmetteur, telle que la catécholamine, est ajoutée à un milieu de culture de base afin d'accroître (ou de supprimer) le développement. Cette étape est utile dans la prodction commerciale d'organismes tels que des bactéries, et elle permet également d'augmenter la production de produits secondaires présentant une valeur commerciale, tels que le glucose ou l'éthanol.
PCT/US1992/005847 1992-03-06 1992-07-07 Composes servant a moduler le developpement d'agents infectieux Ceased WO1993018172A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115299438A (zh) * 2022-08-04 2022-11-08 云南民族大学 多巴胺在抗噬菌体中的应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 101, issued 1984, (Columbus, Ohio, USA), SUKMANSKII et al., "Role of Adrenergic Receptors in the Action of Catecholamines and L-Dopa on Proliferative Processes", see page 126, Abstract No. 144669b; & BYULL. EKSP. BIOL. MED. 1984, 98(7), 89-91, (Russ.). *
CHEMICAL ABSTRACTS, Volume 102, issued 1985, (Columbus, Ohio), DYER et al., "Norepinephrine Amplifies Human Chorionic Gonadotropin-Stimulated Androgen Biosynthesis by Ovarian Theca-Interstitial Cells", see page 105, Abstract No. 179661r; & ENDOCRINOLOGY (Baltimore), 1985, 116(4), 1645-52, (Eng.). *
CHEMICAL ABSTRACTS, Volume 97, issued 1982, (Columbus, Ohio), KHOTIMCHENKO, YU. S., "Effect of Adrenotropic Substances in the Growth and Maturation of Oocytes of the Sea Urchin Strongylocentrotus Nudus", see page 354, Abstract 36514n; & EXPERIENTIA 1982 38(6), 696-7, (Eng.). *
CHEMICAL ABSTRACTS, Volume 97, issued 1982, (Columbus, Ohio), MORGER et al., "Catecholamine Stimulation of Androgen Production by Mouse Interstitial Cells in Primary Culture", see page 156, Abstract No. 157029s; & J. ANDROL. 1982, 3(4), 227-31, (Eng.). *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115299438A (zh) * 2022-08-04 2022-11-08 云南民族大学 多巴胺在抗噬菌体中的应用
CN115299438B (zh) * 2022-08-04 2023-07-21 云南民族大学 多巴胺在抗噬菌体中的应用

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