WO1996039493A1 - Procede et appareil de modification d'interactions ioniques avec des produits chimiques et processus chimiques utilisant des champs magnetiques - Google Patents

Procede et appareil de modification d'interactions ioniques avec des produits chimiques et processus chimiques utilisant des champs magnetiques Download PDF

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WO1996039493A1
WO1996039493A1 PCT/US1995/012343 US9512343W WO9639493A1 WO 1996039493 A1 WO1996039493 A1 WO 1996039493A1 US 9512343 W US9512343 W US 9512343W WO 9639493 A1 WO9639493 A1 WO 9639493A1
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ion
magnetic field
response
resonance
ipr
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PCT/US1995/012343
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Carl F. Blackman
Janie Page Blanchard
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U.S. Environmental Protection Agency
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N2/00—Magnetotherapy
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present invention is directed to a method for using magnetic fields to alter ionic interactions with chemicals and chemical processes.
  • ICR ion cyclotron resonance
  • the ICR model predicts enhanced responses by specific ions when the AC frequency corresponds with the ICR model conditions, which are different for each ion, it does not indicate how the response might vary with different AC flux densities. Thus, the ICR model does not anticipate the distinct response form subsequently predicted for increasing B a c at constant B d c and f.e - Lednev (1991) incorporated Liboff's model, in a limited sense, in his examination of how parallel AC and DC magnetic fields might influence ions bound in ligand structures specific to Ca. + + .
  • the ion parametric resonance (IPR) model differs from Lednev 1 s model in three critical ways: it specifically includes a (-l) n term multiplying the Bessel function prediction, the IPR model Bessel function argument is twice that of the Lednev model, and the IPR model considers a wider range of candidate ions, through an expanded understanding of the role of the ion in creating a biologically significant change.
  • the IPR model considers the potential effects on any unhydrated ion, presumably bound within a molecular structure, that can influence the observed biological response.
  • the molecular structure may be composed of proteins, nucleic acids, or lipids, either singly or in any combination, as long as the structure itself requires an ionic cofactor to function.
  • Extension to unhydrated ions beyond Ca ++ can be inferred in part by the work of Liboff (1985, 1992) and Chiabrera and colleagues, op. ci . Background Art
  • Tissue and cell development have been studied extensively to determine the mechanisms by which maturation, maintenance and repair occur in living organisms.
  • development of a cell or tissue can be considered as a transformation from one state or stage to another relatively permanent state or condition.
  • Development encompasses a wide variety of patterns, all of which are characterized by progressive and systematic transformation of the cells or tissue.
  • tissue and organic development involve complex processes of cellular growth, differentiation and interaction mediated by complex biochemical reactions.
  • development is regulated by genomic expression; at the cellular level, the role of membrane interaction with the complex biochemical milieux of higher organisms is instrumental in development processes.
  • remodeling of tissues or organs is often an essential step in the natural development of higher organisms.
  • a fluctuating magnetic field is tuned to the specific cyclotron resonance frequency of a preselected ion such as Ca ++ or Mg ++ .
  • Liboff et al. in U.S. Patent No. 4,932,951 disclose the use of cyclotron resonance tuning to control the growth rate of non-osseous, non-cartiliginous connective solid tissue.
  • U.S. Patent No. 5,067,940 Liboff et al. disclose a method and apparatus based on cyclotron resonance tuning which allow the growth rate of cartilaginous tissue to be regulated.
  • An even more important use of cyclotron resonance tuning which is of particular significance in the treatment of elderly patients is disclosed in Liboff et al. U.S.
  • Patent No. 5,100,373 which deals with a method and apparatus for treating and preventing osteoporosis, both locally and systemically.
  • Additional patents granted to Liboff and his co-workers in the field of ion cyclotron resonance include U.S. Patents 4,818,697; 4,932,951; 5,045,050; 5,059,298; 5,067,940; 5,077,943; 5,087,336; 5,088,976; 5,100,373; 5,106,361; 5,123,898; 5,143,588; 5,160,591, and 5,193,456. All of the above-cited patents are hereby incorporated by reference in their entirety.
  • the mechanism of interaction postulated for the IPR model is distinct from that of the ICR model by virtue of abandoning a geometric concept and focussing on the ion's role within a molecular structure, such as an enzyme, protein, nucleic acid, and that the IPR model's consideration of candidate ions is therefore broader than is that of the ICR model.
  • n a frequency index, defined as the ratio of the (ion specific) cyclotron resonance frequency to the applied AC frequency.
  • a chemical or biological system enzyme, cell, organ, or organism
  • the present invention provides a method for altering ionic interactions in systems including cells and organisms using magnetic fields.
  • the method involves controlling the orientation and varying the intensity, and fluctuation frequency of paired static and sinusoidally varying magnetic fields in such a way as to create certain magnetic interactions between ions and the molecules with which they are associated.
  • the magnetic fields can be adjusted to control precisely the desired orientation, intensity and fluctuation frequency of the magnetic fields.
  • the desired parameters are calculated from a mathematical model developed to quantify the interactions of magnetic fields with ions and their associated molecules.
  • the method which comprises the present invention is based upon application of this mathematical model.
  • the present invention makes it possible to determine parameters necessary for causing particular ion- molecule interactions and can accurately define the desired magnetic fields.
  • the invention thus can be used for non- destructive characterization and evaluation of chemical and biological systems.
  • the ion parametric resonance (IPR) mathematical model of the present invention examines biological responses to parallel AC and DC magnetic fields and, by specifying the functional influences of all magnetic field parameters, provides detailed predictions of the expected atomic level responses.
  • the influence of the strength (flux density) of an AC magnetic field oscillates between maximal and no effect as the AC field is increased, not in direct proportion to the magnitude of the AC field, but as a function of a Bessel function of the first kind whose argument involves the ratio of the frequency index (defined within) and the magnitudes of the AC and the DC magnetic fields.
  • the static magnetic field that is perpendicular to the AC magnetic field must be reduced to near zero to avoid alternative interaction with the phenomena.
  • the static magnetic field is used for the identification of multiple ions for which this effect occurs, and the multiple chemical systems differentially stimulated concurrently using a single exposure condition, as well as a crucial role for hydrogen ions, which had heretofore been unrecognized.
  • alternative forms of response by a chemical or biological system are possible by adjusting the relative amount of B dc perpendicular to B ac .
  • the addition of a perpendicular B d c can dramatically attenuate or even eliminate the response caused by parallel B a c and B d c .
  • Substituting a B dc perpendicular to the B ac in place of the parallel B dc called for in the IPR model situation, can cause a reversal in the direction of the response in a chemical or biological system.
  • alterations may occur in any process that has an ion as part of a regulatory control mechanism, as in the case of ion gates in some protein channels in cell membranes.
  • biologically active compounds attached to carrier molecules or encased in vesicles designed with ionic cofactors may be released at particular sites in the body when the kinetic interactions of the particular ions with the carrier or vesicle are altered by appropriate exposure to magnetic fields.
  • the biologically active compounds may include an active component complexed with an inactivating agent, which includes an ion cofactor. The compounds are then rendered active or inactive, as desired, when the ionic interaction is changed by appropriate magnetic field exposure conditions with in a specific volume of space.
  • Figure 1 shows the results of Test 1 for inhibition of percent neurite outgrowth stimulated by nerve growth factor in PC-12 cells exposed to 45 Hz sinusoidal magnetic fields between 77 and 200 G(rms) ⁇ 108-283 mG(pk) ⁇ .
  • Figure 2 shows the results of Test 2 for reduced effectiveness of 45Hz sinusoidal magnetic fields between 200 and 468 mG(rms) ⁇ 284-662 mG(pk) to inhibit neurite outgrowth stimulated by nerve growth factor in PC-12 cells.
  • Figure 3 shows the results of Test 3 for the response of nerve growth factor-stimulated PC-12 cells to 45 Hz, sinusoidal magnetic fields under non-resonance conditions.
  • Figure 4 shows the results of Test 4.
  • Figure 4a shows a comparison of neurite outgrowth in cells stimulated by nerve growth factor and exposed to sinusoidal magnetic fields under resonance conditions by AC exposure frequency (45 Hz vs. 25 Hz where the 25 Hz responses are connected by lines) but plotted without regard for the different B dc flux densities that are present in the 45 Hz v. 25 Hz cases.
  • Figure 4b shows the results from each run when the different B dc flux densities are included as part of the independent variable (plotted on the horizontal axis) to indicate the IPR model predicted Bessel function argument.
  • Figure 5 shows the ad hoc fit of an IPR model to acquired data.
  • Figure 5b shows the near constant results across the range of B ac for off-resonance conditions, Test 3.
  • Figure 6 shows a best fit to data using the simplest interpretation of the IPR model: a weighted sum of Bessel function responses selected by ions at or very near resonance.
  • Figure 7 shows an improved fit when the response associated with the hydrogen ion is considered exclusively for the low B ac response, with a conversion to a weighted sum of Bessel functions at a mathematically determined optimal coversion point.
  • This result suggested a special role for the hydrogen ion, that was later confirmed by other experimentalists (Trillo et al., 1994). In that work, the conversion point identified here also showed a critical distinction in the data.
  • Figure 8 demonstrates continued consistency of PC-12 cell responses with IPR model predictions under specific IPR model prescribed combinations of AC and DC magnetic fields.
  • the IPR model fit to the data in Figure 7 was extended to higher values of B ac , where the results of experimental tests at those values were then plotted. This match of the data to such an unusual predicted response form is extremely unlikely to occur by chance.
  • Figure 9 shows that cells prepared in the standard medium, RPMI 1640, exhibited the IPR model anticipated response form whereas those prepared in Iscoves' medium exhibited a minimal response, nearly indistinguishable from an IPR model predicted off-resonance response form, although the magnetic field exposure conditions for each of these test were essentially identical. This result is consistent with the effect of magnetic fields being localized to the cell plasma membrane.
  • Figure 10 demonstrates an alternate biological/chemical system response to IPR model specified conditions.
  • Rat liver cells in culture (clone 9) exhibit gap junctional intercellular communication by transferring a fluorescent dye away from the site of application, which is quantified by scoring the number of rows away from the application point that are stained with the dye.
  • IPR exposure conditions similar to those described for neurite outgrowth in PC-12 cells, the expected U-shaped exposure response is observed.
  • PC-12 measurements show, in a preliminary way, the reasonableness of extending the IPR model to systems other than PC-12 cells.
  • Figure 11 shows the neurite outgrowth response of cells exposed over a field-strength range of 45-Hz AC magnetic fields under various different B dc orientations and flux densities.
  • the mean and 2 • SE of the percent neurite outgrowth (%N0) is plotted for four different DC magnetic field conditions.
  • H(5 trials) is for 366mG perpendicular, ⁇ 2mG parallel DC magnetic fields.
  • H&V(3 trials) is for 366mG perpendicular, 366mG parallel DC magnetic fields.
  • 0.4H&V(3 trials) is for 160mG perpendicular, 366mG parallel DC magnetic fields.
  • V(3 trials) is for ⁇ 2mG perpendicular, 366mG parallel DC magnetic fields.
  • Ionic cofactors and reaction centers and their dynamic interactions driven by thermal motion are critical elements in biological activities. At biologically relevant temperatures the enzyme molecules are immersed in a bath of solute molecules vibrating at infrared frequencies. Thus, it follows that ionic cofactors, their associated enzymatic reaction centers, and their dynamic interactions driven by the ever present thermal bath, are critical elements in biological activities. "Thermal” means the average translational kinetic energy of molecules. Native proteins at biologically relevant temperatures are not static forms, but fluctuate constantly, passing through a variety of similar configurations due to thermal influence. Karplus and Petsko (1990) point out the importance of this kinetic view of proteins by stating that "i would not be surprising if internal motions had been subjected to selective pressure during evolution.
  • thermally driven kinetic motion is an essential element of protein function, with functional selection of specific motions or forms evolvin over time.
  • some enzymes have ligand-bound ions that can impart stability and conformational changes necessary for reaction site to orient to optimal enzymatic activity.
  • Rosenfelder et al (1988) note that different conformational states of a working protein have the same overall structure and function but have varying structural details and rates at which the function is performed.
  • Bialek et al. (1989) suggest that the most important enzyme configurations are those that reflect the optimal compromise between structures with high reaction probability and small strain energy in the protein.
  • Blackman 1994; Blanchard et al. 1994 a,b,c) predicts very distinct responses that are consequences of multiple, independent variables of exposure including the DC, or static, magnetic field flux density as well as the AC magnetic field flux density, frequency, and relative orientation to the DC magnetic field.
  • the IPR mathematical model is the first to assemble them in a coherent, experimentally accessible manner and to provide a clear indication of the expected magnitude of result, relative to that of an unexposed sample, for any given combination of the independent variables.
  • the IPR model in its simplest form, assumes an effect on an enzyme that is complexed with an ionic cofactor to perform its catalytic function in some reaction pathway. Magnetic fields, under conditions described by the IPR model, cause minor but significant changes in the ionic interaction with the enzyme that can alter its rate of reaction. This altered reaction rate can have biological consequences for the whole cell, and consequently for the organisms.
  • the IPR mathematical model is based upon an earlier derivation of the influence of parallel exogenous AC and DC magnetic fields at the atomic level by Podgorestkii and Khrustalev (1964).
  • Podgoretskii's derivation for atomic spectroscopy was extended to biological systems by Lednev (1991) .
  • Lednev's model contained some critical mathematical errors and focussed strictly on a limited set of ions that could be bound to the Ca ++ binding protein.
  • the present inventors have corrected the errors in the Lednev model, extended the set of ions potentially influenced by magnetic fields, and described the expected response form when the energy levels of two or more resonant ions are altered by external magnetic fields.
  • an external DC magnetic field creates a Zeeman splitting of the quantum energy levels of each ion. These split energy levels are then frequency modulated by an external AC magnetic field.
  • the involvement of frequency modulation suggests that the IPR response is distinct from the random effects of amplitude modulated thermal noise.
  • Frequency modulation does not requir addition of kinetic energy to the system. Rather, frequency modulation locally alters the potential energy of the system.
  • the IPR mathematical model indicates that, although the potential energy alteration may be small on the global scale (and certainly less than the overall thermal noise level) , the resultant small changes created loca3.1y in the population distributions may be significant in producing specific biological effects if associated with an ion resonance.
  • a fundamental parameter of the IPR mathematical model is the frequency index, which is the ratio of the ion's characteristic resonant frequency, f c , to the frequency of the AC magnetic field oriented parallel to the DC magnetic field,
  • n f c /f ac (1)
  • f c the characteristic resonant frequency in order to avoid confusion with Liboff's ion cyclotron resonance models.
  • the critical term is n, describing the key relationship between system specific (ion) parameters (q/m) and externally imposed conditions (f ac and B dc ).
  • n defines an ion resonance condition associated with a specific splitting of an energy level, arising from the applied DC magnetic field.
  • the IPR mathematical model examines how the probability of ion transitions to lower energy levels changes when the ion is near resonance. According to the IPR model, the probability of ion transition, p, is given by the equation
  • the IPR mathematical model predicts that when the applied DC field and AC frequency create a resonant environment for an ion, the probability of transitions between energy states associated with that ion will be modified in a deterministic way.
  • the modification for that ion is proportional to a Bessel function whose order is selected by the ion's integer-valued frequency index. Whether the contribution from the Bessel function is additive or subtractive, at least at the atomic level, is also determined by that ratio, with odd integer values for the ratio inverting the sign of the Bessel function because of the (-l) n term. This distinction is expected to be significant primarily at the molecular level since at more complex levels (cellular, organ, or organism) , molecular actions may reinforce or restrain the selected biological/chemical endpoint.
  • the IPR model derives a relationship between the fundamental ICR frequency, f c , and integer multiples of the applied AC frequency that is the inverse of ICR model harmonics.
  • the IPR model explicitly recognizes that a system's response may reflect the combined influence of several different near resonance ions. In the absence of contrary information, ions are assumed to act independently to produce the observed response, and the IPR model predicts that the response will be a linear sum of the individual response functions uniquely characteristic of the ions within the system.
  • the ions at resonance are assumed to be in the unhydrated state. This situation may be found, for example, when transition metal ions are loosely bound by ligands in a molecular structure.
  • Table 1 lists biologically significant ions (compiled from Liboff, 1985, 1992; Liboff and Parkinson, 1991; EPRI, 1990; Abrams and Murrer, 1993; Karlin, 1993; Lippard, 1993; O'Halloran, 1993; Pyle, 1993; Regan, 1993; Thomas et al., 1986) for which IPR model predictions have been made, and shows how the frequency index for each ion changes with variations in either the flux density of the DC magnetic field or the AC magnetic field frequency. This table is not a all inclusive list.
  • Appearance of an ion in Table 1 indicates a potential biological role but does not imply significant activity of any particular ion in a given system.
  • the present inventors have included all valences and have not limited the selection to th oxidation states normally considered biologically relevant, as these may exist momentarily in some biological/chemical system as intermediate states and could be affected by the imposition of a magnetic field.
  • the masses used are for unhydrated ions, as required by the model and supported by the work of Chiabrera and Bianco (1991) ; otherwise, the effective ionic mass could be infinitely variable.
  • the possible frequency indices are 1 (Ca2, Co3, Ni3, Fe3, Mn3) , 3 (Li) , and 19 (hydrogen) .
  • Ions with closely related frequency indices at given values of B d c and f ac can most easily be distinguished at higher B ac flux densities. Effects predicted by the IPR mathematical model are most easily tested when exposure values create resonance conditions for either a single biologically significant ion or only one near-integer-valued frequency index. When the exposure values " create resonance conditions for a variety of ions representing several frequency indices, the resulting response function may become quite complex, requiring an extensive number of exposure test points to sample that response function unambiguously.
  • the enhanced sensitivity predicted by the IPR mathematical model for small changes in B ao as B dc approaches zero is limited by the finite bandwidth of ionic resonances.
  • Control of the AC frequency and the DC field strength so as to create a resonance for an ion suspected to be active in the creation of said biological/chemical action within the biological/chemical system is such that the ratio of the ion's charge to its mass is an integer multiple of the ratio of the angular frequency of the AC magnetic field (2*pi*f ac ) to the flux density of the DC magnetic field.
  • the resonance claimed here is the mathematical inverse of the Ion Cyclotron Resonance already addressed by Liboff et al. Therefore they are the same ONLY when the integer value of the present claim is uniquely one. Further, the ions considered by ICR are a subset of those specified by IPR.
  • An ion may be suspected to be active as a result of previous research (see for example the list of ions identified through other studies as having potential biological significance given as Table 1 of this application) , or it may be identified as a result of testing for other ions (see for example the work of Blackman et al., 1994, that identified hydrogen as a potentially significant ion and the subsequent work by Trillo et al., 1994, confirming a biological role in accordance with the claims of this application) .
  • K 2 K 2
  • Varying B dc by itself would bring different ions on and off resonance, changing their frequency indices in the process. Since the IPR mathematical model predicts a flat response (p - constant) when no ion is at resonance, detuning for all ions through an appropriate selection of the DC flux density tests the IPR model while eliminating one of the variables in which B dc plays a role (no Bessel function is selected, so there is no influence on the argument) . This provides a second critical test of IPR model applicability.
  • Hydrogen provides the limiting case: its charge to mass ratio is the largest possible of all elements and it is a potential biologically significant ion.
  • B d c is chosen and fixed such that the frequency index for hydrogen is well below unity, and the same f ac used in the previous test is maintained in this test.
  • test points should be generated to test the system response over a range of Bessel function arguments (n x 2 x B ac /B dc ) comparable to those tested in the previous case.
  • the predicted system response under these conditions is flat, with no variation across the entire range of AC field values tested (see Figure 5b) . Changes in f ac
  • Test points should then be generated to give the same argument to the Bessel function(s) as in the first test. In essence, this performs the first test at a different frequency.
  • the predicted response function is the same as that predicted for the first test (e.g., U-shaped if the J x term is dominant).
  • the neurite outgrowth (NO) these cells display in response to stimulation by nerve growth factor (NGF) has served as a basis world-wide for investigations of NGF-induced changes in nervous system- erived cells (Levi et al., 1988) since Greene and Tischler (1976) first established the line from a rat adrenal pheochromocytoma.
  • the PC-12 assay system has been shown previously to be differentially responsive to AC magnetic fields. The assay is simple, well established, and widely used among those skilled in the art of neuroscience. Because this system examines functions at the isolated, single-cell level, its simplicity is of considerable advantage to theoreticians studying the interactions of electromagnetic fields with biological systems.
  • This neurite outgrowth assay system is currently used to evaluate neurotransmitted production and second messenger signaling processes and subsequent genomic events, particularly those induced by nerve growth factor stimulation.
  • Previously tests demonstrated that NGF-stimulated NO in PC-12 cells could be inhibited by exposure to 50 Hz magnetic fields during a 22 hour treatment period (Blackman et al., 1993) .
  • the response demonstrated a magnetic field strength dependence across the range of 35-90 mG(rms) ⁇ 3.5 and 9.0 ⁇ T (rms) ⁇ .
  • the inhibition was further shown to be independent of any induced electric field.
  • These exposures consisted of a vertical AC magnetic field with an ambient static DC magnetic field, wherein the DC field contained both vertical and horizontal components. At the time, there was no apparent explanation for the observed changes in NO in PC-12 test cells.
  • PC-12 cells used in these experiments were obtained from the Tissue Culture Facility at the University of North Carolina at Chapel Hill.
  • the cells were primed by growth in RPMI 1640 medium (Gibco 320-1875) , supplemented with 10% horse serum (Gibco 200-650) , and 5% fetal calf serum (Gibco 240-6000) , and 100 units/ml each of penicillin and streptomycin on six-well collagen-coated plates (Costar 3046) in a 5% C0 2 incubator at 37°C, with addition of NGF (50 ng/ml, 2.5 S, Sigma 6009) at plating and every other day for six days.
  • RPMI 1640 medium Gibco 320-1875
  • horse serum Gibco 200-650
  • fetal calf serum Gibco 240-6000
  • the medium was removed from the primed cells and the cells were washed three times with complete medium to remove any remaining NGF.
  • the cells were then removed from the plates by trituration, centrifuged, resuspended, counted, placed into 10% dimethylsulfoxide (DMSO) , frozen, and stored at -80°C in 1 ml volumes of 1 x 10 6 cells/ml. Prior to each experiment, the cells were thawed and rinsed with medium three times, and the contents of one ampoule were placed into 50 ml of medium.
  • DMSO dimethylsulfoxide
  • the collagen-coated petri dishes were prepared by placing 0.45 ml of stock solution ⁇ 5 mg of collagen (Sigma rat tail type VII 8897) in 125 ml of 0.1 M acetic acid) on 60 mm dishes (Costar 3060) . The dishes were then air dried in a sterile hood. Before use, the dishes were rinsed with medium to neutralize the pH. Five milliliter volumes of primed cells, at 2 x 10 4 cells/ml, were plated onto 60 mm collagen-coated petri dishes. At this plating density, the cells covered less than 10% of the growth surface.
  • the cell medium was supplemented with 5 ng/ml of NGF (Sigma 6009) for all but the zero NGF control. This NGF concentration had previously been shown to induce NO in approximately 50% of the cells (Blackman et al., 1993). All dishes in the exposure system contained 5 ng/ml of NGF. To establish the control parameters in each experiment, two dishes with and two dishes without NGF were placed within the control (nonexposed) area in the same incubator. The one exception to this occurred for one trial each of tests 1 and 2, when only one dish was used for each control condition. Magnetic Field Exposure System
  • the PC-12 cells were exposed to prescribed sets of parallel AC and DC magnetic fields while housed in a 5% C0 2 incubator maintained at 37°C. Prior to testing, the ambient AC and DC fields were measured with a Bartington MAG-03 fluxgate magnetometer. The ambient DC magnetic field within the exposure apparatus was 447 mG at an inclination of 60°N (389 mG vertical and 220 mG horizontal) .
  • the ambient 60 Hz magnetic field in the exposure apparatus was 8.8 mG(rms) ⁇ 7.0 mG(rms) vertical and 5.3 mG(rms) horizontal ⁇ , whereas within the shielded area it was 1.2 mG(rms) ⁇ 0.74 mG(rms) vertical and 0.92 mG(rms)horizontal ⁇ .
  • the following description is one of many possible embodiments of the present invention by which controlled magnetic field exposures can be created. This description is included merely for illustration, and not for limitation.
  • a pair of Helmholtz coils consisting of two 100- turn, 20-cm. diameter coils of enameled wire (22 awg; 35 ohms resistance per coil) , aligned coaxially 10 cm apart, were oriented and energized to control the vertical DC and AC magnetic fields (cf. Blackman et al., 1994). As needed, both coils were energized with a direct current to adjust the ambient, vertical DC magnetic field in the sample area. Only the lower coil was energized with AC current to create sinusoidal magnetic fields of decreasing strength on the coil axis as a function of distance above the coil. The six PC-12 samples to be exposed were placed coaxially with the coil center line.
  • a seventh dish was added within the stack to provide the desired range or spacing of B ac .
  • the exposure system consisting of the coil systems and cell samples, was located on a plastic shelf in the upper two-thirds of the incubator space.
  • a Co-netic metal magnetic- field shield (Magnetic Shield Corp.) created a shielded area, also within the incubator, to serve as a control, or unexposed, area. This shield reduced the flux density of the magnetic field generated by the exposure system to less than 1% of ambient (cf. Blackman et al., 1994).
  • the Co-netic shield was in a tube configuration position near the bottom of the incubator space, with the long axis fore and aft to allow for air circulation and ease of positioning the dishes.
  • the PC-12 cells were exposed for 23 hours, beginning within three minutes after plating, to examine responses to four distinct tests, shown in Table 2. (Note that these conditions did not tune for calcium ions.) The observed effects were both consistent with the predictions of the IPR mathematical model and confirmed the validity of extending the ion list beyond calcium. Additionally, the repeatability of the results was checked by running tests 1, 3 and 4 three separate times and tuning test 2 four times. For every exposure, two sets of controls were placed in the shielded area: one set with and one set without NGF.
  • a common reference point was selected, consisting of an AC frequency of 45 Hz for tests 1 - 3 , B d c V of 366 mG (ambient) for tests 1 and 2 , and a horizontal DC flux density (B d c H) reduced to as close to 0 mG as possible (less than 2 mG in all experiments) .
  • the ratio of B d c to f a c is the same as for tests 1 and 2, so that Table 3 remains applicable.
  • Test 3 was designe d specifically for off-resonance conditions for all ions listed in Table 1.
  • Mn(4) , V(4) , Mg ( 2 ) , Li(l) , and H(l) are within 10% of an integer- alued frequency index.
  • Ca(2) and Fe(3) are well off- resonance. Note that, as either the DC field flux density or the AC field frequency changes, the number and closeness of ions to resonance conditions will vary. A more complete list of potentially biologically significant ions is given in Table 1 of this application.
  • ions involved in neurite outgrowth or other biological/chemical changes may involve at least one or more other critical biochemical steps. It is possible that some of the molecular-level actions could oppose each other so that observed biological responses, in contrast to molecular- level responses, may in some instances be small because of potential offsetting influences of different ions. For example, the analysis below suggests that lithium may play a minor opposition role vs. that of the other ions. Because, under the exposure conditions selected from these tests, the frequency index of 1 is common to magnesium, vanadium, and manganese, it is more difficult to determine directly exactly which molecular processes, e.g., enzyme reactions, are affecte in the exposures.
  • the cells were assayed in a blinded fashion from pseudo-random (i.e., non-overlapping) areas near the center of each dish.
  • the neurite outgrowth assays determined the number of cells either with a neurite length greater than the cell body or with neurites containing either a branch or a growth cone (these accounted for approximately 5% of the total cells scored positive) to create a raw cell scoring. All cells were counted within each microscopic field, and at least 200 cells per dish were assayed. However, each set of primed cells can produce a slightly different response to NGF.
  • the AC magnetic field was colinear with the DC magnetic field of 366 mG.
  • a fifth run was conducted at 45 Hz under conditions ⁇ B ac range of 132- 344 mG(r s), or 186-486 mG(pk), generated by energizing the coil with 6.7 mA(rms) ⁇ that overlapped those in tests 1 and 2 to verify continuity of the test results across the range of AC flux densities.
  • the results of Test 2 demonstrate the reduced effectiveness of 45Hz sinusoidal magnetic fields between 200 and 468 mG(rms) to inhibit neurite outgrowth stimulated by nerve growth factor in PC-12 cells.
  • the IPR model predicts a constant response across the AC flux density range chosen for this test ⁇ 7.9-21 mG(rms), 11-29 mG(pk) , 0.405 mA(rms) in the coil ⁇ . This prediction was confirmed by the empirical data shown in Figure 3, replicated three different times. As noted above, under resonance conditions, this range of B ac /B dc produced a U-shaped inhibition response, as shown in Figure 2. The final test of the series evaluated whether the on-resonance results observed for tests 1 and 2 can be obtaine at a different frequency.
  • Figure 4 shows the results of three repetitions of this test in comparison to the combined results of tests 1 and 2.
  • Figure 4a plots the data in terms of the AC flux density of B ae (rms)
  • Figure 4b plots each response a a function of 2 «B ac (pk)/B de , as suggested by the IPR model.
  • These results demonstrate essentially identical cellular responses as a function of exposure when compared in terms of the Bessel function argument, 2»B ac (pk)/B dc , indicated by the IPR model, but not when B a c is used alone as the common point of reference. This result highlights the importance of using the IPR model identified form of Bessel function argument when identifying and comparing results under different exposure conditions.
  • the IPR mathematical model indicates how B d c and the AC frequency select responses from ions based on their charge to mass ratio.
  • the model is unique because it is the first mathematical model to predict specific, distinct responses based on the experimentally controllable variables B a c , B d c and f a c .
  • the experimental results show that full characterization of the independent variables, B ac , B dc and AC frequency, is essential.
  • One of the crucial distinctions between the IPR model and its predecessors is the extension of proposed influence to a variety of ions beyond calcium and magnesium that have shown biological significance. It was discovered that the biological/chemical system itself determines whether any particular ion is sufficiently near resonance to create a change in the selected observable (in this case, neurite development) .
  • the IPR mathematical model suggests that, as a first approximation, each ion functions independently to produce the observed response and that the overall response will be a linear, weighted sum of the individual response functions, unless there is evidence to the contrary.
  • the normalized data were fit using the iterative multivariate secant method to determine least squares estimates of the coefficients (K 2 > x ) of the Bessel functions in the IPR-predicted response form corresponding to frequency indices of 1, 2 and 12: Pit- 100 -
  • the first three tests described above were extended by exposing the PC-12 cells for 23 hours to AC magnetic fields over three higher ranges ⁇ (233-544, 430-1002, or 607-1416 mG(rms) ⁇ with a parallel DC magnetic field of 366 G and a perpendicular magnetic field of ⁇ 2mG (Blackman et al., 1995a).
  • the neurite outgrowth assay was used. After 23 hours' exposure to the magnetic fields, pseudo-random areas near the center of each dish were selected for cell counting. All cells within each microscope field (minimum of 200 cells per dish) were assayed in a blinded fashion.
  • the cell response to higher values of B ac exhibited the predicted oscillatory pattern, but the observed pattern showed a distinct characteristic of the test system of the present invention: the simplest application of the IPR mathematical model predicts cycles of inhibition and enhancement (values greater than control values) of a biological response, whereas in the above-noted experiments an oscillating pattern was observed that cycled between inhibition and control values. This suggests that in the PC-12 system, the control (unexposed) values represent a maximal response of the system under the IPR model specified parallel AC and DC magnetic field exposure conditions used in these experiments.
  • the neurite outgrowth of PC-12 cells under predicted resonance conditions for the hydrogen ion were evaluated using three different combinations of AC and DC magnetic fields.
  • the first set of tests examined the nerve outgrowth response in cells exposed to 45 Hz magnetic field with a B a e ranging from 2.9 to 41.1 mG (rms) in the presence of DC magnetic field of 29.6 mG, over which a U-shaped resonance was obtained.
  • the IPR model predicts a "U-shaped" curve of inhibition of nerve outgrowth as a function of the values of B a c .
  • the neurite outgrowth was analyzed.
  • the results showed that the nerve growth factor stimulation of neurite outgrowth in PC-12 cells was affected as predicted under field conditions in experiments 1 and 3, but not under the conditions of experiment 2, the off-resonance condition.
  • the apparent lack of response to specific B a c values over this range suggests that an additional, perhaps hydrogen specific, mechanism could be involved in the particular response of the PC-12 system.
  • PC-12 cells for exposure to magnetic fields was conducted as described above, with one exception. In addition to experiments exposing six dishes of cells simultaneously, in one set of experiments only three dishes were exposed.
  • the exposure system was housed in a cell culture incubator.
  • the AC and DC fields were verified at the sample locations with a Bartington MAG-03 fluxgate magnetometer and representative values are given.
  • the ambient DC magnetic field within the exposure apparatus was 387 mG at an inclination of 58°N (327 mG vertical and 207 mG horizontal) .
  • the ambient 60-Hz magnetic field in the exposure apparatus was 0.86 mG rms (0.50 mG rms vertical and 0.70 mG rms horizontal).
  • the ambient DC magnetic field was 298 G at an inclination of 82°N (295 mG vertical and 44.1 mG horizontal) .
  • the ambient 60-Hz magnetic field was 0.84 mG rms (0.59 mG rms vertical and 0.60 mG rms horizontal). Comparison experiments between the two incubators showed no difference in cell response, measured as the percent of cells displaying neurite outgrowth, when the coils were not energized.
  • the tests described above included the response of the cells exposed to six flux densities of 45 Hz fields over the range 132-344 mG rms, with the B dc perpendicular ⁇ 2mG/parallel 366 mG. Two additional tests were conducted under these B dc exposure conditions (designated V, for vertica field) . Other flux densities and alignments of B dc with the A field were then tested directly at least three independent times, using B dc :
  • the neurite outgrowth assay used in the above- described experiments was used. These assay procedures for neurite outgrowth are well established (Greene and Tischler, 1976; Greene, 1977; Blackman et al., 1993, 1994) . The results displayed internal consistency between the three to five repetitions of each test. Further, they were consistent with the earlier data where there was an overlap of AC magnetic field intensity.
  • Case H is for perpendicular 366 mG/parallel ⁇ 2mG DC fields.
  • Case V is for perpendicular ⁇ 2mG/parallel 366 mG fields.
  • Case H&V is for perpendicular 366 mG/parallel ⁇ 366 G.
  • Case 0.4H&V is for perpendicular 160 mG/parallel 366 mG. It is apparent from case H compared to case H&V that the perpendicular component of the DC magnetic field dominates the cell response. The analysis of variance of the experimental design is shown in Table 4.
  • a combination of parallel oriented AC and DC magnetic fields can be used to influence a biological system (i.e., chemical mixture, organelle, cell, tissue, or organism) , perhaps through an ion- molecule or ion-enzyme interaction, with the magnetic fields altering the form of that interaction for specific ions.
  • the ions affected by the applied AC and DC magnetic fields are identified according to the mathematical equations given above. It has also been found that the hydrogen ion has a distinct role. The function of the hydrogen ion can, under certain circumstances, suppress the magnetic field influence on the function of all other ions.
  • the IPR mathematical model described above one can investigate and diagnose mechanisms involved in chemical and biological reactions entities such as in an organism, a group of individual cells, and even isolated organelles or chemical mixtures. Since the IPR mathematical model conforms to the predicted behavior of the application of parallel magnetic fields, one can affect the behavior of a group of cells or an organism by subjecting the entities to the influence of parallel magnetic fields. By varying three basic parameters, namely, the AC magnetic field, the DC magnetic field and the AC magnetic field frequency, one can examine on and off resonance cases.
  • the resonance for a given ion is any combination of B d c and f a c that produces a frequency index (n) within 10% of an integer value.
  • the overall response of the biological/chemical entity for example a biological system which could be as simple as a biochemical reaction mixture or could be as complex as an organism, can be changed by exposing to parallel magnetic fields to create a resonance for a selected ion or ions.
  • a resonance for the ion(s) By creating a resonance for the ion(s) , the interaction of the ion(s) with its(their) biomolecular environment(s) (e.g., enzyme, transfer RNA, DNA) changes the overall response of the biological system.
  • This response is predictable across a range of intensity values of B ac (measured as peak, not rms) as defined by equation 3 above. Examples of such response include controlling the rate of tissue growth, stimulating bone growth rate, and preventing or treating osteoporosis.
  • an ion is selected which is characteristic of the chemical reaction to be studied.
  • membrane surface phenomena such as receptor aggregation and involution and membrane components affecting surface molecules.
  • This technique can also be used to evaluate and perhaps alter the function of membrane components, e.g., gap junction intercellular communication (gjic) as well as ion channels and pumps.
  • gjic gap junction intercellular communication
  • Measuring gap junction intercellular communication is also useful in measuring toxic effects on developing organisms which may lead to abnormalities. Processes occurring in the nuclei of cells can be measured and altered, including DNA conformational changes and the action of gene repressors and inducers.
  • RNA's such as zinc fingers
  • enzyme activities that require ionic cofactors protein and composite structures requiring ions for activity, such as calmodulin, cytochromes, and ribosomes.
  • Cellular events can be studied and altered using the techniques of the present invention. Among these are cell differentiation, such as produced by nerve growth factor action. Cell proliferation, such as segregation of chromosomes, can be studied and altered, as abnormal segregation leads to micronuclei. Ions involved in cell secretion processes can be stimulated by IPR model selected parallel magnetic fields; among the cellular components and functions associated with specific ions that can be studied include nerve synapses, immune factors and antibodies, and hormone and endocrine activities.
  • the process of the present invention can also be used to study the action of hormones on cells, e.g., melatonin and growth factor interaction on cell response.
  • hormones on cells e.g., melatonin and growth factor interaction on cell response.
  • the effect of toxic chemicals on cells can be studied by exposing the selected ions in the cells to parallel magnetic fields and measuring the ion's response to the resonance imposed. This technique can also be used for studying the modification of cells by cellular, tissue or organism generated molecules.
  • Chemical and biological processes and reactions can be altered by selecting an ion which is involved in the proces at issue.
  • the process or reaction is exposed to parallel magnetic field in such a manner as to create a resonance for a selected ion or ions, such that the balance between alternative, competing chemical pathways is altered, thus leading to a different mix of products.
  • SUBSTITUTESHEET(RULE26 molecular process and reaction in this manner one can alter the relative dynamics in interactive metabolic network of chemical reactions, such as cellular biochemical cycles.
  • This technology also permits control of where a reaction will occur in a sample or a body by imposing localized resonance conditions on the sample or the body.
  • This process permits alteration of receptor-ligand interactions, including growth factors and antibodies, and changes in ion channel gating actions.
  • imposition of resonance on a selected ion according to the present invention permits altering information at the gene level, such as induction and repression.
  • Molecular information transfer to and status in the cytoplasm can be altered, including RNA processing and activities, as well as protein production and function.
  • An organism's response to chemical environmental agents can be altered by choosing an ion which interacts either with the agents or biochemical target molecules of those agents. The organism is then exposed to parallel magnetic fields so as to create a resonance for the selected ion. The strength and relationship of the magnetic fields are altered s as to diminish the organisms response to opioids or to free radicals. Additionally, the magnetic fields can be chosen so as to reinforce activity in selected locations in the brain to enhance memory retention.
  • the toxicity of chemicals can be altered.
  • Enhancing the activity of a cytotoxic chemical, particularly at a selected site in an organisms can inhibit or treat cancerous cells.
  • the chemica treatment which destroys rapidly growing cancer cells is enhanced by application of the invention to alter the function of gap junctions, ion channels or receptor signaling systems, and by damaging cell division processes. Enhanced damage to cancer cells is also useful in inhibiting metastasis of cancer cells.
  • the process of the present invention can be used in conjunction with conventional chemotherapy in treating cancer.
  • Imposition of resonance on a selected ion can be used to modulate hormonal action on cell processes, the ion being selected based upon the cellular ions involved or upon the ion involved in the hormonal action. Stimulation of ion resonance can also be used to alter tissue and organ system functions, such as learning (acquisition of information) , memory (retention of information), and immune response to challenges.
  • the action of growth factors can be altered by imposing resonance on at least one selected ion in the growth factor stimulated system.
  • An example of this is altering the role of nerve growth factor in maturation and repair of cells.
  • Imposition of resonance on a selected ion in a cell or tissue can be used to alter cell surface events in immunological response, such as challenge from an infectious agent, or stimulation by autoimmune processes.
  • ionic resonance conditions in small selected volumes within a body can be used to activate biologically active compounds directly at a particular site in the body. This is of particular importance in delivery of chemotherapeutic drugs, which are often toxic to healthy cells as well as to tumor cells.
  • the biologically active compound is attached to a carrier molecule or component which has an ionic cofactor necessary for the entrapment of the compound. Imposition of an appropriate magnetic field at the target site can change the ionic interactions, causing the conjugate molecule to release the active compound from the carrier molecule or component thereof.
  • a detectable label may be added to the ionic cofactor to determine when the conjugate has reached the target site. This determines when appropriate fields can be applied.
  • carrier components include molecules and membrane vesicles to transport chemicals.
  • the chemical could be in an inactive state and rendered active at
  • BSTITUTE SHEET fiULE 28 the site by imposition of the resonance on a selected ion complexed with the chemical.
  • a chemotherapeutic drug is contained in liposome-like vesicles which contain ion- controlled pores that open under resonance conditions. Drugs can be engineered to be inactive under normal conditions, but active when they are within a volume in which the resonance condition exists.
  • an ion is selected which is characteristic of the chemical reaction to be studied or altered.
  • the ion is exposed to parallel magnetic fields in such a manner as to create a resonance for the ion, and the degree of change in the chemical reaction thereto desired is controlled by adjusting the applied AC magnetic flux density.
  • a variety of chemical steps in the movement of information from the cell's exterior to the nucleus, via signal transduction processes are susceptible to alteration by magnetic field which affect ionic components in these processes. This alteration of the ionic components is thus used to reveal the parts of the processes that are involved as well as their cellular locations.
  • the ion-related processes that bring instructions from the genes to the cytoplasm and are involved in the assembly and function of molecular components to perform specific cellular functions can conceivably also be examined and altered or controlled using IPR methodology.
  • Imposition of resonance on an ion and subsequent controlled alteration in response using IPR methodology can be used to selectively change chemical/biochemical reactions and thus the interaction of a biological system with its environment. Alteration in specific ion-related biochemical steps in complex, interactive metabolic pathways can cause changes in final reaction products and thus in biological processes, including integrated functions of an organisms. More specific examples of applications of IPR methodology are given below. However, these examples are given solely for purposes of illustration, and are not meant to be exclusive or exhaustive.
  • IPR methodology can be applied to investigate and regulate the functioning of ion channels, which often have transition metal cofactors in gating regions, as well as ion pump processes which actively segregate ions between different cell compartments and the exterior of the cell.
  • Other Processes Other critical processes in the cell that are susceptible to diagnosis and control by IPR methodology are those which involve an ion that can be subjected to resonance.
  • DNA molecules in the nucleus are complexed with molecules that act as structural and control elements for reading the genetic code.
  • Ions including magnesium, manganese and hydrogen, have central roles in providing forces to maintain and alter DNA conformation, and for information processing of genes.
  • biochemical processes in the cytoplasm of cells can be investigated using IPR methodology.
  • Some transfer RNAs use zinc complexes to confer selectivity in function.
  • Hydrogen is important for conformational specificity, and may be examined for the relative stability it confers on the RNA.
  • enzyme activities require ionic cofactors and hydrogen, as do some protein and composite structures, e.g., calmodulin, cytochromes, and ribosomes.
  • IPR methodology can be used to understand and regulate the processes by which chemicals cause abnormalities during development. Further, since intercellular communication is important to maintain homeostasis and prevent abnormal growth in the mature organisms, IPR methodologies can be incorporated into studies investigating and controlling the causes and processes involved with carcinogenesis and other abnormal growth processes.
  • One essential operation of cell proliferation is the segregation of chromosomes into two compartments. This segregation is accomplished by a molecular fiber system that draws duplicate chromosomes to opposite positions before the cell is partitioned in two. The processes producing this movement of chromosomes to opposite poles require the involvement of ions.
  • IPR methodology can be used to establish which ions are involved, which are particularly labile to perturbation by resonant magnetic fields and to precisely regulate those processes via control of the applied parallel AC magnetic field flux density. Data already in the literature on micronuclei formation in human peripheral lymphocytes indicate that this process as well is amenable to investigation and control by IPR methodology.
  • IPR methodology can be applied to localized regions within a body, for selected maximal and minimal perturbation of ongoing reactions and processes. For example, a DC field of 366 mG parallel and ⁇ 2 mG perpendicular to a 45-Hz AC field of 238 mG (rms) would cause a maximal effect for a set of tuned ions. However, by virtue of gradients in AC field flux densities easily created by an exposure apparatus, AC fields of 492 or 919 mG(rms) could be generated that would produce no effect.
  • Receptor-ligand interactions which occur with growth factor stimulation for receptors, can be altered by IPR methodologies.
  • This application has been demonstrated as described above with primed PC-12 cells stimulated with nerve growth factor to produce neurites.
  • Natural and toxic biological processes can be altered by IPR-defined resonant magnetic fields.
  • the toxicity of chemicals can be enhanced, as demonstrated by the further reduction in gap junction intercellular communication in chloral hydrate-treated Clone 9 rat liver cells as described above. Reduction of intercellular communication in chloral hydrated cells is consistent with reduced growth control and thus an increased potential for cancer promotion.
  • IPR resonant fields For example, primed PC-12 cells stimulated with nerve growth factor to produce neurites, and exposed for 23 hours to IPR resonance conditions as described above, produced varying degrees of inhibition of the stimulation depending upon the AC flux density applied, exactly as predicted by the IPR model.
  • IPR methodologies can be expected to be used in certain biological circumstances to enhance nerve regeneration after injury or trauma.
  • the IPR methodology which identifies combinations of AC and DC magnetic fields that can reduce growth control processes, can be used to augment chemotherapy by enhancing the number of rapidly growing cancer cells by altering intercellular communication thereby making them more susceptible to a chemotherapuetic agent.
  • IPR methodology thus can be used either directly or in combination with chemotherapeutic agents to damage cell division processes in a precisely controllable manner, including regulation of enhanced micronuclei formation in human peripheral lymphocytes.
  • hormone action on cell processes can be modulated using IPR.
  • the hormone melatonin has been shown to enhance intercellular communication in C3H 10T1/2 cells, derived from a mouse embryo (Ubeda et al., 1995a).
  • Morphological transformation in this cell line which includes altered intercellular communication, is used to identify potential cancer promoting chemicals.
  • High intensity magnetic fields removed the enhancement in intercelluar communication caused by the melatonin (Ubeda et al., 1995b). Pilot data show the effect also occurs near an optimum in IPR-defined conditions (45 Hz AC at 660 mG(rms), and DC field at 366 mG parallel and ⁇ 2 mG perpendicular to the AC field) .
  • Cell and tissue functions that depend on ion cofactors can be altered by IPR-defined resonant conditions for pertinent ions.
  • the acquisition of information (learning) and the retention for information (memory) are known to be influenced by calcium and magnesium ions, among others. Pilot data are consistent with an IPR-defined resonant control of these ions during rodent performances on tasks designed to evaluate learning and memory.
  • IPR methodologies There are numerous potential applications of IPR methodologies in the neurosciences research and clinical areas.
  • the influence of IPR-defined exposure conditions on cell membrane processes indicate the immune response in numerous categories might be controlled, either positively or negatively, when magnetic fields are tuned for specific ions and the AC flux density adjusted to produce the desired inhibition/stimulation of the selected process.
  • IPR methodologies immune defence against various disease agents or aberrant cells may be subject to alteration by IPR methodologies.
  • drugs and/or delivery systems can be engineered either to preferentially activate or deposit drugs at desired sites within the body using selected ion-related resonance conditions defined by the spatial gradient features of IPR methodology.
  • Ribozymes a distinct class of metalloenzymes. Science 261: 709-714.

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Abstract

Il est possible de modifier, de renforcer ou de supprimer des systèmes chimiques et biologiques, de manière maîtrisée avec précision, en exposant ces systèmes à une combinaison préétablie de champs magnétiques de courant alternatif et continu. Il est possible de réduire au minimum la densité de flux du champ magnétique de courant continu perpendiculaire au champ magnétique de courant alternatif tout en ajustant la densité de flux du champ magnétique de courant continu parallèle au champ magnétique de courant alternatif à la valeur requise pour provoquer la résonance d'un ion particulier ou de plusieurs ions particuliers. Il est possible d'ajuster la densité de flux du champ magnétique de courant alternatif de façon à produire un niveau maîtrisé de réponse dans un système chimique ou biologique.
PCT/US1995/012343 1995-06-06 1995-09-27 Procede et appareil de modification d'interactions ioniques avec des produits chimiques et processus chimiques utilisant des champs magnetiques WO1996039493A1 (fr)

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WO1999020345A1 (fr) * 1997-10-17 1999-04-29 Axel Muntermann Dispositif de magnetotherapie
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EP0966988A1 (fr) * 1998-06-24 1999-12-29 Santi Tofani Appareil et méthode permettant d'empêcher la survie de cellules pathologiques

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