IHINIHIIIHHHHHHHIII US005104639A United States Patent (19) 11 Patent Number: 5,104,639 Matson (45) Date of Patent: Apr. 14, 1992

54 METHOD FOR BOLOGICAL TESTING of Blood Products with Time and Attempted Identifica AND/OR DEVELOPNG tion of the Spasmogens, Stroke 12(6): 775-80 (1981). PHARMACEUTICALS FORTREATMENT OF Clin. Chem 30/9, pp. 1477-1488 (1984) Matson et al., DSORDERS USING "n-Electrode Three-Dimensional Liquid Chromatog ELECTROCHROMATOGRAPHY raphy with Electrochemical Detection for Determina 75 Inventor: Wayne R. Matson, Ayer, Mass. tion of Neurotransmitters'. Journal of Liquid Chromatography, 6(2), pp. 375-381 (73) Assignee: ESA, Inc., Bedford, Mass. (1983) Yamaguchi "Liquid Chromatographic Determi 21 Appl. No.: 274,505 nation of Excretion Patterns of Urinary Phenolic Com 22) Filed: Nov. 21, 1988 pounds'. Biological Markers in Psychiatry and Neurology, Proc. Related U.S. Application Data of a conference at Ochsner Clinic, New Orleans (1982), (60) Division of Ser. No. 797,615, Nov. 13, 1985, Pat. No. Schildkraut, "Biochemical Discrimination of Sub 4,863,873, which is a continuation-in-part of Ser. No. groups of Depressive Disorders Based on Differences in 670,483, Nov. 13, 1984, abandoned, which is a continu Catecholamine Metabolism' see pp. 23-33. ation of Ser. No. 579,401, Feb. 17, 1984, Pat. No. La Nouvelle Presse Medicalle, 9, pp. 2061-2063 (1980) 4,511,659, which is a continuation-in-part of Ser. No. Devynck et al., "Dosage rapide des Catecholamines 472,387, Mar. 4, 1983, abandoned, and a continuation Plasmatiques Pour le Diagnostic d'urgence des Pheo in-part of Ser. No. 425,183, Sep. 28, 1982, abandoned, which is a continuation of Ser. No. 111,917, Jan. 4, chromocytomes'. 1980, Pat. No. 4,404,065. Journal of Clinical Endocrinology and Metabolism, 50(5), pp. 857-861 (1980), Gullner et al., "Correction of 51 Int. Cli...... G01N1/00; G01N 33/48; Increased Sympathoadrenal Activity in Bartter's Syn G01N 25/08; A61K 31/00 drome by Inhibition of Prostaglandin Synthesis'. 52) U.S. C...... 424/2; 424/520; 424/530; 424/531; 424/545; 424/570; 73/61.1 (List continued on next page.) C; 436/63; 436/150; 436/161; 204/153.1; 514/1 Primary Examiner-Richard L. Raymond (58) Field of Search ...... 424/1.1, 2, 531, 520, Assistant Examiner-Gary Hollinden 424/530,545,570; 436/63, 150, 161; 73/61.1 C; Attorney, Agent, or Firm-Hayes, Soloway, Hennessey 204/153.1, 411, 412; 514/1, 2 & Hage (56) References Cited 57 ABSTRACT U.S. PATENT DOCUMENTS A method for establishing a treatment protocol for 4,046,634 9/1977 Mercer ...... 435/16 correcting an abnormal condition or disease in a living 4,215,09 7/1980 Rohenstroth-Bauer ...... 514/21 4,233,031 11/1980 Matson et al...... 23/230 organism is described. The method comprises the steps 4,338,811 7/1982 Miyagi et al...... 73/23.1 of analyzing body fluids from normal and abnormal 4,404,065 9/1983 Matson ...... 204/1 T individuals to generate analysis patterns of the normal 451 1,659 4/1985 Matson ...... 436/150 and abnormal fluids, comparing the patterns to deter 4,863,873 9/1989 Matson ...... 436/63 mine differences in the patterns, and establishing a treat ment protocol for the abnormal individual through OTHER PUBLICATIONS chemical or metabolic therapy which treating will nor Guy, Michael N. et al., Analysis of Angiotensins I, II III malize the abnormal pattern. and Iodinated Derivatives by High Performance Liquid Chromatrography, Analytical Biochen, 1.12: 272-7 (1981). Okwusaba, F. et al., Changes in Vasoactive Properties 23 Claims, 9 Drawing Sheets

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OTHER PUBLICATIONS Psychopharmacology (n.Y.) pp. 275-328 (1976) Schild Archives of General psychiatry, 35, pp. 1427-1433 kraut et al., "Biochemistry of Effective Disorders' (1978) Schildkraut et al., "Toward a Biochemical Clas CA85(25) 190161d Abstract only. sification of Depressive Disorders". Psychopharmacology (Berlin), 56(3), pp. 327-333, British Medical Journal (1969) 2, 153-155, Marks et al., (1978) Schildkraut et al., "Opiates, Catecholanines, Be "Application of Urine Analysis to Diagnosis and Treat havior, and Mood" 89:53498n Abstract only. ment of Heroin Addiction'. Journal of American Geriatr. Soc., 25(7), pp. 289-298 Clinical Pharmacology and Therapeutics, 7(6), pp. (1977) Meyer et al., “Neurotransmitter Precursor 727-739 (1966) Anton et al., “The Effect of Resperine Amino Acids in the Treatment of Multi-Infarct De on Catecholamine Metabolism and Behavior in Re mentia and Alzheimer's Disease' Index Medicus Jour tarded Children'. nal Announcement: 7709 Abstract only. U.S. Patent Apr. 14, 1992 Sheet 1 of 9 5,104,639

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5,104,639 1. 2 to rigorous interlaboratory testing, the current values METHOD FOR BOLOGICAL TESTING AND/OR for normal and abnormal neurotransmitter levels must DEVELOPNG PHARMACEUTICALS FOR be taken with unspecified and probably wide limits of TREATMENT OF DSORDERS USENG confidence. ELECTROCHROMATOGRAPHY Although the analysis of single neurotransmitters or metabolites from a complex biochemical pathway has This is a divisional of co-pending application Ser. No. been shown to correlate with a number of disorders 797,615 filed on Nov. 13, 1985, U.S. Pat. No. 4,863,873, utilizing statistical analysis over a large number of sam issued on Sept. 5, 1989, which is a continuation-in-part ples, the analytical level of a single neurotransmitter in of 670,483 filed Nov. 13, 1984 abandoned, which in turn 10 an individual sample, with a few exceptions, has had is in part a continuation of my copending application relatively low clinical diagnostic utility. Essentially the Ser. No. 06/579,401, filed Feb. 17, 1984, (now U.S. Pat. state of the field of biochemical correlates of disorders is No. 4,511,659 issued Apr. 16, 1985), which is in part a that while between large populations of normal and continuation-in-part of my application Ser. No. 472,387 abnormal individuals a correlation generally can be filed Mar. 4, 1983 (now abandoned), and in part a con 15 determined for a particular biochemical, the scatter that tinuation of my copending application Ser. No. 425, 183 results from both analytical and biochemical phenom now abandoned filed Sept. 28, 1982 which is, in turn, a ena typically does not permit the level of a particular continuation of my application Ser. No. 111,917 filed biochemical to be utilized diagonostically for a particu Jan. 4, 1984 (now U.S. Pat. No. 4,404,065 issued Sept. lar single individual. Nor may a single biochemical 13, 1983). 20 value be utilized for the rational prescription or devel This invention relates to analytical systems, i.e. appa opment of a pharmaceutical for that individual. This is ratus and methods for qualitatively and quantitatively not particularly surprising in that both the levels and testing materials in solution. The invention has particu effects of a particular neurotransmitter are modified by lar utility for the detection and determination of biologi a number of other neurotransmitters in the same, or cally active materials such as neurotransmitters and 25 parallel metabolic pathways. If, for instance, 5-HT (se other neurochemical substances in brain tissue, cerebro rotonin) is to be used as a diagnostic tool for depression, spinal fluid, plasma, serum, saliva, urine and the like, suicidal tendencies, or schizophrenia, it would be neces such as catecholamines, their precursors, cofactors and sary and perhaps provide a route to definitive diagnosis their metabolites. The invention is uniquely capable of and pharmaceutical specification or development, to generating analytical values and patterns of a large 30 simultaneously determine the approximately 40 other number of compounds of biological, diagnostic and compounds that derive from tryptophan and signifi pharmaceutical significance and of describing complex cantly effect the indolaminergic neuronal system's ac metabolic pathways of such substances from single tivity. small samples of biological materials and the use of such In recent years, LCEC (Liquid Chromatography values and patterns for rational development of pharma 35 with Electrochemical Detection) has become a con ceuticals and will be described in connection with such non tool for the determination of catecholamines bio utility although other uses are contemplated. genic amines and their metabolites in biological fluids. There is an extensive body of literature relating ab Because of sensitivity limitations (typically 20-50 pg) normalities in neurotransmitters, precursors, and metab and the complexity of biological samples, both separa olites to degenerative, neuropsychiatric and behavioral tion and concentration steps typically have been neces disorders, hypertension and certain carcinomas. See, for sary. Heretofore, plasma catecholamine analysis typi example, Schildkraut et al in The Brain, Biochemistry cally required three steps. First, the sample is collected and Behavior, Proceedings of the Sixth, Arnold O. and the catecholamines separated and concentrated, for Beckman Conference in Clinical Chemistry, pages example, using the alumina extraction procedure of 47-68. Although the potential role of these compounds 45 Anton and Sayre (See A. H. Anton and D. F. Sayre, J. in a number of significant disorders has been estab Pharmacol, Exp. Ther, 138 (1962), p. 360-375). The lished, their routine analysis has not yet achieved wide analytes, norepinephrine, epinephrine and dopamine, spread clinical use. Two problems in the clinical utility along with the internal standard DHBH(dihydroxyben of neurotransmitter measurements are related to the zylamine), then are separated chromatographically, and economic and technical limitations of current technol SO finally detected electrochemically. Typical sample size ogy. First, there is felt to be a high degree of inter requirements are 1.0 ml plasma or serum. In routine laboratory and intersample uncertainity in quantitative clinical use, there have been numerous problems with values. Second, it has been difficult to measure enough conventional techniques (alumina adsorption, ion ex of the known metabolically related compounds of a change and extraction) due to a large number of poorly particular neurotransmitter to fully describe its bio 55 understood variables in the overall analysis system of chemical significance in an individual sample, or to sample acquisition, storage, preparation and sensor re detect, identify and measure unusual neurotransmitter sponse. These problems have quite likely confused the s-an important aspect of basic research in various dis relationships that may exist between levels and distribu ease states that is presently very expensive and special tion of the catecholamines and various physiological ized. and behavioral phenomena and disease states. While a number of interlaboratory technique inter In the analysis of complex biological materials such as comparisons for a variety of neurotransmitters have blood, serum and cerebrospinal fluids which may con been carried out, there has been no comprehensive tain numerous different constituents, the important (e.g. study within and among different techniques and labo abnormal) metabolites such as neurotransmitters to be ratories for neurotransmitters in typical samples of in 65 identified may be present in only parts per trillion. terest. In the absence of such studies, given the com While a chromatographic column can achieve macro plexity of the analytical problem and the historically separation of the various constituents it may not pro wide variation whenever an analyte has been subjected vide adequate spatial (in time) separation of the ex 5,104,639 3 4. tremely small portion of metabolites of interest from the use of a much larger relative surface area for reaction. much larger percentage of the many other compounds And, finally, and most important tot he eventual devel coeluted from the column at the same time as the metab opment of array and gate cells, a coulometric electrode olites of interest. Many of these interfering coeluted by virtue of its essentially 100% efficiency allows se materials are electrochemically active but electrochen quential oxidation and/or reduction of compounds at ically irreversible while many metabolites such as neu successive-in-line detectors. The improved sensitivity rotransmitters are both electrochemically active and of the detection system as discussed in my aforesaid electrochemically reversible. It has been found that the U.S. Pat. No. 4,511,659, particularly where two or more analytical problems of reliable measurements of neuro active testing electrodes follow the screening electrodes chemicals and related compounds are complicated by O has given the ability to do direct injections of serum the fact that interferences with conventional or prior filtrates and has also allowed the generation of repro technologies are disorder related. This was discussed in ducible patterns of compounds with catecholamine like my prior publication, (Matson et al, Clinical Chemistry, electrochemical behavior of a large number of resolv Vol. 30, No. 9, 1984) for dopamine, dopac and seratonin able components. This provides the possibility of per measurements in directly analyzed brain extract and 15 forming patten recognition for the diagnosis or perhaps cerebrospinal fluid for normal, schizophrenics and Alz even predictive diagnosis, of various disorders or dis heimers. Recent work has indicated that even for the ease States. widely used and accepted technique of alumina extrac The present invention in broad aspect provides a tion for plasma catecholamines that inferences may be system for resolving and detecting hundreds of con disorder specific. Higher values for Norepinephrine 20 pounds in a single sample at femtogram levels whereby (NE) and Epinephrine (EP) were observed following to provide a small molecule inventory or metabolic alumina extraction and analysis of a single energy con pathway pattern of a individual. The small molecule ventional electrochemical detector than for a three cell inventory may be considered to reflect the underlying redox detector on neonatal stress blood samples. Analy activity and distribution of the enzymatic pathways of sis of the neonate extracts on the sixteen channel chemi 25 an individual and hence reflect an operational measure cal imaging system revealed several unexpected com of the genone determining those enzymes. The small punds that are potential interferences including dihy molecule inventory of an individual may thus be used to droxyphenylacetic acid (DOPAC), 3-hydrox determine the health state of the individual and/or to ykynurenamine (3-OHKYA), and 3-hydroxy diagnose disease states. Correlation of the patterns from anthranilic acid (3-OHAN). These compounds have not 30 a plurality of individuals provides an understanding of been detected in normal adult plasma alumina extracts. the mechanisms of disorders or disease states or condi In my aforesaid U.S. Pat. No. 4,511,659, there is pro tions and in turn provides a rational route to pharmaco vided an electrochemical detection system comprising a logical development leading to treatment, cure or sup plurality of coulometrically efficient electrochemical pression of such disorders, disease states or conditions. cells, in series, for sequentially oxidizing and reducing 35 More particularly, in accordance with the present selected substances in a sample solution under con invention, sample solutions (e.g. body fluids) are elec trolled conditions prior to measurement on a down trochemically tested for both normal and abnormal stream testing electrode or electrodes. individuals or conditions, patterns are generated for said More specifically, in accordance with the invention normal and abnormal individuals or conditions, the provided in my aforesaid U.S. Pat. No. 4,511,659, a patterns compared and differences identified, and the sample solution (e.g. a body fluid) is passed through a identified differences utilized to design a specific treat suitable chromatographic column and the eluant is ment protocol. streamed in contact with a series of electrochemically In one aspect of the present invention provides a isolated, in-line coulometric electrodes operated under method for developing pharmaceuticals for disorders in conditions so as to establish a series of "gates' for the 45 living organisms and the pharmaceuticals so produced. sequential oxidation and reduction of substances in the The method comprises the steps of analyzing body sample solution whereby to screen (remove) selected fluids from normal and afflicted (abnormal) individuals interfering and electrochemically irreversible sub to generate analysis patterns representative of constitu stances contained in the sample solution, while passing ents of the metabolic pathways of said individuals; con selected electrochemically reversible products for de paring said patterns to determine differences in said tection and measurement on a downstrean electrode. patterns representative of (a) the presence or absence of The gate electrode series is followed in-line by one or specific compounds in said patterns, or (b) differences in more, preferably an array of six or more coulometric the concentration of said compounds in said patterns; measuring electrodes, each formed of porous electrode and introducing into a pharmaceutically acceptable base material such as fritted graphite, fritted carbon or 55 carrier therefor one or a mixture of chemical con other conductive fritted material, for detecting and pounds known to act at sites within said metabolic path measuring the electrochemically reversible compounds ways to (a) block or potentiate those metabolic path of interest (e.g. neurotransmitters). ways identified as abnormal by the pattern, or (b) sup As reported in my aforesaid U.S. Pat. No. 4,511,659, ply or retard those unique compounds identified as there are several beneficial effects of this approach to 60 abnormal in said patterns. electrochemical analysis. Long-term drift in response is The invention in another aspect provides a method effectively eliminated by acquiring essentially 100% of for developing pharmaceuticals for disorders in living the signal. The capability of analyzing essentially 100% organisms and the pharmaceuticals so produced. The of a material allows the assay of compounds of un method comprises the steps of analyzing body fluids known purity by relating then to the basic principles of from normal and afflicted (abnormal) individuals to electrochemical reaction embodied in Faraday's Law. generate analysis patterns representative of individuals' Poisoning of the electrode, a dominant problem with electrochemically active small molecule inventory, electrochemical sensors, is effectively eliminated by the comparing said patterns to determine differences in said

5,104,639 5 6 patterns representative of (a) the presence or absence of tential as well by time of elution, and comparing said specific electrochemically active compounds in said measurements thus obtained with measurements of nor patterns, or (b) differences in the concentration of said mal and abnormal body fluids to provide identification compounds in said patterns; and introducing into a and relevant concentrations of constituents coeluting pharmaceutical carrier one or a mixture of chemical from the column. compounds known to act at sites within metabolic path Yet another aspect of the invention provides a ways of said organisms to (a) block or potentiate those method of identifying abnormalities in the small mole metabolic pathways identified as abnormal by the pat cule inventory of an individual comprising the steps in tern, or (b) supply or retard those unique compounds sequence of: identified as abnormal in said patterns. 10 determining the presence and relative concentration The invention also provides a method for identifying of electrochemically active constituents in the meta pharmacological materials for treating living organisms, bolic pathways containing such abnormalities by pass which method comprises the steps of analyzing body ing a sample containing said body fluid sequentially fluids from normal and afflicted (abnormal) individuals through a liquid chromatograph column for achieving to generate chemical analysis patterns representative of 5 time-space separation of the constituents eluting from the electrochemically active small molecule inventory the column and an electrochemical detection apparatus contained in said normal and abnormal body fluids; comprising a plurality of coulometric cells arranged in comparing said patterns and identifying differences in series; said patterns representative of (a) the presence or ab maintaining the coulometric electrodes of said cells at sence of specific compounds in said patterns, or (b) 20 different potentials, said cells operating at progressively differences in the concentration of said compounds in varying potentials along the path of flow of the eluant the metabolic pathways of said individuals; and select through the cells, said plurality of cells being sufficient ing from chemical compounds known to act at sites in number to separate by measuring potentials those within said metabolic pathways one or a mixture of relevant constituents coeluted from the chromato compounds to (a) block or potentiate those metabolic 25 graphic column at any instant of time and recording the pathways identified as abnormal by the pattern, or (b) results of such coulometric measurements so as to sepa supply or retard those unique compounds identified as rate said measurements by measuring potential as well abnormal in said patterns. as by time of elution and comparing the said relevant In yet another aspect the invention provides a concentrations thus obtained with known concentra method of identifying an abormality in a body fluid of 30 tions of normal or abnormal body fluids and thereby an individual which comprises the steps of determining identify changes of rates through the various metabolic the presence and relative concentration of electrochem pathways of said relevant constituents as well as un ically active compounds contained in said body fluid by known metabolites and abnormal precursors for normal passing a sample containing said body fluid sequentially metabolites. through a liquid chromatographic column for achieving 35 The invention also provides a method of determining time-space separation of materials eluting from the col the relative concentration of principal constituents in umn and an electrochemical detection apparatus for the metabolic pathways of an animal by passing a sam electrochemically testing a sample containing electro ple containing body fluid from said animal sequentially chemically active materials in solution, the detection through a liquid chromatograph column for achieving apparatus comprising a plurality of coulometric cells 40 time-space separation of the constituents eluting from arranged in series, maintaining the testing electrodes of the column and an electrochemical detection apparatus said coulometric cells at different potentials, said cells comprising a plurality of coulometric cells arranged in operating at progressively varying potentials along the Series; path of flow of the eluant through the cells, and record maintaining the coulometric electrodes of said cells at ing the results of coulometric measurements from said 45 different potentials, said cells operating at progressively cells so as to separate said measurements by measuring varying potentials along the path of flow of the eluant potential as well as by time of elution, and comparing through the cells, said plurality of cells being sufficient the said relative concentrations thus obtained with in number to separate by measuring potentials those known concentrations of normal or abnormal body relevant constituents coeluted from the chromato fluids. graphic column at any instant of time, and recording the Also provided in accordance with the present inven results of such coulometric measurements so as to sepa tion is a method for identifying an abnormality in a body rate said measurements by measuring potential as well fluid of an individual which comprises the steps of de as by time of elution. termining the presence and relative concentration of Also provided in accordance with the present inven electrochemically active compounds contained in said 55 tion is a method for treating a disorder in a living organ body fluid by passing a sample containing said body ism which method comprises the steps of analyzing fluid sequentially through a liquid chromatographic body fluids from normal and afflicted (abnormal) indi column by step gradients for achieving time spaced viduals to generate analysis patterns representative of separation of the materials eluting from the column and constituents of the metabolic pathways of said individu an electrochemical detection apparatus for electro 60 als; comparing said patterns to determine differences in chemically testing the eluant from said column, the said patterns representative of (a) the presence or ab detection apparatus comprising a plurality of coulomet sence of specific compounds in said patterns, or (b) ric cells arranged in series, maintaining the coulometric differences in the concentrations of said compounds in electrodes of said cells at different potentials, said cells said patterns; introducing into a pharmaceutically ac operating at progressively varying potentials along the ceptable carrier therefor one or a mixture of chemical path of flow of the eluant through the cells, recording compounds known to act at sites within said metabolic the results of coulometric measurements from said cells pathways to (a) block or potentiate those metabolic so as to separate said measurements by measuring po pathways identified as abnormal by the pattern, or (b) 5,104,639 7 8 supply or retard those unique compounds identified as measuring) cells are employed after the gate cells and abnormal in said patterns; and administering to said the outputs of the test cells are processed by a central living organism said identified compounds in said phar processing unit; maceutically acceptable carrier so as to (a) block or FIG. 7 is a flow chart of an embodiment of disease potentiate those metabolic pathways identified as ab 5 detection and treatment design method according to the normal by the pattern, or (b) supply or retard those present invention; unique compounds identified as abnormal in said pat FIG. 8 is a block diagram of an electrochemical appa tes. ratus adapted for the practice of the method shown in Finally, the invention provides a method of treating a FIG. 7; and disease state in an animal, comprising the steps in se 10 FIG. 9 is a voltage/time resolution pattern obtained quence of identifying abnormalities in a body fluid of in accordance with the present invention. said animal by determining the relative concentration of Further understanding of the features and objects of the principal constituents in the metabolic pathways the present invention will be had from the following containing such abnormalities by passing a sample con detailed description of one preferred embodiment of the taining said body fluid sequentially through a liquid 15 invention which illustrates an electrochemical neuro chromatograph column for achieving time-space sepa transmitter pattern generation and resolution and detec ration of the constituents eluting from the column and tion of the principal constituents or agents contained in an electrochemical detection apparatus comprising a cerebrospinal fluid and affecting motion sickness. It will plurality of coulometric cells arranged in series; be understood, however, that the process of the present maintaining the coulometric electrodes of said cells at 20 different potentials, said cells operating at progressively invention may be advantageously employed for detect varying potentials along the path of flow of the eluent ing and resolving of various other electroactive metabo through the cells, said plurality of cells being sufficient lites in a biological sample solution, including but not in number to separate by measuring potentials those limited to neurotransmitters, precursors and metabo relevant constituents coeluted from the chromato 25 lites, hormones, transmitting compounds, peptides, cer graphic column at any instant of time and recording the tain vitamines, pterins, purines, certain drugs and me results of such coulometric measurements so as to sepa tabolites and other materials that mediate metabolism. rate said measurements by measuring potential as well More particularly, the present invention in one aspect as by time of elution and comparing the said relevant provides a method by which sample solutions for both concentrations thus obtained with known concentra normal and abnormal individuals and conditions are tions of normal or abnormal body fluids and thereby tested, principal constituents or agents resolved and identifying changes of rates through the various meta detected, patterns indicating the presence and concen bolic pathways of said relevant constituents as well as tration of such constituents or agents generated, and the unknown metabolites and abnormal precursors for nor differences between the patterns determined whereby nal metabolites, and identify those compounds respon 35 compounds suspected of causing said abnormalities or sible for said changes of rates, and administering to said conditions may be identified, and a treatment protocol animal said identified compounds so as to provoke a established through chemical or metabolic therapy, to change of rate through said metabolic pathways of said normalize the (abnormal) pattern, or block or potentiate individual substantially to approach that found in said those metabolic pathways identified as abnormal by the normal fluids. pattern, or supply or retard those unique compounds Yet other objects of the invention will in part appear identified as abnormal in the pattern, or utilize changes obvious and will in part appear hereinafter. The inven in the patterns during therapy and the concentration of tion accordingly comprises the process comprising the the therapeutic drugs and metabolites to modify the several steps and relation of one or more of such steps course of the therapy. with respect to each of the others, all of which are 45 The first step of the process is to analyze the sample exemplified in the following detailed description, and solutions for normal and abnormal individuals or condi the scope of application as will be indicated in the tions to generate patterns for comparison. The electro claims. chemical detection described in my aforesaid U.S. Pat. For a fuller understanding of the nature and objects No. 4,511,659 is particularly adapted for this purpose. of the present invention, reference should be had to the As reported therein such apparatus is capable of re following detailed description taken in combination sponding to and differentiating between electrochemi with the accompanying drawings wherein: cally reversible species in a sample solution and for FIG. 1 is a side elevational view, in cross-section, of discriminating against species in a sample solution that an electrochemical detection apparatus useful in accor are electrochemically irreversible. Sample solutions dance with the process of the present invention; 55 from normal and abnormal individuals are flowed FIG. 2 is a block diagram of the electrical controls through a chromatographic column and the eluant is and functions of the electrochemical detection appara passed through a series of electrochemically isolated tus of FIG. 1; cells or "gates' operated at potentials resulting in se FIGS. 3, 3A & 3B is a time/potential plot of a chro quential oxidation and reduction of various species con matogram obtained in accordance with the present 60 tained in the sample solution. These gates are followed invention; by an array of measuring electrodes for measuring elec FIG. 4 represents the major metabolic pathways of trochemical activity of compounds of interest. The tyrosine (TYR); strategy of analysis discriminates against both back FIG. 5 represents the major metabolic pathways of ground current from the mobile phase itself and re tryptophan (TRP); moves (screens) compounds that are irreversible, while FIG. 6 is a schematic diagrammatic drawing of a electrochemically reversible species of interest, e.g. the detection system useful in accordance with the process catecholamines are cycled back and forth from oxidized of the present invention wherein a plurality of test (or to reduced state at various potentials whereby to en

5,104,639 9 10 hance the sensitivity and specificity of downstream 20... 34A are hydraulically connected to one another detection and measuring electrodes. via stub tubing members 32 which together with cells 20 For convenience of illustration the function of the ... 34A define a fluid flow path between inlet 14 and gate cells is described as "removing' or "screening” the outlet 16. Each electrochemical cell 20 . . . 34A com electrochemically irreversible compounds from the 5 prises a three electrode system consisting of at least one eluant. This is not what actually happens; these con working electrode 34a, 34b, 34c, 34d, 34e, 34g, 34h (T1 pounds are merely electrochemically altered by the gate ... T16) respectively; at least one counter electrode 36a, cells so that the potential existing on the following de 36b, 36c, 36d, 36e, 36f 36g, 36h, respectively; and, at tecting electrode(s) will not change the oxidation state least one reference electrode 38a, 38b, 38c, 38d, 38e, 38f, of the "removed' compound and therefore the "re 10 38g, 38h, respectively. Electrochemical cells 20... 34A moved" compound will not be detected and coulomet are fixedly positioned within enclosure 12 by suitable rically measured. It is as if the "removed" compound means (not shown). were not present in the eluant. It is no longer electroac Each working electrode 34a... h (T1... T6) is in the tive under the detection conditions. form of a flat disc formed of a porous electrode base The gate/array cell principal was evaluated using a 15 material such as fritted graphite or fritted carbon or cell configured typically as shown in FIGS. 1 and 2. In other conductive fritted materials. Most of these prefer essence, a gate/array cell is designed to set up windows ably have a relatively large area to volume ratio to give for determination of selected compounds in the cate large half times (up to 90) at the flow rates contem cholaminergic systems and in the indolaminergic sys plated and with the electrochemically active materials tem. These assays include Homogenistic Acid (HGA), 20 of interest. Half time is the time required for half of a 3-hydroxy-L-tyrosine (L-DOPA), Norepinephrine quantity of a compound to react at an electrode. Allow (NE), Dihydroxyphenylacetic acid (DOPAC), Epi ing a reaction to proceed for two halftimes causes 75% nephrine (EP), Dopamine (DA), 3-Methoxy, 4-Hydrox reaction, 5 half times 97% reaction. yphenylglycol (MHPG), and 3-Methoxytyramine In order to maximize response of the porous working (3-MT) to describe the catecholaminegic system, and 25 electrodes the electrode base material was pretreated in Tryptophan (TRP), 5-Hydroxytryptophan (5-HTP), accordance with the teachings of my copending U.S. 5-Hydroxytryptophal (5-HTOL), Serotonin or 5 Pat. No. 4,976,994 issued Dec. 11, 1990 to load the Hydroxytryptamine (5-HT), 5-Hydroxy Indole Acetic secondary porosity of the electrode base material with a Acid (5-HIAA), and Homovanillic Acid (HVA) to selected non-electroactive material (i.e. under antici describe the serotonergic system. 30 pated sensor conditions) to reduce background noise The electrochemical gate/array cell should have a and enhance selectivity to aminergic compounds. The minimum of 8 gates or arrays to effect the desired reso overall process was as follows: A piece of commercially lution and preferably will have a minimum of 16 gates available fritted graphite was impregnated by a mixture or arrays. Preferably the analysis is conducted in three of 80% C-48 paraffin and 20% 0.9163-ML density poly steps. The first step comprises an open array analysis in 35 ethylene containing 1.00 grams per liter sodium salt of which the potential on the array is set at relatively wide naphthalene sulfonic acid at 110' C. and cycling be testing increments. Generally the open array evaluation tween vacuum and 1000 psi pressure. Electrodes were is performed in two passes, a first pass in which the cut from the impregnated fritted graphite material and materials of interest are dissolved in a low organic mod these electrodes were treated to remove the impregnat ifier, e.g. a mobile phase having for example 0 to 1 ing material blocking the frits by heating at 100' C. for percent MeOH and CH3CN, and a second pass in which five minutes under a vacuum of 750-760 mm HgAbs. the materials of interest are dissolved in a high organic Thereafter the treated electrode material was rinsed modifier, typically 30 to 60 percent or more MeOH, and with 50-50 tetrahydrofuran-hexane. The resultant elec CH3CN in lower proportions. This provides a first cut. trode material which contained approximately 0.01% Thereafter, compounds of potential interest based on by weight of sorbed napthalene sulfonic acid and ap analysis of the data are evaluated as before, but using a proximately one percent by weight of high molecular tighter array setting the electrodes to relatively low weight organic material, were assembled in the flow potential increments over a specific region. As before cell detector of a type illustrated in FIG. 1, of my afore the analysis is done in two passes-i.e. in a low organic said U.S. Pat. No. 4,511,659. modifier and in a high organic modifier. Finally, se 50 Also provided are electrical connections 40a, 40b, lected components then are analyzed under REDOX 40c, 40d, 40e, 40f 40g 40h for connecting working elec conditions with oxidation followed by reductive assay. trodes 34a . . . h, (T ... T6) respectively, to potential Analyzing the sample in this manner provides ex controls 42a, 42b, 42c, 42d, 42e, 42?, 42g, 42h, respec tremely high resolution and specificity. tively, for applying selected working potentials to the Referring to the details of FIGS. 1 and 2, there is 55 various working electrodes 34a... h. electrical connec shown a preferred embodiment of the electrochemical tions 44a, 44b, 44c., 44d, 44e . . . 44h, for connecting detection apparatus useful in accordance with the pro counter electrodes 36a . . . h, respectively, to potential cess of the present invention, indicated generally at 10. controls 46a, 46b, 46c, 46d, 46e . . . 46h (T1 . . . T16) Electrochemical detection apparatus 10 comprises a respectively for applying selected counter potentials to hollow, liquid-tight enclosure indicated generally at 12 60 the various counter electrodes; and electrical connece and having an inlet 14 and an outlet 16 found in a pair of tions 50b, 50b, 50c, 50d, 50e, 50f, 50g, 50h, respectively, end plates 17 and 18, respectively. Enclosure 12 is for connecting reference electrodes 38a . . . h, respec formed of a liquid-impervious, rigid, electrically insulat tively to a potential control 52 for applying a reference ing chemically inert material such as unplasticized poly potential to the various reference electrodes 38a. . . h. vinylchloride, polytetrafluoroethylene, fluorohydro 65 Counter electrodes 36a... h. and reference electrodes carbon resin or the like. Disposed within enclosure 12 38a... h preferably comprise inert metal terminals such are sixteen electrochemically isolated electrochemical as platinum or palladium wire. Alternatively, reference cells 20, 20A, 22, 22A... 34,34A. Electrochemical cells electrodes 38a... h may comprise silver/silver chloride 5,104,639 11 12 reference electrodes or the like. The counter electrodes after, the cell was flushed at 1 ml/min for 30 minutes 36a . . . h. and reference electrodes 38a . . . h preferably with 10/10/80 THF hexane acetonitrile. are arranged in pairs to bracket an associated working Further understanding of the principles and advan electrode 34a . . . h. tages of the present invention may be had by reference In order to minimize or reduce diffusion effects on to the following examples which are based upon elec the counter and reference electrodes, the electroactive trochemical analysis employing an electrochemical ap surfaces of the metal teminal counter and reference paratus made in accordance with FIGS. 1 and 2 and electrodes were coated with a permeable membrane in comprising eight electrochemically isolated electro accordance with the teachings of my copending U.S. chemical cells 20. . .34A. The working electrodes 34a Pat. No. 4,976,994 issued Dec. 11, 1990. The overall O . . . h. each comprise fritted graphite discs, treated as process was as follows: above described, each having a working area of approx Palladium wires were spray coated with Teflon S to imately 4 cm2 (90 halftimes). Electrode 34h has a work build up a coating of approximately 0.0002' thickness. ing area of approximately 0.3 cm2 (4 half times). (Teflon S is a trademark of E. I. DuPont de Nemours Counter electrodes 36a . . . h. and reference electrodes Company for a synthetic tetrafluorohydrocarbon.) The 15 38a . . . h comprise inert metal (palladium) wire termi coated wires thus prepared were dipped sequentially in nals treated as above described. commercially available Teflon etching solution, con Coerebrospinal fluid samples from motion-sick, non centrated HNO3/H2SO4 (50:50 by volume) and tetrahy sick and motion-sickness resistant cats were analyzed. drofuran in a sonifier bath for approximately 1 minute in The initial hypotheses were: each to develop a semipermeable membrane on the wire 1. Some compound or compounds are produced surface. The Teflon coated/etched palladium wires within individual animals during motion stimulus that were cut and assembled as the counter and reference causes the enetic response in susceptible animals; or electrodes in accordance with the teachings of my 2. Some difference in the small molecule inventory or aforesaid U.S. Pat. No. 4,511,659. chemical patterns exists in susceptible vs. non-suscepti As will become clear from the following description 25 ble animals. electrochemical cells 20.... 32A act as gate electrodes Initially, conditions of maximum resolution and sensi for discriminating and screening interfering species, tivity of known compounds suspected of having a rela while electrochemical array cells 34 and 34A contain tionship to notion sickness were considered. Thereaf the measuring electrodes. In order to achieve high noise ter, conditions were defined for maximum resolution of discrimination against pressure spikes and voltage fluc 30 unknown compounds or compounds as appear to be tuations the measuring electrode should have a rela different among sick, non-sick and motion-sickness re tively small electrode area and thus a relatively small sistent animals. Cerebrospinal fluid samples were di number (e.g. 4) of reaction half-times as compared with rectly injected in a mobile phase comprising 5% the various upstream gate electrodes, MeOH, 2.5% CH3CN and 0.1M NaH2PO4 adjusted to In order to maximize bandspreading and reduce sig 35 pH 3.00 with H3PO and 650 mg/1 octane sulfonic acid nal-to-noise ratio the electrochemical cell (20. . .34A) sodium salt utilizing a Brownlee RP18 3 cm. Guard was pretreated to optimize the flow path therethrough Column and into a Waters C18. Bondapak Analytical in accordance with the teachings of my copending U.S. Column. Mobile phase modifications were carried out Pat. No. 4,753,714 issued June 28, 1988. As taught for the following samples, under the following operat therein, a melted polymer mixture comprising 67 vol ing parameters (all voltage values in millivolts): ume percent C4s hydrocarbon and 33 volume percent REDOX analysis of the samples was performed (oxi 0.916 density polyethylene, heated to 98' C. was dation followed by step-wise reduction of the products pumped into the cell at 800 psi. Thereafter, water for detection) under detector conditions chosen for heated to 98 C. was passed through the cell at 0.1 maximum resolution of NE, EP, L-DOPA, DOPAC ml/min at 20 psi. The cell was then cooled from 98' C. 45 and DA on T1, HGA on T5 and T6 and indoles on Ts, to ambient (25 C.) over a 15 minute time period by T7 and Ts and the results recorded in Table I. continuing the flow of cooler and cooler water. There Detector: GC -350, CC - 400, T1 -30, T2 -60, T3 -140, T4 - 150, Ts-200, T6-230, T7-310, T8-350.

TABLE I n/mL in CSF. Sample No. 2-201 2-203 2-239 2-243 2-342 2-554 MHPG 8.20 7.10 8.00 8,80 7.60 S.30 NE (LT 0.15) 0.30 0.24 (LT 0.20) (LT 0.20) (LT 0.20) HGA 1.02 4.80 5.30 4.40 4.80 440 L-DOPA 0.66 .10 0.90 0.89 0.89 0.67 EP 1.83 210 1.87 2.00 1.75 60 OOPAC 7.97 7.36 10.0 8.23 9.60 740 DA (LT 0.38) (LT 0.30) (LT 0.30) (LT 0.30) (LT 0.30) (LT 0.30) SHTOL 1.07 1.25 1.30 1.21 1.65 I.14 SHAA 162.00 16200 19000 23.00 23.00 68.00 5HTP - - - HVA 1600 112.00 31.00 12.S.00 135.00 109.00 3MT (LT 0.90) (LT 0.90) (LT 0.90) (LT 0.90) (LT 0.90) (LT 0.90) SHT 2.67 0.53 2.00 (LT 0.43) 0.60 (LT 0.43) T7 - 5.20 44.00 74.00 4.00 45.50 4.50 T8 At k' is (GT 100) 24.00 7.00 8.50 4.50 3.S.0

5,104,639 13 14 TABLE I-continued ng/mL in CSF Sample No. 2-201 2-203 2-239 2.243 2-342 2554 nAT2 k' = 49.00 21.00 (LT 3.00) 3.00 (LT 3.00) (LT 3.00) 0.93 AT5 k = (GT 100) 47.00 --- 21.00 12.00 9.00 0.93 nAT6 "Average two analysis Coelutes with HGA k = 1.71 d LT = less than GT = greater than Table II shows the result of standards analyzed under TABLE III-continued these conditions. Partial List of Abbreviations for Chemical Compounds and Typical Ranges Found With Preliminary Gradient TABLE II Work in Cerebrospinal Fluid (CSF) RESPONSE na OF STANDARD 20 ng/mL. 8 9 10 X SD CV 2 Typical Values or Range in T3 - T5 MHPG 13.8 4.2 14.4 13.86 0.332 2.40 9 CSF (Feline T3 NE 6.0 6.2 6.2. 6.04 0.33 2.21 9 ventricular) F T6 HGA 9.5 9.0 9.5 9.30 0.422 4.53 10 F T3 L-DOPA 10.0 10.0 9.5 9.80 0.258 2.63 10 25 T3 EP 4.0 3.6 3.6 3.62 0.175 4.84 10 glyoxylic acid T3 - TT4 DOPAC 29.8 31.0 30.8 30.82 0.553 1.79 10 DOPAC dihydroxyphenylacetic acid" 8-30 T3 DA 3.4 3.4 3.2 3.32 0.193 5.82 0 DHQ 4,8-dihydroxyquinoline" T7 - T85HTOL 18.5 18.5 7.0 14.83 0,750 S.05 6 DMPA 3,4-dimethoxyphenyiacetic 18.37 0.250 1.33 4. acid T7 - 8 SHIAA 910 92.0 92.0 78.75 .290 1.64 6 30 DMPEA 3,4-dimethoxyphenyl 91.38 0.650 0.7 4 ethylamine T3 - T4 HVA 7.2 7.2 7.2 7.30 0.254 3.48 10 DA dopamine" 0.08–0.17 EP ... epinephrine" 0.05-0.32 EPIN epinine" -- In the initial high resolution/selectivity Redox condi FER fetulic acid -- FAN formylanthranilic acid tion several anamolous peaks were noted some of which 35 FKY formy kynurenine" are listed by their k' and electrode number in Table I. GA gentistic acid" 0.8-2.0 Consequently, a more open 16 channel array from 0 to GSH glutathione" 50-70 1600 millivolts without a gate cell preceding it was HIST histanine HIS histidine utilized to define the more complete structure of profile HGA homogentisic acid 0.5-3.0 of the preliminary samples. A series of form mobile 40 HQ hydroquinone phase compositions of increasing organic modifier of HVA homovanillic acid S0-250 0.5, 5.0, 1.5 and 30% methanol was used. Approxi HVOL homovanillyl alcohol 0.5-1 mately 300 components were isolated in the various HVRA homoveratric acid HOR hordenine -- mobile phases. Several of these components appeared to 3-OHAN 3-hydroxyanthranilic acid 0.1-0.2 vary as a function of emesis. Qualitative identification of 5 5-HIAL 5-hydroxyindoleacetaldehyde' these components was made by comparison of poten 5-HIAA 5-hydroxyindoleacetic acid' 150-350 3OHKYA 3-hydroxykynurenamine tial/time locations of compounds from Table III. 3OHKY 3-hydroxykynurenine 0.3-1.0 TABLE III 6-OHMEL 6-hydroxymelatonin' 3-HPA 3-hydroxyphenylacetic acid" Partial List of Abbreviations for Chemical Compounds and 50 5-HT 5-hydroxytryptamine" 0.1-10 Typical Ranges Found With Preliminary Gradient 5-HTP 5-hydroxytryptophan' 0.2-0.6 Work in Cerebrospinal Fluid (CSF) 5-HTP- 5-hydroxytryptophol .5-3.0 ng/mL. ol Typical Values I indole or Range in IAA indoleacetic acid' CSF (Feline IET indole ethanol ventricular) F 55 IPA indolepyruvic acid F IS isatin Acet acetaminophen' KYA kynurenic acid' ANA anthranilic acid NM KYN kynurenine 1.0-2.0 ASC ascorbic acid 100-300 LD L-dopa 0.2-4.0 BEND B-endorphin' LENK leucine enkephalin' THBDHB biopterin 3-30 60 MEL nellatonin BUF bufotinene' MN metanephrine LT 0.01 CAF caffeic acid 0.1- MENK methionine enkephalin' CAT catecho 7-7 S-MGA 5-methoxygentisic acid CCK8 cholecystokinin 8 MHMA 3-methoxy-4-hydroxymandelic CCK4 cholecystokinin 4 -- acid CYSH cysteine' 6.5 MHPG 3-methoxy-4-hydroxyphenyl DHBAC 3,4-dihydroxybenzoic acid 1-8 glycol 0-40 OOMA dihydroxymandelic acid 2-6 MHPG- MHPG sulfate conjugate 100-140 OOPEG dihydroxyphenylglycol 0.2-1.0 SO DHPGA 3,4-dihydroxyphenyl 5-MAA 5-methoxyindoleacetic acid" 5,104,639 15 16 TABLE III-continued emesis from the same stimulus. Several other possible Partial List of Abbreviations for Chemical Compounds and mechanisms could be postulated from the initial data Typical Ranges Found With Preliminary Gradient and several other possible abnormalities in the cerebro Work in Cerebrospinal Fluid (CSF) spinal fluid data were observed in the preliminary study. ng/mL. Among these mechanisms were possible problems asso Typical Values or Range in ciated with the 150 ul sample size acquired. Therefore, CSF (Feline in a follow-up study 50-80 ul samples were used and ventricular) F conditions were set up to maximize the total informa F tion that could be obtained from a single sample. A step 3-M 3-methoxytyrannine 0.020 gradient procedure combined with a sixteen channel 3-MD 3-O-methyldopa array cell was utilized to describe the majority of the MGA methylgentistic acid MHST methylhistamine components of the tyrosine and tryptophan pathways HPA In-hydroxylphenylacetic acid on cerebrospinal fluid samples. Four separate mobile nHMA n-hydroxymandelic acid phase solutions were prepared as follows: HPPA m-hydroxyphenylpyruvic acid 15 OCT m-octopamine Mobile Phase I comprising 0.5 volume percent TYRA m-tyrannine 1.0-3.0 MeOH, 1 g/1 disodium octane sulfonic acid, 0.1M TYR n-tyrosine 5.0-10-0 NaH2PO4 adjusted to pH 3.00 with H3PO4; NASHT N-acetylserotonin' 0.5-2.5 NM5-HT N-methyl-5-hydroxytryptamine Mobile Phase II comprising 0.5 MeOH, 150 mg/ DMSH NN-dimethyl-5-hydroxyindole' disodium octane sulfonic acid, 0.1M NaH2PO4 adjusted DMTR NN-dimethyltryptamine to pH 3.00 with H3PO4; DMTR-P NN-dimethyltryptamine phosphate Mobile Phase II comprising 1.0 volume percent iso NE norepinephrine" 0.2-1.5 NMN nornetanephrine' LT 0.05 propanol alcohol, 0.5 volume percent MeOH, 175 mg/1 oHTA o-hydroxyphenylacetic acid disodium octane sulfonic acid, 0.1M NaH2PO4 adjusted YRA o-tyrannine to pH 3.00 with H3PO4; and oTYR o-tyrosine" 25 Oc octopamine' 0.05-1.5 Mobile Phase IV comprising 5 volume percent iso pCOU p-counaric acid -- propanol alcohol, 0.5 volume percent MeOH, 275 mg/1 pHMA p-hydroxymandelic acid disodium octane sulfonic acid, 0.1M NaH2PO4 adjusted pHPAA p-hydroxyphenylacetic acid 4-10 pHPLA p-hydroxyphenylactic acid 0-10 to pH 3.00 with H3PO4. pHPPA p-hydroxyphenylpyruvic acid 30 The overall procedure is as follows: PAC phenylacetic acid NM First the column was swept with Mobile Phase IV. PA phenylalanine NM Mobile Phase I was then flowed through the column for PAE phenylethylamine NM PLA phenylactic acid NM one minute. 200 microliters comprising a mixture of 1.0 PPA phenylpyruvic acid NM mg/ml ascorbic acid and 0.5 mg/ml. EDTA was in PAL pyridoxal" 35 jected into the column and Mobile Phase I was flowed PALPS pyridoxal-5-phosphate PYRA pyridoxamine through the column for an additional five minutes. At PYRASP pyradoxamine-5-phosphate" the six minute mark, the mobile phase was changed to PYR pyridoxine -- Mobile Phase II and flow continued for an additional QA quinaldic acid eighteen minutes. Sample solution was injected into the SYN synephrine -- column at the 24 minute mark. At the 35 minute mark RPA tryptamine 1.0-1.8 TRP tryptophan 800 the mobile phase was shifted to Mobile Phase III and at YRA tyramine O.S.-10 the 60 minute mark the mobile phase was shifted to TYR tyrosine 1500 U uric acid 600 Mobile Phase IV. Mobile phase flow was continued for VA vanilic acid twenty-five minutes, the mobile phase was then shifted VMA ' vanillyinandelic acid 0.2-0.3 45 back to Mobile Phase I and the sequence repeated with - indicates observed but not quantified additional samples. A typical voltage/time resolution pattern for this method is shown in FIG. 9. iadicates compound to be included in study A double blind analysis of 106 samples from enesis - indicates not preseat susceptible, enesis moderately susceptible and enesis From this work the thirty-nine compounds listed in 50 non-susceptible animals was performed. Thirty-nine Table IV were decided upon as candidates for involve components under controlled test were analyzed in nent in the emesis event. each sample and separated from 150 additional un From the preliminary data, one mechanism was pos known components utilizing the electrochemical imag tulated for emesis and emesis blocking as a guide to the ing system of the present invention. Column tempera sample acquisition and analysis protocols to be used in a SS ture was maintained at 23.5 C. plus or minus 0.1 C. following study. The mechanism postulated was in the The results of the precision of the method as derived resting cat NE and HGA exist at very low free levels in from coanalyzed standard is shown in Table IV. Of the protein bound pools of approximately 1 and 4 ng/mL, thirty-nine components, approximately thirty were respectively. Stimulus causes the free NE to spike due present and unambiguously identified in all samples and to neuronal emission. Referring to FIGS. 4 and 5, this 60 submitted to extensive statistical analysis. emission is mediated by several other neurotransmitter pathways, including the serotonergic and adrenergic TABLE IV and possibly by B6 compounds (which are probable C variable peaks in the second array condition). The free Compound CV NE causes the enesis response and is then deteriorated DHPAA 4. MHPG-SO 7.4 by adsorption into the bound pool. Free HGA spikes TB 4.3 simultaneously either by emission or release from the DOMA 6.16 pool and persists acting as a blocking agent for further ooc 4,81

5,104,639 17 18 viduals, essentially the entire complex metabolic path TABLE IV-continued ways of such substances, and the identity of naturally C Compound CV occurring substances or chemical adjuncts for treating a pHMA 2.37 disorder or related diseases. This permits, for example, U 2,32 those compounds identified as blocking agents, or pre NE 4.60 cursors of said blocking agents, to be administered di WMA 5.37 rectly to an individual, or dietary modification which MHMA 8.23 MHPG 3.71 will cause the individual to produce internally the de OCT 6.2 sired blocking agents may be administered. This also HGA 8.3 10 EP 5.43 permits the exact definition of differences in metabolic DHBAC 2.91. pathways between classes of individuals and allows the NM 3.80 selected administration of compounds which enhance LD 6.31 or retard the appropriate segment of the metabolic sys DOPAC 5.21 tem. This provides a key to the rational identification, SYN 10.4 15 GA 4.31 development, manufacture and/or administration of pHPLA 6.21 existing or new pharmacological drugs and treatment 3-OHAN 9.85 protocols. Thus, in order to treat a disorder resulting DA 7.62 3-OHKY 7.95 from a diagnosed chemical imbalance such as a neuro TYR 6.34 logical disorder the physician or veterinarian need pHPAC 6.2 20 merely administer to the patient sufficient blocking nTYR 6.01 MN 5.72 agents, substances, or precursors thereof to make up for 5HTO 6.7 a deficiency in the naturally occurring blocking agent EPIN 5.71 or substance or administer the appropriate metabolic TYRA 6.84 inhibiting substance to modify the metabolic pathways 5-HIAA 3.74 25 HVOL 4.80 described using the chemical imaging system above HVAVA 3.01 described. Moreover, the ability to determine the pre 5-HTP 4.54 cise chemical nature of a neurological disorder, e.g. in 3-MT 6.2 SHT 4.03 comparison with a normal individual, is an extremely CAF S.12 30 important and desirable test, and may permit early iden TRP 5.01 tification and classification of individual disorders, and a treatment protocol with those naturally occurring The potential/time locations of the analytes in the compounds, substances or precursors which have a tyrosine (TYR) and tryptophan (TRP) metabolic path specificity for blocking, mediating or otherwise elimi ways (FIGS. 4 and 5) were plotted on X-and-Y time and 35 nating the disorder. potential axes as shown in FIG. 9. Individual analytical In other words, the present invention permits normal values were obtained by comparison of the magnitude izing information from precursors and metabolites of of a standard at the XY locations for the samples. From the neurotransmitters or other chemicals identified as the data produced on 106 samples for 28 compounds, active in disorders such that algorithms for the com significant differences between emesis susceptible and pounds measured can be developed. These algorithms enesis non-susceptible individual animals were found in can be utilized to compensate for biological variability the Noradrenergic, Dopaminergic, Serotonergic and in individuals, products and metabolism and refine the Purine metabolic systems. The compounds quantitated assessment of the effect of an individual neurotransmit reflect the various pathways affecting the Dopamine ter or pathway. Thus in the enesis case entire metabolic metabolites and others. These values were considered in 45 pathways when considered as a whole were shown to terms of an algorithm to reflect the possible concentra be different between susceptible and non-susceptible tions of MHPLA (3-methoxy-4-hydroxy phenyl lactic individuals, intervention guided by the chemical imag acid) to the HVA concentration, 3-MT to increasing the ing pathway definition to make the susceptible path DOPAC concentration, and DHPE (dihydroxyphenyl ways approach the non-susceptible was efficatious in ethanol) and MHPE or HVOL (3-methoxy-4-hydrox 50 suppressing emesis. It should also be recognized that the yphenyl ethanol) to decreasing the DOPAC concentra patterns obtained may show specific presence or ab tion: sence of compounds in a particular disease state. For From this information an appropriate compound to example, as discussed in my aforesaid U.S. Pat. No. block the motion induced emesis was deduced, and the 4,511,659, a significant binary different between cere compound was successfully tested on moderately eme 55 brospinal fluid samples from patients with dementia of sis susceptible animals. the Alzheimer type and normals was found in the appar It can be seen the present invention permits determi ent oxidation states of the neurotransmitters serotinin or nation of the precise identity of causitive agents, e.g. 5-hydroxy tryptamine (5HT), and its precursor 5 neurotransmitters responsible for motion-sickness, and hydroxy tryptophan (5HTP). Assuming these com allows the manipulation of metabolic pathways which 60 pounds to have causative effect in Alzheimers, by appear to have the effect of blocking motion sickness. blocking the generation of these compounds through Determination of the precise identity of these com metabolic pathways, the potential toxic effects of such pounds provides a valuable adjunct both to the diagno compounds may be mediated or eliminated. Having sis of the causative conditions or syndromes leading to identified those specific compounds which appear to a disorder, and also can provide the physician, veteri 65 reflect an underlying condition, one skilled in the art narian or pharmacologist with an analysis tool for de rationally may determine the point in the metabolic scribing essentially the complete small molecule or pathway in which to intervene in order to eliminate or substance inventory of an individual or group of indi block the generation of potentially toxic materials. 5,104,639 19 20 Blocking agents have an established and valued place cells, and also enhance separation of compounds that in symptomatic treatment of various allergic reactions co-elute. or disease. The present invention is based in part on the FIG. 6 is a block diagram of a system for processing realization that there are certain naturally occurring such dual signals. The elements illustrated in FIG. 6 are blocking agents in living organisms. In certain instances given the same manbers as the equivalent elements in disease or disorder is caused by an imbalance or insuffi FIGS. and , ciency of blocking agents. In other instances such as Through the use of peak signatures, there have been stress, e.g. due to motion sickness, blocking agent pro detected in the analysis of cerebrospinal fluid and brain duction may be insufficient, or simply too slow to pro tissue several peaks whose "signatures' were clearly not duce the desired blocking effect from the initial insult. 10 identical to that of the standard compound normally The present invention in part provides a method for observed at that retention time. Prior art detection identifying those individuals susceptible to a particular methods would not have allowed for the resolution of disorder, and those particular blocking agents or pre such co-eluting compounds and would have resulted in cursors thereof, or other substances or compounds, erroneous data. This new technology has clearly pro which advantageously may be administered to that 15 individual whereby to enhance or retard an appropriate vided a much needed method for determining the exis segment of that individual's metabolic pathway. Thus, tence of co-eluting compounds and has virtually elimi for example, while classical blocking drugs are pharma nated the misidentification of neutrotransmitters. cological antagonists that appear to act by occupying Additionally, two-dimensional pattern diagrams, i.e. receptors on the effector cell, to the exclusion of agonist electrochemical fingerprints of various selected materi molecules without themselves initiating a response, als, can be generated, similar to two-dimensional pat blocking agents identified in accordance with the pres tern diagrams achieved by standard chromatography ent invention necessarily need not react in a similar techniques by suitably integrating signals on various manner. The important criteria is that the so-called electrodes. In such case, samples of known substances blocking agent or other substance employed in therapy may be flowed through the electrochemical apparatus, produces in the individual a normalized or near normal 25 and two-dimensional pattern diagrams in the form of ized reaction. voltanmograms representing the selected substances Unlike artificial drugs the blocking agents or other generated using the apparatus and procedures above substances or compounds identified in accordance with described. The resulting pattern diagrams may be the present invention are naturally occurring in the stored in a suitable central processing unit for subse body of the individuals. Accordingly the usual side 30 quent pattern matching and identification. effects, contraindications, etc. as normally may be expe The process thus described also may be advanta rienced with administration of artificial drugs may not geously employed for directly analyzing body fluids result in administration of blocking agents or other such as urine or blood for the purpose of diagnosing substances or compounds identified in accordance with diseases or predisposition to diseases of a subject and the present invention. 35 establishing pharmaceutical protocols (sequence, type One skilled in the art will recognize the aforesaid and concentration of pharmaceuticals) for treating such invention is susceptible to modification. Thus, for exam diseases, thus providing unexpected and marked advan ple, while the electrochemical apparatus has been illus tages over the many step chromatographic analysis trated as having sixteen electrochemically isolated gate techniques reported by A. B. Robinson and L. Cauling, Marray cells, any number of electrochemically isolated in the paper entitled "Techniques of Ortho-Molecular electrochemical-gate/array cells may be employed in Diagnosis" in Clinical Chemistry, Vol. 20, No. 8, 1974, series to achieve the desired results. Moreover, reaction pages 967-965, and by Miyagi et al, in U.S. Pat. No. half-times may be modified by manipulating individual 4,338,811. cell volumes which in turn may reduce certain kinds of noise. Also, two or more sensing electrode cells may be Referring to FIGS. 7 and 8, a sample fluid may be employed downstream of one another to detect and directly streamed into the electrochemical detection measure additional groups of compounds of interest. apparatus 10 as described in detail supra, where sub For example, after a four-electrode gate sequence to ... stances of interest may be separated and detected in the eliminate irreversible substances and define the upper manner previously described. Individual peaks in the and lower potential limits, an array of sensors (for exam SO output signal from the detector 10 may then be inte ple 10 to 16), if desired, including also additional gate grated in an integrator S4, and the output signal from electrodes, could be arranged in an increasing oxidative the integrator 54 applied to a central processing unit 56 and then reductive modes. This will effectively display which is referred to as a CPU hereinafter as described the current voltage curves for eluting compounds for by Mijagi et al. In the CPU 56, a two-dimensional pat 55 term diagram representing the relation between the peak both their oxidative and reductive modes. The current areas and the retention times provided by the input from each electrode will produce one set of simulta signal is formed according to a preset program. When neous equations as follows: peak matching (shown in FIG. 7) is required, a refer iAC)--C). CC) . ence chromatogram is read out from a memory 58 such as a magnetic tape or a magnetic disk, and the two where A, B and Care constants defined by the potential dimensional pattern diagram above described is formed and the nature of the compounds a, b and c and where after the CPU 56 judges whether or not the retention Ca, C. C. . . . are the concentrations of a, b and c. It time of each peak in the subject's chromatogram coin would then be possible to solve and display, for as many cides with the retention time of the corresponding peak coeluting components as there are sensors in the array, in the reference chromatogram and carries out peak providing that there is not an absolute identity in the matching when coincidence is not detected. signature or current voltage curve. Emiploying an array Then, the data of the upper and lower limits of nor of sensors in sequence would enhance sensitivity of the mal persons are read out from the memory 58 upper 5,104,639 21 22 limit pattern diagram and lower limit pattern diagram of Still other changes and advantages will be obvious to the normal persons in the CPU 56. The pattern diagram one skilled in the art. of the data of the subject is then compared geographi I claim: cally with these upper and lower limit pattern diagrams 1. A method for developing a treatment protocol for in the CPU 56 so as to judge whether the subject is 5 correcting an abnormal condition or disease in a living normal or abnormal. The result of judgment is dis organism which method comprises the steps of: played on a display unit 60 and recorded by a hard copy supplying body fluids that contain electrochemically unit 62. As described hereinbefore, the process of the active constituents which are representative of the comparison and judgment may be displayed when so metabolic pathways of normal and abnormal or desired. 10 diseased individuals of the living organism; As shown in FIG. 8, the process of disease diagnosis analyzing the samples to detect the individual electro is completed when the subject is judged to be normal. chemically active constituents representative of the When, on the other hand, the subject is judged to be metabolic pathways of said individuals by passing a abnormal, the CPU 56 retrieves disease data in a manner sample containing body fluid from said living or as described hereinbefore from the file of classified 15 ganism sequentially through (a) a liquid chromato diseases stored in the memory 58. On the basis of the graph column for achieving time-space separation thus retrieved disease data, the CPU 56 forms the upper of the constituents eluting from the column and (b) limit pattern diagram and lower limit pattern diagram of an electrochemical detection apparatus comprising the disease and compares geographically these pattern a plurality of coulometric cells arranged in series; diagrams with the subject's pattern diagram so as to 20 maintaining the coulometric electrodes of said cells at select the corresponding disease or analogous disease. different potentials, said cells operating at progres Further the CPU 56 calculates the correlation coeffici sively varying potentials along the path of flow of ent between the pattern diagram of the subject and the eluant through the cells, said plurality of cells being average-valve curve of the selected disease and identi sufficient in number to separate by measuring po fies the chemical deficiency of the subject, and thus a 25 tentials of those relevant electrochemically active treatment protocol. The result of selection of the corre constituents coeluted from the chromatographic sponding disease or analogous disease, the result of column at any instant of time, and recording the calculation of the correlation coefficient and/or the results of such coulometric measurements so as to process of disease diagnosis by comparison of the pat separate said measurements by measuring potential tern diagrams, and the treatment protocol, are displayed 30 as well as time of elution; on the display unit 60 and recorded by the hard copy comparing the representative electrochemically ac unit 62. When it is necessary to modify some of the data tive constituents concentrations thus obtained with on the basis of doctor's other observations in the course representative electrochemically active constituent of judgment of the normality or abnormality or in the 35 concentrations of other living organisms and course of disease selection, necessary data are applied thereby identifying any differences between the from a manipulator panel 64 to the CPU 56 for the various metabolic pathways of said representative purpose of modification of the data. electrochemically active constituents as well as The invention has been described for use in analyzing unknown metabolites; Tyrosine and Tryptophan derived neurotransmitters, identifying those compounds which effect the pres precursors and metabolites vis-a-vis their role in mo ence and concentration of the representative elec tion-induced emesis. It will be understood, however, trochemically active constituents of the metabolic that the invention advantageously may be used to pathways; and diagonose and characterized disorders, and to charac administering to said organism said compounds terize and establish pharmaceutical protocols for a vari 45 which provoke a change in said metabolic path ety of environmentally induced, inherited and/or de ways and representative electrochemically active generative disorders such as Alzheimer's Disease, Par constituents substantially to approach that found in kinson's Disease, Huntington's Disease, ALS, MS and body fluids of normal organisms. senile dementias; behavioral disorders such as depres 2. A method according to claim 1, wherein said body sion, suicide, hyperactivity, weight control, schizophre 50 fluid contains a blocking agent, and including the step of nia, learning disabilities, memory retention, alcohol and adjusting the metabolic pathways by administering a substance abuse and dependency and sleeplessness; eme measured quantity of said blocking agent to said living sis in pregnancy or following chemotherapy; post oper organism. ative anesthesia response; and hypertension and cardio 3. A method according to claim 2, and including the vascular involvement which are given as exemplary. 55 step of provoking a change in the metabolic pathways One skilled in the art will recognize that the apparatus of said living organism by administering a chemical and method above-described advantageously may be compound which promotes autogenetic production of employed for resolving mixtures of a large number of said blocking agent in said living organism. electroactive organic substances among which are men 4. A method according to claim 1, wherein said body tioned unsaturated hydrocarbons, azides, triazines and 60 fluid comprises serum. phenothiazines, amino acids, amines and amides, phe 5. A method according to claim 1, wherein said body nols, aromatic OH, quinolines, quinones, imines, olefins, fluid comprises cerebrospinal fluid. ketones, aldehydes, esters, olefinic esters, ethers, or 6. A method according to claim 1, wherein said body ganometallics, diazo compounds, nitro compounds, and fluid comprises plasma. halogens. Electroactive organometallic compounds in 7. A method according to claim 1, wherein said body association with macro molecules also can be resolved fluid comprises urine. using the electrochemical apparatus and method above 8. A method according to claim 1, wherein said body desired. fluid comprises saliva. 5,104,639 23 24 9. A method according to claim 1, wherein said tissue metabolic pathways of normal and abnormal or comprises brain tissue. diseased individuals of the living organism; 10. A method for selecting a pharmaceutical for treat analyzing the samples to detect the individual electro ing abnormal or diseased living organisms which chemically active constituents representative of the method comprises the steps of metabolic pathways of said individuals by passing a supplying body fluids that contain electrochemically sample containing body fluid from said living or active constituents which are representative of the ganism sequentially through (a) a liquid chromato metabolic pathways of normal and abnormal or graph column for achieving time-space separation diseased individuals of the living organism; of the constituents eluting from the column and (b) analyzing the samples to detect the individual electro 10 an electrochemical detection apparatus comprising chemically active constituents representative of the a plurality of coulometric cells arranged in series; metabolic pathways of said individuals bypassing a maintaining the coulometric electrodes of said cells at sample containing body fluid from said living or different potentials, said cells operating at progres ganism sequentially through (a) a liquid chromato sively varying potentials along the path of flow of graph column for achieving time-space separating 15 eluant through the cells, said plurality of cells being of the constituents eluting from the column and (b) sufficient in number to separate by measuring po an electrochemical detection apparatus comprising tentials of those relevant electrochemically active a plurality of coulometric cells arranged in series; constituents coeluted from the chromatographic maintaining the coulometric electrodes of said cells at column at any instant of time, and recording the different potentials, said cells operating at progres results of such coulometric measurements so as to sively varying potentials along the path of flow of separate said measurements by measuring potential eluant through the cells, said plurality of cells being as well as time of elution; sufficient in number to separate by measuring po comparing the representative electrochemically ac tentials of those relevant electrochemically active tive constituent concentrations thus obtained with constituents coeluted from the chromatographic 25 representative electrochemically active constituent column at any instant of time, and recording the concentrations of other living organisms and results of such coulometric measurements so as to thereby identifying any differences between the separate said measurements by measuring poten various metabolic pathways of said representative tials as well as time of elution; electrochemically active constituents as well as comparing the representative electrochemically ac unknown metabolites; tive constituent concentrations thus obtained with identifying those compounds which effect the pres representative electrochemically active constituent ence and concentration of the representative elec concentrations of other living organisms and trochemically active constituents of the metabolic thereby identifying any differences between the pathways; and various metabolic pathways of said representative 35 selecting one or a mixture of chemical compounds electrochemically active constituents as well as known to (a) block or potentiate selected metabolic unknown metabolites; pathways identified by the analysis, or (b) supply or identifying those compounds which effect the pres retard selected molecules within said metabolic ence and concentration of the representative elec pathway. trochemically active constituents of the metabolic 19. A method according to claim 18, wherein said pathways; and body fluid comprises a fluid selected from the group selecting one or a mixture of chemical compounds consisting of serum, cerebrospinal fluid, plasma, urine known to (a) block or potentiate selected metabolic and saliva. pathways identified by the analysis, or (b) supply or 20. A method according to claim 18 wherein said retard selected molecules within said metabolic 45 body fluid derives from a body tissue. pathway. 21. A method according to claim 20 wherein said 11. A method according to claim 10, wherein said body tissue comprises brain tissue. sample contains an electrochemically active blocking 22. A method for treating an abnormal condition or agent, and including the step of electrochemically ex disease in a living organism which method comprises amining said sample to identify said blocking agent. SO the steps of: 12. A method according to claim 10, wherein said supplying body fluids that contain electrochemically body fluid comprises serum. active constituents which are representative of the 13. A method according to claim 10, wherein said metabolic pathways of normal and abnormal or body fluid comprises cerebrospinal fluid. diseased individuals of the living organism; 14. A method according to claim 10, wherein said 55 analyzing the samples to detect the individual electro body fluid comprises plasma. chemically active constituents representative of the 15. A method according to claim 10, wherein said metabolic pathways of said individuals bypassing a body fluid comprises urine. sample containing body fluid from said living or 16. A method according to claim 10, wherein said ganism sequentially through (a) a liquid chromato body fluid comprises saliva. graph column for achieving time-space separation 17. A method according to claim 10, wherein said of the constituents eluting from the column and (b) body fluid is derived from a body tissue. an electrochemical detection apparatus comprising 18. A method for identifying pharmaceutically active a plurality of coulometric cells arranged in series; compounds for testing living organisms for correcting maintaining the coulometric electrodes of said cells at an abnormal condition or disease, which method con 65 different potentials, said cells operating at progres prises the steps of: sively varying potentiais along the path of flow of supplying body fluids that contain electrochemically eluant through the cells, said plurality of cells being active constituents which are representative of the sufficient in number to separate by measuring po

si ig. 5,104,639 25 26 tentials of those relevant electrochemically active metabolic pathways of said animals by passing the constituents coeluted from the chromatographic samples containing body fluid from said animals column at any instant of time, and recording the sequentially through (a) a liquid chromatograph results of such coulometric measurements so as to column for achieving time-space separation of the separate said measurements by measuring potential constituents eluting from the column and (b) an as well as time of elution; electrochemical detection apparatus comprising a comparing the recorded results thus obtained with plurality of coulometric cells arranged in series; representative electrochemically active constituent maintaining the coulometric electrodes of said cells at concentrations of other living organisms and different potentials, said cells operating at progres thereby identifying any differences between the O sively varying potentials along the path of flow of various metabolic pathways of said representative eluant through the cells, said plurality of cells being electrochemically active constituents as well as sufficient in number to separate by measuring po unknown metabolites; tentials of those relevant electrochemically active identifying those compounds which effect the pres constituents coeluted from the chromatographic ence and concentration of the representative elec 15 column at any instant of time, and recording the trochemically active constituents of the metabolic results of such coulometric measurements so as to pathways; separate said measurements by measuring potential selecting one or a mixture of chemical compounds as well as time of elution; known to (a) block or potentiate selected metabolic comparing the recording results thus obtained with pathways identified by the analysis, or (b) supply or 20 like coulometric measurements of body fluids taken retard selected molecules within said metabolic from other individual species of said animals and pathways and introducing said compounds into a thereby identifying any differences between the pharmaceutically acceptable carrier; and various metabolic pathways of said representative administering to said living organism a pharmaceuti electrochemically active constituents as well as cally effective amount of said selected chemical 25 unknown metabolites; and compounds in said pharmaceutically acceptable identifying those compounds which effect the pres carrier. ence and concentration of the representative elec 23. A method for treating a disease state in an animal trochemically active constituents of the metabolic which method comprises the steps of analyzing body pathways; and fluid between animals by: 30 administering to said animals those of said com supplying body fluids that contain electrochemically pounds which provoke a change in said metabolic active constituents which are representative of the pathways and representative electrochemically metabolic pathways of said animals; active constituents substantially to approach that analyzing the samples to detect the individual electro found in body fluid or other animals. chemically active constituents representative of the 35 k E s

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65 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : slo , 639 DATED April 14, 1992 INVENTOR(S) : Wayne R. Matson It is Certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: "Claim lo, col. 23, line 6, after "fluids", insert --samples--." "Claim l8, col. 23, line 67, after "fluids", insert --samples--." "Claim 22, Col. 24, line 5l, after "fluids", insert --samples--." "Claim 23, col. 26, line 34, "or" should be --of--."

Signed and Sealed this Twenty-second Day of February, 1994 Attest: (a (e?ol

BRUCE LEHMAN Attesting Officer Commissioner of Patents and Trademarks