WO1992014149A1 - Dispositif et procedes d'utilisation de l'hemozoine - Google Patents

Dispositif et procedes d'utilisation de l'hemozoine Download PDF

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WO1992014149A1
WO1992014149A1 PCT/US1992/000819 US9200819W WO9214149A1 WO 1992014149 A1 WO1992014149 A1 WO 1992014149A1 US 9200819 W US9200819 W US 9200819W WO 9214149 A1 WO9214149 A1 WO 9214149A1
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birefringent
hemozoin
marker
sample
cells
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PCT/US1992/000819
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English (en)
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David W. Sammons
Robert M. Nalbandian
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Arizona Technology Development Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to diagnostic an therapeutic methods employing hemozoin, a metabolite of protozoa parasites in the genus Plasmodium .
  • Plasmodiu parasite Four species of the Plasmodiu parasite are known to infect humans with malaria. Plasmodium viva causes tertian malaria; Plasmodium malariae , quartan malaria; Plasmodium ovale , ovale malaria; and Plasmodium falcipazum . cause the most severe, and sometimes fatal, form of the disease, know as pernicious, subtertian, malignant or estivoautumnal malaria.
  • the life cycle of the Plasmodium organism is well known. See, e.gr., Kissane, J.M. , ed. , Anderson ' s Pathology, 9th ed. , 446-447, 1990.
  • Sporozoites of the malarial parasite invade and incubate i liver cells. The sporozoites evolve in and eventually lyse the liver cells, after which they are free to invade the red blood cells. Within the erythrocyte the parasite ingests hemoglobin and converts the heme portion to malarial pigment. Robbins, S.L., et al. , Pathologic Basis of Disease , 2 ed. , 459-462, 1979.
  • the most widely used diagnostic method is microscopic examination of thick and thin blood films to determine the existence of stained malarial pigment. This method typically requires about thirty to forty-five minutes in the preparation and examination of each sample and is associated with an error rate of about twenty-five percent.
  • malarial pigment and "hemozoin.”
  • the term "malarial pigment” to be a brown-black amorphous matrix which is the consequence of hemoglobin digestion by the asexual Plasmodium parasite in the stages of maturing trophozoites, schizonts, merozoites, and gametocytes.
  • Jamjoon, G. , JRev. Infect . Di ⁇ . , 10:1029-1034, 1988 has proposed malarial pigment serves as a nutrient and energy source through the gametocytes for the parasite in the sexual stages within the female Anopheles host.
  • hemozoin shall mean a linear polymer of oxidized heme molecules consisting of ferriprotoporphyrin IX with a +3 valence for the iron atom in the high spin mode, a having an adherent investing coat of protein. Hemozoin is bo birefringent and paramagnetic.
  • crystalline hemozoin shall mean hemozoin absent the investing coat protein. Crystalline hemozoin is also both birefringent a paramagnetic.
  • Jamioon suggests the utility of dark field microscopy for th detection of all forms of malarial parasites, including, the non pigment containing ring forms, in unstained blood films.
  • Th principal advantage offered by the dark field microscopic metho proposed by Jamioon. is its ability to detect ring forms of th malarial parasites which contain much smaller amounts of aggregate hemozoin than the more evolved, highly pigments schizonts an gametocytes.
  • the dark field process proposed by Jam oon requires however, that the thin film sample be prepared on a long cove slip, inverted, and mounted in glycerol while being raised from th slide by the support of two smaller cover slips. While thi arrangement avoids artifacts present on the surface of the slid and the mounting medium, by rendering them out of focus, it i cumbersome and inconvenient.
  • Jamioon suggests that the propose dark field microscopic diagnostic method is more sensitive tha routine bright-field methods due to faster scanning of relativel larger volumes of blood under lower magnifications (X 250 to 400) .
  • the spot hybridization techniques of Franzen are tim consuming and require a DNA probe. While these techniques are o value in detecting malarial infection, they are not commonl available in third world countries where malaria is most prevalen and are not easily adapted to rapid screening off multiple samples.
  • the second property of hemozoin which has recognize diagnostic value is its paramagnetic susceptibility.
  • Melville, D., et al., Nature , 255:706, 1975 employed a magnetic separato consisting of a polyethylene cylinder having an inflow and outflo arrangement mounted between the poles of a conventiona electromagnet.
  • Stainless steel wire having a circular cros section and diameter of 251m was randomly packet into the cylinder.
  • a magnetic field of 1.75 T (17.5 kgauss) was used and an estimate magnetic field gradient of 8 X 10 T/m was established close t the wires.
  • a sample of whole human blood was introduced into th cylinder. It was determined that approximately 70% of the re cells applied to the column were retained until the field was removed.
  • Fairla b, et al., Mol . Biochem. Parasit. , 12:307-312, 198 proposed a magnetic method for purification of hemozoin.
  • Fairlamb, et al. uses the paramagnetic susceptibility of hemozoi to separate hemozoin from lysed red cells. The lysed cells were then loaded onto a magnetic separation apparatus.
  • the describe magnetic separation apparatus consisted of a circular C-type Alnic permanent magnetic which generates a field strength of 0.7 Tesl between the poles.
  • the separation chamber was packed wit stainless steel wool and inserted between the poles of th magnetic; an induced magnetic field is established in the stee wool.
  • the lysed cells were run through the separation chamber an exposed to the magnetic field.
  • the paramagnetic hemozoin magnetically binds to the steel wool in the presence of the magnetic field. Removal of the separation chamber from the magnetic field demagnetizes the steel wool and releases much of the magnetically bound hemozoin.
  • hemozoin oftentimes confusingly termed malarial pigment
  • the characteristics of birefringenc and paramagnetic susceptibility attributed to hemozoin are, i fact, attributable to the crystalline structural arrangement of th components comprising hemozoin.
  • th "structure of malarial pigment" consisted of a complex of hemati and a nitrogenous compound.
  • hemozoi is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces and hydrophobic interaction, but not by covalent bonding.
  • Transmission electron-microscopy of isolated digestive vacuoles of the parasite reveals a population of "roughly spherical, 1.5 to 2 lm (diameter) organelles, each with a classic lipid bilayer membrane surrounding a striking array of crystals.”
  • the parasitized erythrocytes are characterized knob protrusions of the erythrocyte membrane and an underlyi cytoskeleton. These cytoskeletal knobs cause cytoadherence betwee the erythrocyte membrane and the vascular endothelium. Miller, e al. These knobs seem to bind specifically to an endothelia receptor, proposed to involve thrombospondin, which, in turn, lead to accumulation of the red cells within the visceral and cerebra capillaries. Kissane, J.M. , ed. , Anderson r s Pathology, 9th ed.
  • Miller, et al. discloses the involvement of malarial protein of about 300 kDa, which has been identified on th surface of a P. falciparum infected red cell, as a possibl cytoadherent group based upon its reactivity to antisera sensitivity to tryptic cleavage at the surface of the red cel coupled with loss of cytoadherence, and that knob-less variant which are not cytoadherent do not express this surface protein
  • knob-less variant which are not cytoadherent do not express this surface protein
  • Widder, K. , et al., J. Pharm . Sci . , 68:79-82, 1979 was the first group to synthesis human seru albumin microspheres, average diameter of 1 lm, in which magnetit and doxorubicin were entrapped.
  • Wider, et al. demonstrated tha the magnetic microspheres, when injected intravascularly, could b localized to a desired target site in vivo and in the presence of an externally applied magnetic field.
  • Hemozoin's characteristics of birefringence and paramagneti susceptibility render it an ideal candidate for incorporation int a drug delivery system. A use which has heretofore not been know or suggested. Because Qf the structure of hemozoin postulate herein, the crystalline hemozoin molecule offers a multitude o available binding sites, which may be linked to pharmacologicall active compounds, such as chemotherapeutic agents, to affinit ligands, to monoclonal antibodies or to DNA probes, in accordanc with well-known methodologies. The characteristics o birefringence and paramagnetic susceptibility, further permi diagnostic and therapeutic screening of body fluids or tissue to aid in determining the targeting and incorporation of the pharmacologically active compounds into the targeted site.
  • pharmacologicall active compounds such as chemotherapeutic agents, to affinit ligands, to monoclonal antibodies or to DNA probes
  • the present invention provides a diagnostic method which employs hemozoin or crystalline hemozoin bound to a targeting carrier, as a birefringent and paramagnetic label.
  • the present invention also provides a related therapeutic method which employs hemozoin or crystalline hemozoin bound to a pharmacologically active substance, and bound to a targeting carrier
  • th present invention includes a purification methodology for purifyin hemozoin by degrading the proteinaceous material associated wit hemozoin, to yield crystalline hemozoin, which is substantiall free of an associated protein sheath.
  • the present invention further presents a novel means for usin hemozoin in the development and screening of monoclonal antibodie and DNA gene probes. Because of its birefringent and paramagneti properties, hemozoin is an ideal label for monoclonal antibodie and DNA probes.
  • the cellular internalization of monoclona antibodies, as well as their cellular specificity and bindin affinity can be readily determined based upon magneti concentration of hemozoin bound cells and subsequent ligh microscopy under polarized light to determine birefringence.
  • Hemozoin either in crystalline form or with anommen protein coat, therefore, acts as a label and can be attached t monoclonal antibodies, affinity ligands or bioactive substances, and will replace conventional fluorescent labels, radioactiv labels, and chromogen labels as presently used in immunoassays and immunohistochemistry.
  • hemozoin may be used as a paramagnetic label to impart paramagnetic properties to nonmagnetic cells and bioactive substances. The imparted paramagnetic property permits the separation of cell sub-populations for diagnostic assays or in automated flow-through instrumentation, such as flow cytometry.
  • the birefringent characteristics of a labeled cell or bioactive substance either alone, or in combination with known fluorescent, radioactive or chro ogenic markers, will facilitate differential speciation of cell sub-populations.
  • the present invention presents a novel kit for magnetic separation of hemozoin bound cells or cellular material in a sample or for diagnosing malarial infection b detecting the presence of hemozoin complex in a blood sample.
  • Thi kit permits, for the first time, accurate diagnosis of malaria infection at the ring or early trophozoite (non-hemozoi containing) stage.
  • the present invention also presents a novel malaria therapeutic modality which is based upon blocking parasitemi degradation of hemoglobin into hemozoin, and, hence, destroying th parasite by obstructing the catabolism of hemoglobin or by blockin the aggregation of heme into hemozoin, thereby allowing the letha heme to injure the parasitic membrane and causing the parasite t self destruct.
  • Figure 1 is a scanning electron micrograph, at 30,000 magnification, of aggregated hemozoin molecules with anommen protein coat.
  • Figure 2a is a scanning electron micrograph, at 250,000 magnification, of a single hemozoin molecule showing theommen protein coat and the underlying crystalline hemozoin.
  • Figure 2b is a negative stain scanning electron micrograph at 200,000 X magnification, of a hemozoin preparation treated wit 4M urea illustrating the investing protein coat removed from th underlying crystalline hemozoin molecule.
  • Figure 3a is a diagrammatic representation of the deduce molecular structure of a single hemozoin molecule.
  • Figure 3b is a diagrammatic representation of the deduce three dimensional molecular structure of a crystalline hemozoi polymer.
  • Figure 4 is a transmission electron micrograph, at 30,000 X magnification, of a malarial infected erythrocyt showing the development of intra-erythrocytic cytoskeletal knobs.
  • Figure 5 is a perspective view of a magnetic separation apparatus in accordance with the present invention.
  • Figure 6 is a diagrammatic illustration of an automated hematology multi-parameter analyzer which has been extended to diagnose malaria in accordance with the present invention.
  • Figure 7a is a micrograph illustrating binding specificity of hemozoin-linked CTMO-1 monoclonal antibody to mouse erythrocytes under light microscopy.
  • Figure 7b is the micrograph of Figure 7a taken under polarized light.
  • Figure 8a is a micrograph illustrating binding specificity o hemozoin-linked rabbit anti-mouse monoclonal antibody to mous erythrocytes under light microscopy.
  • Figure 8b is the micrograph of Figure 8a taken under polarize light.
  • Figure 9 is a micrograph illustrating binding specificity o hemozoin-linked CTMO-1 monoclonal antibody to mouse B cells unde polarized light microscopy.
  • glycine-rich proteins may for sheets between adjacent ferriprotoporphyrin molecules, which ar trapped by noncovalent hydrophobic attraction. These glycine-ric sheets may act as spacer molecules which give rise to the 10A (lnm) periodicity noted by Moore, et al.
  • Alternative ligands oriented perpendicular to the plane of the pyrrole groups of each ferriprotoporphyrin IX molecule in the hemozoin molecule may include oxo, hydroxo, aquo, amide, phospho or disulfide.
  • FIGs 2a and 2b are scanning electron micrographs taken by the inventors. As illustrated in Figures 2a and 2b, the ferriprotoporphyrin IX polymer has an intimately adherent investing coat of laminar proteins, from which there is a protruding portion of the underlying crystalline hemozoin. The molecular structure of crystalline hemozoin, underlying the investing coat of protein.
  • ITUTE SHEET offers numerous available binding sites at each of the thir hydrogen atoms and eight hydrocarbon chains, consisting of fo methyl, two vinyl and two propionate chains, on each heme molecu within the polymer.
  • Hemozoin may be isolated from infected cel according to any one of a known variety of methodologies describ above, each of which are hereby incorporated by reference.
  • Fairlamb. et al. discloses the extraction of malari pigment from infected red cells by saponin lysis a centrifugation. The resulting pellet is ground in a morta Ground samples are withdrawn and diluted with buffer a centrifuged. The supernatant is loaded onto a magnetic separati device. Paramagnetic hemozoin in the sample binds to the ste wool in the magnetic separator. Removal of the magnetic fie releases the bound hemozoin for collection.
  • Removal of the investing protein coat increases t aggregation of the resulting crystalline hemozoin. Removal of t investing protein coat is achieved by treating it with pronase, mixture of proteolytic enzymes, at 37° C. in 0.1M TRIS-HC1 buffe at pH7.5. Due to its hydrophobic nature, crystalline hemozoin ha a marked tendency to aggregate in the presence of water.
  • crystalline hemozoin may be maintained in sodiu dodecyl sulfate (SDS) , non-ionic detergents, or other cationic o anionic detergents except deoxycholate, may be associated wit uniformly charged species, which are readily disassociated, suc as by cations, or a uniform protein coating, such as albumin, ma be imparted on the crystalline hemozoin in accordance with know methodologies.
  • SDS sodiu dodecyl sulfate
  • non-ionic detergents or other cationic o anionic detergents except deoxycholate
  • deoxycholate may be associated wit uniformly charged species, which are readily disassociated, suc as by cations, or a uniform protein coating, such as albumin, ma be imparted on the crystalline hemozoin in accordance with know methodologies.
  • Figure 3a represents the deduced structure of a singl crystalline hemozoin molecule, i.e., the ferriprotoporphyrin I polymer.
  • the structure illustrated in Figure 3a was deduced fro the known hemozoin's known characteristics of paramagneti susceptibility, birefringence and linear structure
  • Ferriprotoporphyrin IX is known to have unique valence electro orbital configurations in the 3d subshell, which are characterize by five unpaired outer orbital electrons, each with identica orbital spins. These orbital spins are algebraically additive i each planar molecule of ferriprotoporphyrin IX and, therefore, throughout the linear polymer. This unique electron arrangeme is in compliance with both the Pauli Exclusion Principle and Hund' Rules.
  • Each heme molecule contains thirty hydrogen atoms and eigh hydrocarbon chains, consisting of four methyl, two vinyl and tw propionate chains. It is believed that hydrophobic interaction and hydrogen bonds, mediated by the methyl, vinyl and propionat side chains of the tetrapyrrole ring of the ferriprotoporphyrin I molecule, may act with each other and the protein coat to furthe serve to stabilize the linear hemozoin polymer.
  • each crystalline hemozoin molecule consists of a plurality o ferriprotoporphyrin IX polymers aligned in a three-dimensiona array.
  • a plurality of ferriprotoporphyrin IX polymers form sheet in the X and Y axes, and overlying sheets of th ferriprotoporphyrin IX polymers stack to form the Z axis of th crystalline hemozoin.
  • the arraye ferriprotoporphyrin IX polymers are thought to be axially aligne through the central Fe in each ferriprotoporphyrin IX molecule.
  • the ferriprotoporphyrin IX polymer are thought to be laterally adjacent. The entire crystalli hemozoin molecule is stabilized by interactions between the si chains of each ferriprotoporphyrin IX molecule.
  • a preferred diagnostic and therapeuti strategy includes diagnosing Plasmodium falciparum infection b administering sub-therapeutic dosages of a membrane active compoun to a subject suspected of malarial infection. The subject's bloo levels are monitored to determine whether there is any shedding o the Plasmodium parasite into the blood stream. Once Plasmodiu infection is confirmed, therapeutic dosages of at least on membrane active compound may be given to have malariacidal effect.
  • a therapeutically effective dosage level will promote de polymerization or blockade of polymerization of ferriprotoporphyri to hemozoin, which will increase intra-erythrocytic levels of heme resulting in killing of the infected erythrocytes prior to maturit of the infection.
  • Plasmodium falciparum tissue cultures It has been found in Plasmodium falciparum tissue cultures, that treatment with 0.6M urea or 0.01M to 0.02M Piracetam exhibite malariacidal activity. Tables 1 and 2 detail the results of a fou day test of the malariacidal action of urea and Piracetam o Plasmodium falciparum . Plasmodium falciparum was plated into RPMI liquid growth medium with human serum and human red cells. The original samples were divided into two groups, Group 1 fo treatment with Urea and Group 2 for treatment with Piraceta (NOOTROPIL, UCB, Brussels, Belgium) .
  • Group 1 was divided into fiv titers, a control, 0.2M, 0.4M, 0.6M,and 0.8M;
  • Group 2 was divide into seven titers, a control, 0.02M, 0.04M, 0.06M, 0.08M, 0.1M an 0.2M. After twenty-four hours from inoculation, parasitemia dat was collected for four additional consecutive twenty-four hou periods.
  • Urea therapy of malaria will not precipitate the clinica paroxysms of malarial crisis.
  • the elevated and maintained BU levels will kill the parasites in situ within the erythrocyte which, in turn, will be removed from the systemic circulation b the reticulo-endothelial system.
  • No pyrogenic or foreig substances are introduced into the blood, as occurs when th Plasmodium schizont stage perforates and releases merozoites int the blood precipitating a malarial crisis.
  • UREAPHILE Abbott Laboratories
  • urea in 5% glucose/water
  • BUN levels are regularly monitored until a BUN level of about 200 mg/% is achieved.
  • the 200 mg/% BUN level maintained and the subject is observed until the clinical malari paroxysms have abated.
  • Diuresis is managed by water a electrolyte balance regimen.
  • crystalli hemozoin-pharmacologically active substance complex may be boun to monoclonal antibodies or affinity ligands as an antigen-specifi targeting carrier.
  • pharmacologically active substances such as chemotherapeut agents
  • EBA-175 Plasmodium falciparu erythrocyte binding antigen
  • Orlandi, et al demonstrated by comparative immunoprecipitations that EBA-175 i immunologically distinct from other well characterized merozoit surface antigens.
  • These other merozoite surface antigens included the 130 kDa glycophorin binding protein identified by Perkin an Pf200, the major merozoite surface glycoprotein which gives ris to several soluble processed products which interact with ligand at the surface of erythrocytes.
  • monoclona antibodies have been devised which are specific to Plasmodiu falciparum .
  • examples of other known clinically relevant monoclona antibodies which have been developed include monoclonal antibodie with specificity for malignant mammary tumor, malignant melanoma breast cancer, cervical carcinoma, lung tumor, measles virus
  • At least one of crystalline hemozoin having bound o chemically linked pharmacologically active substances, crystallin hemozoin formed with a uniform investing coat of protein, to whic there are linked pharmacologically active substances, crystallin hemozoin having chemically linked pharmacologically substances an formed into or in association with a microsphere or microcapsule is bound to a selected monoclonal antibody for targeted deliver of the pharmacologically active substance.
  • the chemical linke between the crystalline hemozoin and the pharmacologically activ substance is stable until the complex binds to the surfac receptors of a target cell and is taken up by pinocytosis o membrane transport.
  • an affinit ligand specific for selected receptor types may be bound to a least one of crystalline hemozoin having bound or chemically linke pharmacologically active substances, or crystalline hemozoin forme with a uniform investing coat of protein, to which there are linke pharmacologically active substances, crystalline hemozoin havin chemically linked pharmacologically substances and formed into o in association with a microsphere or microcapsule.
  • Widder & Senvei hereby incorporated by reference, note tha IgG and F(ab) 2 fragments have been covalently linked to liposome via oxidized glycolipids present in the bilayer of the liposome. Where F(ab) 2 specific for red cells were employed, a high degree of specificity for red cells was observed in vitro . Monoclonal antibodies were rendered hydrophobic and linked to the liposome with subsequent in vitro specificity, or were covalently linked v phosphatidylethanolamine in the liposome. Widder & Senve however, recognized that increased binding to the target cell do not ensure greater efficacy of the associated antitumor drug, ev in vitro.
  • U.S. Patent No. 4,885,172 disclosed that t glycoprotein streptavidin, which is a common biochemical linke can be coupled to any type of liposome, for the purpose of dr delivery.
  • streptavidin may additionally coupled to monoclonal antibodies of proteins, such Immunoglobulin G, to bioactive agents, including drugs, f therapeutic purposes, or dyes for diagnostic uses.
  • the hemozoin polymer may b bound to other macromolecules, such as dyes, e .g. , acridine orange to assist in the diagnosis of disease states or for imagin applications.
  • dyes e .g. , acridine orange
  • methyl, vinyl and propionate groups on eac ferriprotoporphyrin IX molecule within the hemozoin polymer serv as binding sites for attaching the polymer to carbohydrates of th heavy chain of monoclonal antibodies, amino groups on the heavy o light chains of monoclonal antibodies, to sulfhydryl groups on th monoclonal antibodies, amino groups, carboxylic acid groups o phosphate groups of the nucleic acids or their derivatives of gen probes, or reactant moieties, such as sulfhydryl groups, of variety of pharmacologically active substances.
  • Binding hemozoi or crystalline hemozoin to a monoclonal antibody, liposome or t an affinity ligand, as well as to a drug permits verification o the uptake of the drug by the targeted cell by i) magneti separation of the targeted cells and ii) light microscopy of th separated cells under polarized light to determine the presence o birefringent hemozoin.
  • Hemozoin may also be bound t radionucleosides for medical imaging, chemotherapy and/o radiography.
  • hemozoin or crystalline hemozoin wit a monoclonal antibody or affinity ligand, and a drug, under th present invention, therefore, readily facilitates testing for th internalization of the monoclonals, specificity and affinity of th monoclonals, internalization and membrane trafficking in the cell and targeted delivery of the monoclonals, affinity ligands an associated drugs, both in vitro and in vivo.
  • hemozoin, crystalline hemozoin, or complexed hemozoin will als serve as a paramagnetic and birefringent label for screening o monoclonal antibodies, purification of monoclonals and isotypin of monoclonal antibodies.
  • hemozoin, crystalline hemozoin, or complexe hemozoin may be used to give magnetic susceptibility to non magnetically susceptible cells.
  • hemozoin an crystalline hemozoin facilitate use of the molecule as a extracorporeal diagnostic marker when linked to monoclona antibodies which are cell or species specific.
  • a class of birefringent markers which may be non-magnetic or naturally or derivitized to have magnetic properties such as diamagnetism or paramagnetism.
  • hemozoin and crystalline hemozoin are but one example of potential members of this class.
  • birefringent materials for in vitro diagnostic use is characterized by either optical or electric birefringence and have available binding sites for binding to monoclonal antibodies, affinity ligands or liposomes. Where a particular birefringent material has magnetic properties, it will have additional utility as a means for separating bound cells from a cell sample in the presence of an applied magnetic field, which permits sequestration and concentration of the cell sample.
  • birefringent materials bound to cell or species specific monoclonal antibodies, affinity ligands, liposomes, gene probes etc. can replace or supplement the use of fluorphores in fluorescence microscopy.
  • Fluorescence microscopy is a widely used, expensive diagnostic technique which requires the diagnostici have relatively expensive system requiring a the generation of filtered specific high frequency stimulating wave length of lig in the u.v. range, and a series of filters which sequentially pa longer wave lengths of light to avoid injury to the observer.
  • use of birefringent markers requires only the use of polarizing filter with a light microscope.
  • optically or electrically birefringent marke coupled with site directed monoclonal antibodies, gene probe affinity ligands, etc. facilitates diagnosis of the presence neoplastic cells, circulating cellular elements, i . e erythrocytes, leukocytes, platelets, cell surface antigen bacteria, fungi, parasites, viruses, histoarchitectural tiss components, e . g. , elastin, collagen, amyloid varieties, endotheli cells, reticuloendothelial cells, or the like.
  • the presence of particular molecular structure in a sample may be verified und polarized light microscopy, irrespective of whether the sample a wet cell preparation, frozen tissue section, formalin or alcoh fixed tissue section, thin or thick blood smears, such as f malaria or sickle cell diagnosis, and pap smears.
  • Non-exclusi examples of current diagnostic tests which will benefit from u of birefringent markers include i) malaria diagnosis, whi currently employs thick and thin blood smears; ii) blood groupi and subgrouping, which is currently accomplished by agglutinati techniques; iii) HLA typing, which is currently performed fluorescence microscopy; iv) paternity testing, which is mo commonly accomplished by agglutination techniques; v) diagnost coagulopathy which is evidenced by the presence or absence platelet/endothelial surface active factors; and pap smears.
  • birefringent materials suitable for use diagnostic markers include polyurethane fibers, polypropylene, nylon beads or fibers frozen agarose gel, amylose complexes with polar and non-pol compounds which form crystalline complexes, polyethylene or ult high molecular weight-low molecular weight polyethylen polyethylene blend gels, polyamide spherulites an poly(aminocarboxylie acid) type polyamide spherulites, fibrin polydiethylsiloxane, proteoglycan, hyaluronic acid, collagen, variety of polypeptides, including, without limitation, poly- ⁇ benzyl- -glutamate, sodium dodecyl sulfate complexes o transferrin, bovine serum albumin, fumarase, ovalbumin chymotrypsinogen, pepsin, ⁇ -lactoglobulin, hemoglobin or myoglobin Stein, et al
  • a marker with paramagnetic properties may be naturally paramagnetic, such as hemozoin, crystalline hemozoin, or fibrin, or may b derivitized by attachment of the birefringent marker to a magneti bead or marker, such as by attachment to a magnetized particle, as taught by U.S. Patent No. 4,910,148 to Kvalheim, disclosing cellular attachment to magnetic particles; PCT application No. WO 9101368 to Mechaelsen, disclosing hapten and anti-hapten bound to agarose beads and magnetic particles; PCT application No.
  • WO 9006045 to Homes disclosing attachment of nucleic acid probes to paramagnetic particles for hybridization assays and oligonucleotide sequencing
  • PCT application no. WO 9006044 to Homes disclosing production of cDNA by contacting mRNA with an magnetic support
  • Homes PCT application WO 9006042 disclosing the use of magnetic particles for carrying a 5• attached DNA probe during the polymerase chain reaction, each of which is hereby incorporated by reference.
  • the present invention affords those in th art a significant advance over the current methods of choice fo in vitro diagnostics, i . e . , fluorescence microscopy.
  • Monoclonal antibodies which are either species-specific and/o stage-specific for each species, for a known disorder, are prepare according to known techniques.
  • the monoclonal antibodies are mixe with either crystalline hemozoin, or hemozoin prepared with substantially uniform investing protein coat to form a hemozoin monoclonal antibody complex.
  • a blood sample is drawn from subject.
  • the blood sample is mixed with the hemozoin-monoclona antibody complex and the mixture is passed through a magneti separation column in the presence of a magnetic field.
  • the colum is washed with buffer to remove loosely entrapped material, and th column is removed from the magnetic field.
  • Bound hemozoin-labele monoclonal antibodies are collected and examined under a ligh microscope using polarized light to determine the presence of an birefringent hemozoin. The diagnosis is scored.
  • a hemozoin-gene probe complex is prepared by mixing a selecte gene probe with crystalline hemozoin or crystalline hemozoin havin a substantially uniform investing protein coat.
  • a blood sampl is drawn from a subject.
  • the erythrocytes are separated from th blood sample by centrifugation.
  • the erythrocytes are lysed b saponin lysis to release Pla ⁇ modium, if any, in the sample.
  • DN in the sample is isolated and bound to non-magnetic affinity beads.
  • E SHEET Double stranded DNA bound to the affinity beads is denatured b heat, and the denatured DNA is mixed with the hemozoin-gene prob complex.
  • the hemozoin-gene probe complex is hybridized to DNA, an the hybridized DNA is passed through a magnetic separation colum exposed to a magnetic field. Washing with buffer removes debri from the column. The column is removed from the magnetic field an the hemozoin bound DNA, if any, is collected.
  • the collected DN may then be processed according to known amplification techniques
  • EXAMPLE 3 A hemozoin-gene probe complex is formed as described i Example 2. Cells or cell cultures are collected and lysed b saponin lysis. The cellular DNA is bound to cellulose affinit paper, and heat denatured. The bound cellular DNA is exposed t the hemozoin-gene probe complex and the DNA-hemozoin-gene probe i hybridized. The cellulose affinity paper is washed to remove non specifically bound DNA, and the paper is examined for the presenc of birefringent hemozoin.
  • a cell membrane-specific recognition protein is selected, e.g. , Pla ⁇ modium falciparum 175 kDa erythrocyte binding antigen (EBA-175) , is bound to either crystalline hemozoin or crystalline hemozoin having a substantially uniform investing protein coat.
  • a pharmacologically active agent e . g. , a pro-drug, is bound to the hemozoin.
  • the hemozoin, recognition protein, drug complex is injected intravenously into a subject, and after twenty-four hours, a blood sample is taken. The blood sample is passed through a magnetic separation column in the presence of a magnetic field.
  • the column is washed to elute the column of debris, and the column is removed from the magnetic field and magnetically bound red blood cells are collected.
  • the collected sample is viewed by light microscopy under polarized light to determine the efficacy of the drug delivery system by determining the localization of birefringent hemozoin within the sample.
  • EXAMPLE 5 Hemozoin was purified from a lysate of Plasmodium falcipar refrigerated at 4°C. The lysate was centrifuged for 25 min.
  • a second group was immunized i.p. with 0.2 ml each of solution consisting of the hemozoin pellet from the lysate and 0 ml CTMO-1 monoclonal antibody (5 ⁇ g/ml, Lederle) , an antibody to surface component of ovarian or breast cancer.
  • the mixture w stored overnight at 4°C, then centrifuged at 40,000g for 15 mi at 20°C.
  • the resulting pellet was re-suspended with the 0.1 CTMO-1 monoclonal antibody for 1 hour, centrifuged under identic conditions and re-suspended in with 0.1 ml of CTMO-1 monoclon antibody.
  • the re-suspension was stored overnight at 4°C.
  • a third group was immunized i.p. with 0.2 ml each of solution consisting of the hemozoin pellet from the lysate and 0 ml of rabbit anti-mouse erythrocyte (RAME) monoclonal antibo
  • Magnetic separation apparatus 10 consists generally of a steel-jacketed permanent magnet 12 which defines a pole gap 14.
  • a separation chamber 20 is disposed within the pole gap to expose the separation chamber 20 to the magnetic flux generated by the magnet.
  • the separation chamber 20 is packed with magnetic beads or iron filings 16 to a packing density which permits fluid flow through the separation chamber 20. It is preferable to employ a separation chamber 20 having a generally rectilinear or elliptical cross-section.
  • the use of magnetic beads and/or iron filings enormously increases the magnetic surface area and, therefore, the resultant magnetic dipoles between the pole gap, when compared to the prior art technique of random packing of stainless steel wire.
  • the separation chamber 20 by configuring the separation chamber 20 to have a generally rectilinear or elliptical cross-sectional shape, the peripheral areas of the chamber, i.e., those closer the chamber wall, a packed with a volume of magnetic beads and/or iron filings, whi closely corresponds to that in the more central areas of t chamber. In this manner, a minimal pole gap distance may maintained and, therefore, the maximal magnetic flux density, whi varies inversely with the size of the pole gap, is achieved.
  • a separation chamber e.g., about 1-101
  • magn having a high magnetic flux it is possible to sequest paramagnetic hemozoin or paramagnetic beads within the magnet field without packing the separation chamber with magnetic bea or filings. In this manner, it is solely the magnetic field whi sequesters the paramagnetic particles within the separati chamber.
  • the magnet 12 may be configured as a C-type magnet, b according to the preferred embodiment contemplated for t invention, magnet 12 consists of a pair of rectilinear magnets with the pole gap 14 being maintained by bilateral steel shims 18 Each magnet 12 is contemplated to have dimensions of 6.5 cm x 1. cm x 1.5 cm, with a pole gap of 1.5 mm being maintained by th bilateral steel shims 18. This provides a very compact, high flu magnet which is well suited to field use in remote areas wher automated diagnostic equipment is unavailable. Those skilled i the art will understand that these stated dimensions ar representational, and are not to be construed as limiting the scop of the invention.
  • rare earth alloys e . g. , neodymium vanadium alloy
  • a steel jacketed rare earth alloy permanent magnet and a flattene separation chamber packed with magnetic beads and/or iron filings This combination dramatically increases the magnetic flux densit within the small area of the separation chamber lumen, and afford a strong magnetic environment for sequestration of paramagneti material flowing through the separation chamber.
  • a diagnostic kit consisting o the magnetic separation apparatus 10, as described above, plurality of pre-packed separation chambers 20, packed wit magnetic beads and/or iron filings, and an external peristalti pump (not shown) to provide a fluid flow force to move a sampl through the separation chamber.
  • This will further be provide with appropriate washing buffer solutions, which may be added t the sample and which will be used to wash the separation chambe of extraneous non-magnetically attached material.
  • the buffers wil be provided in the range of pH 6-9, and may, for example consis of borate, phosphate, carbonate or the like. Buffer concentrations will generally range from about 0.01 to about 0.5M.
  • the technicia will provide a light microscope properly fitted with a polarized light source, for examination and diagnostic scoring of the sample.
  • an automated flow-through diagnostic test system may be constructed, utilizing techniques already existent in the art, for the diagnosis of the presence of hemozoin labeled material in a sample.
  • Coulter counters as disclosed in U.S. Patent No. 2,656,508, count blood cells and differentiate types of blood cells, and may be easily adapted to provide counts of hemozoin- labeled cells within a sample.
  • an automated system consisting of an improved automated continuous flow hematology multi-parameter analyzer as illustrated with reference to Figu 6.
  • the hematology multi-parameter analyzer 50 of the inventi provides a three-mode blood analysis system.
  • the analyzer 50 of the invention is capable of performing standa blood analysis, including several parameters of white blood coun red blood count, and platelet data and providing a print out that data.
  • a manifolded system consisting of a primary three-w manifold 52 and a secondary three-way manifold 54.
  • Prima manifold 52 bifurcates or directs a specimen blood flow 56 receiv from an input source to either of the standard blood analysis mo 58 or to a malarial diagnostic mode 60.
  • malarial diagnostic mode 60 provides input to t secondary three-way manifold 54.
  • Secondary three-way manifold 5 in turn, bifurcates or directs a specimen blood flow 56 to eith of an outlet mode for external intact cellular analysis 62 or a lysed blood cell mode 64 for detecting the presence of hemozo polymers in the sample. Samples withdrawn from the outlet mode are ultimately utilized for external microscopic slide examinati of the malarial cells.
  • analytical sub-systems 7 which consist of appropriate sensors and reagents for the detecti of structural and/or molecular features or components of hemozoi
  • analytical sub-systems 70 may consist of birefringence sensor having a polarized light source for examinin the sample for the presence of hemozoin.
  • the birefringence senso will be coupled to a computer programmed with a birefringenc pattern recognition library, which will record valid patter signals on an attached printer.
  • analytical sub systems 70 may consist of a ferrimagnetic electron spin resonanc and a magnetometer which will sense the presence of the Fe characteristic of ferriprotoporphyrin IX, thereby also determinin the presence of hemozoin in the sample.
  • analytica sub-systems 70 may consist of the magnetic separator devic illustrated in Figure 6, described above, adapted for use in a automated system by providing an electromagnet controlled by a D coil, which can be pulsed on and off in response to sensing th presence of a sample in the separation chamber, to provide sequestering and separation of paramagnetic particles or hemozoi in the sample, and having associated washing and bufferin solutions to clear the separation chamber for a subsequent sample Nomarski optics, gene probes and/or monoclonal antibodie techniques, as described above, may also be employed as discret analytical sub-systems 70.
  • birefringent and/o paramagnetic markers other than hemozoin or crystalline hemozoin may be used with the manual separation and/or automatic diagnosti system as markers in conjunction with gene probes, monoclonal antibodies, affinity ligands or other cell-specific materials.

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Abstract

L'invention se rapporte à des procédés diagnostiques et thérapeutiques utilisant des marqueurs biréfringents d'un type caractérisé par une biréfringence optique ou électrique, tel que l'hémozoïne, métabolite de parasites protozoaires du genre Plasmodium. L'invention décrit également des modalités diagnostiques et thérapeutiques utilisant des marqueurs biréfringents combinés en complexe avec un porteur de ciblage spécifique aux cellules et actif pharmacologiquement, tel qu'un anticorps monoclonal, un ligand à affinité sélective, un liposome ou élément similaire, où l'hémozoïne sert d'abord d'indicateur paramagnétique facilitant l'imagerie ou la séparation magnétique et, ensuite, de marqueur biréfringent déterminant la fixation du complexe à un site ciblé. L'invention décrit enfin des modalités thérapeutiques pour le traitement de l'infection paludéenne.
PCT/US1992/000819 1991-02-01 1992-01-31 Dispositif et procedes d'utilisation de l'hemozoine WO1992014149A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US766,622 1985-08-19
US64968991A 1991-02-01 1991-02-01
US649,689 1991-02-01
US76662291A 1991-09-26 1991-09-26

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WO2016066754A1 (fr) * 2014-10-29 2016-05-06 Université de Mons Détection du paludisme
US9400279B2 (en) 2004-12-09 2016-07-26 Osaka University Detection/measurement of malaria infection disease utilizing natural immunity by hemozoin induction, screening of preventative or therapeutic medicine for malaria infection disease, and regulation of natural immunity induction
CN113358709A (zh) * 2020-03-04 2021-09-07 郑有忠 疟疾检测方法和设备

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9400279B2 (en) 2004-12-09 2016-07-26 Osaka University Detection/measurement of malaria infection disease utilizing natural immunity by hemozoin induction, screening of preventative or therapeutic medicine for malaria infection disease, and regulation of natural immunity induction
US10172927B2 (en) 2004-12-09 2019-01-08 Osaka University Detection/measurement of malaria infection disease utilizing natural immunity by hemozoin induction, screening of preventative or therapeutic medicine for malaria infection disease, and regulation of natural immunity induction
WO2016066754A1 (fr) * 2014-10-29 2016-05-06 Université de Mons Détection du paludisme
CN107003311A (zh) * 2014-10-29 2017-08-01 蒙斯大学 疟疾检测
US20170336408A1 (en) * 2014-10-29 2017-11-23 Universite De Mons Malaria detection
US10473657B2 (en) 2014-10-29 2019-11-12 Magnetrap Malaria detection
CN107003311B (zh) * 2014-10-29 2020-08-11 蒙斯大学 疟疾检测
CN113358709A (zh) * 2020-03-04 2021-09-07 郑有忠 疟疾检测方法和设备

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