EP0621894A1 - Heme polymerase et procede pour traiter le paludisme - Google Patents

Heme polymerase et procede pour traiter le paludisme

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Publication number
EP0621894A1
EP0621894A1 EP93902801A EP93902801A EP0621894A1 EP 0621894 A1 EP0621894 A1 EP 0621894A1 EP 93902801 A EP93902801 A EP 93902801A EP 93902801 A EP93902801 A EP 93902801A EP 0621894 A1 EP0621894 A1 EP 0621894A1
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EP
European Patent Office
Prior art keywords
enzyme
heme
agent
malaria
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP93902801A
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German (de)
English (en)
Inventor
Andrew F. G. Slater
Anthony Cerami
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Picower Institute for Medical Research
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Picower Institute for Medical Research
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Publication date
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Publication of EP0621894A1 publication Critical patent/EP0621894A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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

  • Malaria is caused by a protozoan parasite of the genus Plasmodium. of which four species are known to infect humans: P. falciparum. P. vivax. . ovale and P. malariae. Infection is transmitted by female mosquitos of the genus Anopheles when, during a blood-meal, parasites are inoculated by the insect into a susceptible human host. Malaria transmission is indigenous in approximately 102 countries of the world and about 2.7 billion people (i.e., greater than 50% of the world's population) live in areas endemic for the disease.
  • malarial parasites invade erythrocytes where they grow, mature and divide. Typically, this division is characterized by a synchronized 48 hour incubation period which causes an afflicted subject to suffer a series of high fevers. In milder cases, disruption of red blood cells leads to anemia; in more severe cases, red blood cells are trapped within small vessels of the brain and cerebral malaria ensues, which in turn results in stroke and ischemia, followed by death.
  • hemoglobin is utilized by the parasite as a nutrient source. Hemoglobin comprises about 89% of the total cell protein and greater than 99% of the cytoplas ic protein. Since the malarial parasite has a limited capacity to synthesize amino acids de novo or to incorporate them exogenously, the abundant host cell hemoglobin is broken down to
  • hemozoin the black pigment associated with malaria
  • the food vacuole large lysosomes that serve as digestive vacuoles in the proteolysis of ingested erythrocyte hemoglobin
  • the host red cell ruptures, merozoite-form parasites emerge, and the pigment-filled vacuole (known at this stage as the "residual body") is fully excised from the differentiated parasite.
  • the residual body is eventually scavenged by resident tissue macrophages and the pigment is accumulated in the liver, spleen, and brain of an infected patient. Hemozoin can remain in these organs indefinitely, even after the infection has been cleared. Although the process of hemoglobin degradation and hemozoin formation is of central importance to the parasite, it has not yet been successfully characterized at the molecular level.
  • quinoline-containing antimalarial compounds accumulate in the acid food vacuoles of the parasite, the toxic mechanism of these drugs against the parasite, and more particularly the molecular basis for the specific vulnerability of growing intraerythrocytic malarial parasites to quinoline-containing drugs, has remained unascertained.
  • the primary function of the food vacuole is the proteolysis of ingested red cell hemoglobin to provide the growing parasite with essential amino acids. This breakdown of hemoglobin in the food vacuole releases heme which, were it left to accumulate in a soluble form could damage biological membranes and inhibit a variety of parasite enzymes.
  • hemozoin (historically also known as "malaria pigment") .
  • Hemozoin crystals form in the food vacuoles of the growing intraerythrocytic parasites concomitant with hemoglobin degradation, and remain there until the infected red cell bursts during schizogony.
  • the present invention includes the discovery of a previously unknown heme polymerase activity. This polymerase has been identified and characterized from extracts of Plasmodium falciparum trophozoites and found to be inhibited by quinoline- containing drugs, such as chloroquine and quinine.
  • quinoline- containing drugs such as chloroquine and quinine.
  • the present invention provides an explanation of the quinolines' highly stage-specific antimalarial properties.
  • the subject invention further provides means for discovering and testing new anti-malarial agents, especially for use in treating quinoline-resistant malaria.
  • new anti-malarial agents especially for use in treating quinoline-resistant malaria.
  • Another object of the subject invention is to provide a method for treating malaria by inhibiting the action of heme polymerase to minimize the damage caused by malarial parasites.
  • damage is minimized by reducing or eliminating infection through the hampering of heme polymerizing activity, effectively causing the parasites to die.
  • the subject invention provides a purified or isolated heme polymerizing enzyme characterized in that it is inhibited by quinolines, such as quinine, quinidine and chloroquine, and is further characterized in that enzymatic activity decreases at a Ph greater than about 6.5.
  • quinolines such as quinine, quinidine and chloroquine
  • An agent for selectively binding with the heme polymerase is provided.
  • This agent may be an alternate substrate or may directly act on the enzyme and may be employed in a method of inhibiting the polymerization of heme.
  • a prophylaxis or treatment of malaria may be accomplished.
  • a method of treating malaria which comprises contacting a heme polymerizing enzyme with a substrate other than heme.
  • This method can be adapted for treating quinoline-resistant malaria and comprises administering to a subject afflicted with quinoline-resistant malaria an inhibitor of a heme polymerizing enzyme.
  • a method of testing a compound for its effectiveness in treating malaria comprises contacting the compound to be tested, a heme polymerizing enzyme.
  • the measuring comprises assaying the amount of product produced.
  • the subject invention provides nucleic acids for probing and disabling the formation of the heme polymerase enzyme.
  • Figure 1A A bar graph showing hemozoin formation (measured as nmoles heme) formed from trophozoite extracts (0.5 mg protein) incubated overnight at 37°C in 500 Mm sodium acetate pH 4.8 in the presence or absence of 400 ⁇ M hematin. Results (mean +/-sem) from 11 separate experiments are shown.
  • Figure IB A bar graph showing hemozoin formation (measured as CPM from 1 C heme) formed from a trophozoite extract (65 ⁇ g protein) or a red cell extract (1.2 mg protein) incubated for 9 hr as above (see Figure 1A) in the presence or absence of 140 ⁇ M l C-hematin. Incorporation or radiolabel into hemozoin was determined in triplicate (mean +/-sem) .
  • Figure 2 A Fourier-transform infrared spectrum of the hemozoin product after an enzyme assay described in Experiment 2.
  • T is transmittance.
  • FIG. 3A A graph of hemozoin formation (pmoles heme) vs. time (mins) for an initial characterization of heme polymerase under conditions described in Experiment 3. Each data point shows mean +/- sem for triplicate assays.
  • Figure 3B A graph of the rate of hemozoin formation (pmoles heme/hr) vs. amount of protein (ng) in the enzyme extract for a characterization of heme polymerase under conditions described in Experiment 3. Each data point shows mean +/- sem for triplicate assays.
  • FIG. 3C A graph of the rate of hemozoin formation (pmoles heme/hr) vs. pH for characterization of the heme polymerase under conditions described in Experiment 3.
  • Sodium acetate (o) , citrate phosphate (•) or sodium phosphate ( ⁇ ) (200 mM) were used as buffers [control incubations in the absence of enzyme are also shown;
  • Figure 4A - A graph showing the % inhibition of heme polymerase vs. chloroquine concentration (mM) when assayed with S c ⁇ heme as a substrate under conditions described in Experiment 4.
  • Figure 4B A graph showing the % inhibition of heme polymerase vs. chloroquine concentration (mM) when assayed with a hemoglobin substrate under conditions described in Experiment 4.
  • FIG. 4C A graph showing the % inhibition of heme polymerase vs. drug concentration (mM) in a 14 C-heme assay under conditions describe in Experiment 4.
  • the drugs used were quinine (A), quinidine (•) and 9-epiquinine ( ⁇ ).
  • Figure 5 A graph showing chloroquine % inhibition of purified, soluble heme polymerase. Enzyme activity is inhibited 50% at 30 mM chloroquine, which compares with 70 mM chloroquine when enzyme activity in the crude membrane fraction of a parasite extract is assayed.
  • the inventors have discovered that malaria parasites detoxify heme in a unique way; i.e., heme is polymerized by a previously unknown polymerase to form hemozoin.
  • the inventors have also successfully isolated an enzyme from Plasmodium falciparum having heme-polymerase activity.
  • human parasites in the genus Schistosoma CS. mansoni. S. iaponicum are known to accumulate a pigment with similar properties to that of Plasmodium hemozoin while infecting man (Homewood, Jewsbury & Chance, Comp. Biochem. Physiol. , 43B: 517-523 (1972)). It is therefore likely that heme polymerase activity is present in these organisms, and that it would be inhibited by similar compounds to those inhibiting the malaria polymerase.
  • -10- parasites exhibit a heme polymerizing activity at such time, and that this activity is inhibited by the quinoline drug.
  • Isolated, purified and partially purified heme polymerizing enzymes may be incorporated into a number of biochemical assays. These assays include, but are not limited to, assays useful in screening anti-malarial agents. The assays may be performed in vivo or in vitro. By using an in vitro assay, a practical malaria drug screening system will finally be possible.
  • the heme polymerizing enzyme of the present invention would be contacted with the drug in question in the presence of a substrate under suitable conditions. (An illustrative incubation being at 37°C, in a buffer such as 200 mM citrate phosphate at pH 5.5). The effectiveness of the drug at inhibiting the enzyme would then be evaluated by measuring the amount of decrease in product formation. Methods of detecting the product are described hereinbelow. Drugs that effectively inhibit the heme polymerizing enzyme are likely candidates for treating malaria.
  • Agents which selectively inhibit the newly discovered heme polymerase are also envisioned. Such agents may function by inhibiting the enzyme directly or may compete with or disable the substrate which may be heme or some other suitable substrate.
  • An inhibiting agent may also be an alternate substrate which would likely contain a porphin macrocycle with either modified side chains or a different metal chelate.
  • One or more inhibiting agents may be used to inhibit polymerization of heme jLn vivo or in vitro. If the contacting of the heme polymerase with an inhibiting agent occurs in vivo, it may serve as a prophylaxis or treatment for malaria.
  • the agent is introduced in a physiologically/ pharmaceutically acceptable carrier.
  • physiologically acceptable carrier may be either liquid or solid and may include, but are not limited to, saline, sterile water, dextrose solutions, talcs, stearic acid, clay, sugars, salts, etc.
  • the mode of administering the agent for treating malaria may include, but is not limited to, injection (intravenous, intramuscular, subcutaneous, etc.), oral administration, transdermal application, inhalant, or suppository.
  • the invention may also be useful for treating quinoline- resistant malaria by aiding in identifying non-quinoline inhibitors and alternate or blocking substrates of heme polymerase.
  • the agent for interfering with heme polymerase activity will be introduced in a physiologically acceptable carrier.
  • -12- Other agents may be produced which specifically bind with heme polymerase, such as antibodies or other molecules (e.g., substrates) .
  • Specific binding agents may be used in assays, morphological studies and to treat malaria and related conditions.
  • Nucleic acids corresponding to the heme polymerase may also be identified and produced which could then be used as genetic probes or in the manufacture of anti-sense genetic material. Further, identification of the genetic material that encodes for all or segments of the active site of the heme polymerizing enzyme may be used in designing new drugs. It is to be understood that the nucleic acids may be DNA or RNA depending on the conditions of use.
  • any of the enzymes, polymers and agents described in the present application may be packaged as a kit.
  • a drug screening kit might contain a labeled substrate, a heme polymerase and a buffer.
  • the trophozoite extract catalyzed the conversion of heme into hemozoin ( Figure IB) , while in similar experiments, extracts of either uninfected red blood cells or macrophages did not. Solutions of either denatured hemoglobin or albumin (both having a high non-specific affinity for heme) were also negative for heme polymerase activity in this assay.
  • the malaria trophozoite extract is also capable of utilizing hemoglobin as the source of heme for hemozoin formation (see Figure 4B) . This is significant as it is not known whether the physiological substrate in the food vacuole is hemoglobin bound-heme, free heme, or heme associated with another vacuolar heme-binding protein.
  • trophozoite extract is separated by centrifugation into a soluble protein-rich fraction and a pellet containing predominantly membranes and hemozoin, heme polymerase activity is only found in the pellet.
  • trophozoites are extracted in the presence of either 0.5% CHAPS or 1% TRITON X-100, although the activity was destroyed following extraction in 1% SDS.
  • Activity declines rapidly during storage of the pellet at -20°C, although pretreatment of the pellet fraction with either protease E or protease K (1 mg. ml" 1 in 50 mM Tris»HCl, pH 7.4) did not affect enzyme activity.
  • hemozoin/membrane pellet is replaced in the assay with a ten-fold excess of either synthetic hemozoin or P. falciparum hemozoin purified of any protein or lipid contaminants, activity is not present. Accordingly, a component of the freshly prepared trophozoite pellet distinct from the hemozoin itself is responsible for heme polymerase activity.
  • the pellet fraction obtained from homogenized trophozoites was used to further characterize heme polymerase activity.
  • Product formation was linear with time (Figure 3A) and amount of protein added (Figure 3B) .
  • Activity increased slightly as the ionic strength of the buffer was raised, although considerable product formation occurred in all samples (50 mM - l.o M sodium
  • Chloroquine inhibits heme polymerase activity present in an extract of P. falciparum trophozoites (IC 50 ⁇ 120 ⁇ M, Figure 4A) . This inhibition occurs at a pH and drug concentration similar to that estimated to be achieved in the malaria food vacuole in treated patients.
  • 3-methyl chloroquine an antimalarially active congener of chloroquine
  • 8-chloroquine inactive as an antimalarial
  • Chloroquine also inhibits heme polymerase when a hemoglobin-rich red cell lysate was used as the source of heme for the enzyme ( Figure 4B) .
  • quinoline antimalarial amodiaquin which unlike chloroquine does not form a complex with free heme, was also found to inhibit heme polymerase (IC 50 - 250 ⁇ M) . This suggests that enzyme inhibition is not caused by a substrate-drug interaction.
  • quinidine IC 50 - 90 ⁇ M
  • quinine IC 50 - 300 ⁇ M
  • epiquinine IC 50 > 5mM
  • chloroquine may reach millimolar concentrations in the food vacuole of a susceptible malaria trophozoite. This probably inhibits heme polymerase, thereby disrupting the ordered conversion of hemoglobin-bound heme into hemozoin. Heme is known to be highly toxic for proteases, and its presence would therefore be expected to rapidly block further hemoglobin degradation by malarial proteases. This may explain the specific vulnerability of growing intraerythrocytic malaria parasites. to quinoline-containing drugs. A detailed understanding of the mechanisms of action of heme polymerase opens the possibility of designing new classes of antimalarial agents, both as direct inhibitors of the polymerase and as alternative substrates to preoccupy the polymerase.
  • Experiment 1 determined hemozoin formation in an extract of malaria trophozoites.
  • Trophozoite extracts 0.5 mg protein
  • Trophozoite extracts 0.5 mg protein
  • results (mean +/- sem) from 11 separate experiments are shown in Figure 1.
  • a trophozoite extract (65 ⁇ g protein) or a red cell extract (1.2 mg protein) was incubated for 9 hr as above in the presence or absence of 140 ⁇ M 14 C-hematin. Incorporation of radiolabel into hemozoin was determined in triplicate (mean +/- sem) .
  • P. falciparum clone HB-3 was cultured in A+ human erythrocytes. Synchrony was maintained by sorbitol treatment. Trophozoites were harvested by saponin lysis, washed twice in phosphate buffered saline (PBS) pH 7.0 and stored at -70°C. Trophozoite pellets were resuspended in PBS pH 7.0 with 1.5 mM MgCl 2 ⁇ 1 mM PMSF, 1 mM 1,10 phenanthroline, 0.1 mM leupeptin and 50 ⁇ M pepstatin, and mechanically disrupted with a Dounce homogenizer. A red cell extract was prepared by homogenizing 100 ⁇ l washed, packed A+ human red cells in 10 ml PBS as above.
  • PBS phosphate buffered saline
  • Experiment 2 determined the Fourier-transform infrared spectrum of the hemozoin product after an enzyme assay.
  • Heme polymerase in a trophozoite extract (2.3 mg protein, 47 moles hemozoin-associated heme) was assayed with heme as described in Experiment 1(a). The product remaining after SDS- bicarbonate and protease treatment was washed three times in deionized water (dH 2 0) and lyophilized. KBr pellets were prepared, and the spectrum acquired for 30 cycles in a Fourier- transform infrared spectrometer (Perkin Elmer model 1980) . A control spectrum obtained from a trophozoite extract alone was very similar to that shown, except that the peaks were less intense.
  • Experiment 3 was an initial characterization of heme polymerase determining (a) time dependent enzyme activity, (b) concentration dependent enzyme activity, and (c) pH dependent enzyme activity.
  • Experiment 4 evaluated the inhibition of heme polymerase by quinoline-containing antimalarial drugs. The following were determined: (a) effect of chloroquine on heme polymerase activity assayed with 14 C-heme as substrate, (b) effect of chloroquine on heme polymerase assayed with a hemoglobin substrate, and (c) comparison of quinine, quinidine and 9- epiquinine as inhibitors of heme polymerase in the 14 C-heme assay.
  • Trophozoite extracts and 14 C-heme were prepared as described in Experiment l; leupeptin and pepstatin were omitted from the trophozoite extraction buffer for the experiments shown in (b) .
  • Washed A+ human red cells were lysed in three volumes of dH 2 0, and membranes removed by centrifugation at 25,000 x g for 30 min.
  • Results are expressed as % inhibition relative to hemozoin formation in a drug-free control, and show either mean +/- sem for triplicates (a,c) or single assays (b) .
  • Heme polymerase was purified as follows:
  • P. falciparum trophozoites were homogenized in 20 mM bis Tris propane pH 7.3 ("buffer A") in the presence of protease inhibitors and centrifuged at 25,000 x g for 40 min at 4°c. The supernatant was removed, and the pellet resuspended by brief sonication in buffer A containing 1 M NaCl and 10% acetonitrile. The sonicate was diluted in three volumes acetonitrile and shaken for 3 hours at 4°C, and then centrifuged as above. The upper phase of the supernatant was collected, and the pellet reextracted under identical conditions.
  • the soluble upper phases were pooled, diluted in buffer A to 10% acetonitrile, and loaded onto a Mono Q 5/5 fplc column (Pharmacia) .
  • Bound proteins were eluted with a linear gradient from 40 mM to 1 M NaCl.
  • Fractions containing heme polymerase activity were pooled, diluted in one volume buffer A to 5% acetonitrile, and loaded onto a phenyl superose 5/5 fplc column (Pharmacia) .
  • Bound proteins were eluted with a linear gradient from 5% to 90% acetonitrile.
  • Fractions containing heme polymerase activity were pooled, and constitute the material referred to as purified enzyme. Purifications and activities are expressed in Table 1. Enzyme activity is expressed as cpm of 14 C heme incorporated into pigment during the assay.
  • Phenyl superose active fractions (l* 47 >47000 )203
  • Soluble heme polymerase was purified as described in Experiment 5. Enzyme was eluted from a phenyl superose fplc column, and assayed in the presence of increasing concentrations of chloroquine. Heme polymerase enzyme activity is graphically expressed in Figure 5 as % inhibition relative to activity recorded in the absence of chloroquine.
  • Sensitivity of purified, soluble heme polymerase to boiling and protease treatment was determined as follows:
  • Soluble heme polymerase was purified as described in Experiment 5. Enzyme eluted from a phenyl superose fplc column was assayed either before or after boiling at 100°c for 15 mins (see Table 2) . Enzyme activity is expressed as both cp of I4 C heme incorporated into pigment during the assay and as % inhibition of activity relative to that of untreated heme polymerase enzyme. The results of this experiment are expressed in Table 2.

Abstract

L'invention se rapporte à une enzyme hème polymérase. La polymérase est une enzyme critique dans la biologie du paludisme. En utilisant cette nouvelle polymérase, on rend possible des titrages permettant de développer des médicaments antipaludiques. En plus de fournir une clé pour les études pharmaceutiques futures et pour les caractérisations biochimiques du paludisme, cette invention rend possible les traitements antipaludiques, y compris contre les paludismes résistant à la quinoline. En outre, en utilisant des techniques inhibant la polymérase, telles que l'inactivation de la polymérase ou en offrant un substrat alternatif, on obtient des résultats de pointe importants en matière de thérapie, de prophylaxie et de recherche contre le paludisme.
EP93902801A 1991-12-31 1992-12-29 Heme polymerase et procede pour traiter le paludisme Withdrawn EP0621894A1 (fr)

Applications Claiming Priority (3)

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US815400 1985-12-31
US81540091A 1991-12-31 1991-12-31
PCT/US1992/011279 WO1993013197A1 (fr) 1991-12-31 1992-12-29 Heme polymerase et procede pour traiter le paludisme

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WO1998003661A2 (fr) * 1996-07-19 1998-01-29 Arch Development Corporation Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan
US20120077206A1 (en) 2003-07-12 2012-03-29 Accelr8 Technology Corporation Rapid Microbial Detection and Antimicrobial Susceptibility Testing
WO2005027714A2 (fr) 2003-07-12 2005-03-31 Accelr8 Technology Corporation Biodetection sensible et rapide
US10254204B2 (en) 2011-03-07 2019-04-09 Accelerate Diagnostics, Inc. Membrane-assisted purification
EP2683831B1 (fr) 2011-03-07 2015-09-23 Accelerate Diagnostics, Inc. Systèmes de purification rapide de cellules
ES2744630T3 (es) * 2012-09-10 2020-02-25 Accelerate Diagnostics Inc Hemocultivo del mismo día con microscopia digital
US9677109B2 (en) 2013-03-15 2017-06-13 Accelerate Diagnostics, Inc. Rapid determination of microbial growth and antimicrobial susceptibility
US10253355B2 (en) 2015-03-30 2019-04-09 Accelerate Diagnostics, Inc. Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing
AU2016243656A1 (en) 2015-03-30 2017-11-09 Accelerate Diagnostics, Inc. Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing

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US5021426A (en) * 1990-02-26 1991-06-04 Merck & Co., Inc. Method of traeting malaria with cyproheptadine derivatives

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AU3424793A (en) 1993-07-28

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