WO1997002246A1 - Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires - Google Patents

Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires Download PDF

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Publication number
WO1997002246A1
WO1997002246A1 PCT/US1995/008460 US9508460W WO9702246A1 WO 1997002246 A1 WO1997002246 A1 WO 1997002246A1 US 9508460 W US9508460 W US 9508460W WO 9702246 A1 WO9702246 A1 WO 9702246A1
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compound
cells
formula
leukocytes
asp
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PCT/US1995/008460
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Carol Lambert
Ronnie C. Mease
John G. Mcafee
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Research Corporation Technologies, Inc.
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Priority to PCT/US1995/008460 priority Critical patent/WO1997002246A1/fr
Priority to CA002225861A priority patent/CA2225861A1/fr
Priority to US08/673,798 priority patent/US5840859A/en
Priority claimed from US08/673,798 external-priority patent/US5840859A/en
Publication of WO1997002246A1 publication Critical patent/WO1997002246A1/fr

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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0455—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • Lipophilic fluorescent membrane permanent dyes have been used for over 15 years to measure membrane potential in both resting and activated neutrophils and lymphocytes in vitro.
  • the Upophilic chelates m In-oxine, ⁇ n In- tropolone, and 99m Tc-HMPAO (hexamethylpropylene amine oxime) are used clinically to label mixed leukocytes for detection of focal inflammatory lesions.
  • Leukocytes labeled in vitro with ""Tc-HMPAO or the m In-chelates are still the most widely accepted means of imaging inflammation. See, E. Lantto, Scand. J. Gastroent, 29 Supp, 203, 11 (1994).
  • Tc- HMPAO is generally preferred for detecting acute abdominals sepsis, inflammatory bowel disease (IBD), soft tissue sepsis and osteomyelitis. This is in spite of the fact that 99m Tc-HMPAO is less stable both in vitro and in vivo than either of the ⁇ n In radiopharmaceuticals, and that with ""Tc-HMPAO, there is diffuse abdominal radioactivity, gall bladder uptake, and renal excretion of an unidentified polar species containing 99m Tc as early as three hours post injection. Because of this 99m Tc leakage from labelled cells, 99m Tc-HMPAO is of no use in assessing urinary tract infections.
  • 99m Tc-HMPAO leukocytes have lower absolute uptake in abscesses and lower target to background ratios than cells labeled with ⁇ n In-oxine. See, J.G. McAfee et al, Eur. J. Nucl. Med.. ⁇ ,353 (1987).
  • m In-leukocytes are preferable for imaging chronic infection, renal sepsis, fevers of unknown origin, and intraabdominal abscesses in communication with the bowel lumen (A.M. Peters et al., J. Nucl. Med.. 33, 65 (1992)). Quantification of whole body retention of radioactivity with In-labeled granulocytes and of fecal excretion of radioactivity with m In-labeled neutrophils have been used as methods by which to quantify IBD and Crohn's Disease. S.H. Saverymuttu et al., Gastroent.. 85_, 1333 (1983). This quantification is not generally possible using ""Tc-HMPAO unless early SPECT imaging is performed using the procedure of Weldon fScand. J. Ent.. 29 Supp 203, 61 (1994)).
  • mammalian cells such as blood cells, associated with inflammation, infection, malignancies, and related pathologies.
  • the present invention provides compounds which can effectively radiolabel cellular membranes, methods of using them, and intermediates for the preparation thereof.
  • Preferred compounds of the invention are (aminostyryl)pyridinium salts of formula (I):
  • Det is an organic group comprising a detectable radioisotope
  • n is 4-16, preferably 6-10
  • Z " is one equivalent of a biologically acceptable cation, e.g., (I) is the pyridinium salt of an inorganic or organic acid which does not interfere with the ability of the compound of formula (I) to penetrate and label the membranes of target cells and is not toxic to the cells.
  • X is a radioisotope of iodine, i.e., 123 I, 125 I, or 131 I
  • Det is a chelating group comprising one equivalent of a metallic radioisotope such as m In or 99m Tc, chelated by a polycarboxylic acid.
  • the compounds of formula (I) are preferably employed in vitro, in combination with a pharmaceutically acceptable carrier or vehicle, to label populations of mammalian cells, such as blood cells, including mixed leukocytes or lymphocytes.
  • mammalian cells such as blood cells, including mixed leukocytes or lymphocytes.
  • the labelled cells such as the leukocytes or lymphocytes, localize at a site of inflammation, infection, malignancy, or the like, thus enabling the imaging of said site, for diagnostic purposes or to enable the effective targeting of therapeutic agents.
  • compounds (I) and (II) are the pyridinium salts of an inorganic or organic acids, i.e., Z " is halide, sulfate, carbonate, phosphate, bicarbonate, acetate, citrate, tartarate, maleate, malate, propionate, and the like.
  • canine mixed leukocytes and mononuclear cells were labeled in high yield (80-90%).
  • Canine mixed leukocytes labeled with one compound of the invention, 125 I- or 131 I-4b. show a higher degree of localization in a sodium urate induced abscess in the dog model than do lu In-oxine labeled mixed leukocytes.
  • the compounds of formula (I) can exhibit one or more of the following utilities: a. Mixed leukocytes labeled with the radioiodinated compounds can be reinj ected into the donor for the detection of either acute or chronic sites of inflammation/infection by diagnostic imaging techniques. b. Autologous lymphocytes labeled with the radioiodinated compounds can be used for in vivo lymphocyte tracking and clinical imaging of lymphatic malignancies. c. The present compounds can replace ⁇ n In-oxine as the preferred agent with which to label cultured lymphocytes for imaging metastatic melanoma prior to and after adoptive immunotherapy. d. Any isolated cell population can be radiolabeled using the present compounds for in vitro testing or tracking in vivo. e.
  • Autologous lymphocytes labeled with the present compounds can be used to detect the lymphocytic infiltration of the pancreas which occurs prior to and during the early stages of Type I Diabetes Mellitus. Diagnostic imaging with the labeled lymphocytes could be used to assess the effectiveness of different drugs for treating Type I Diabetes during the early stages of the disease. f. Autologous lymphocytes radiolabeled with the present compounds can be used to detect lymphocyte localization in transplanted organs as a means of early detection of the host's rejection of the transplanted tissue. More specifically, mixed leukocytes labeled with "Tc- containing compounds of formula (I) can be used to image sites of acute inflammation.
  • Nonspecific abdominal and renal uptake typical of 99m Tc-HMPAO should be reduced using 99m Tc chelated by a compound of formula (I).
  • Mixed leukocytes labeled with m In-, 131 I-, or 123 I- containing compounds of formula (I) can be used to image sites of chronic inflammation.
  • autologous lymphocytes labeUed in accord with the present method could be used for in vivo lymphocyte tracking and clinical imaging of lymphatic malignancies.
  • the present compounds may replace m In-oxine as the preferred agent with which to label cultured lymphocytes for imaging metastatic melanoma prior to adoptive immunotherapy.
  • Det is an organic group bound to, or which can bind to, or chelate a radioisotope.
  • chelating compounds or chelators are such molecules as EDTA, DTPA or DCTA or analogs or homologs thereof, or the compound of the formula:
  • This fo ⁇ nula depicts a cyclohexane-based metal chelator which may be attached to the pyridinium ring N through positions 4 or 5, or through alkyl group R 3 and which carries from 1 to 4 metal or nonmetal cations, monovalent cations, or the alkaline earth metals.
  • each individual cyclohexane-based molecule may carry up to 4 metal cations (where both R 3 groups are CH 2 COOM).
  • R 3 groups are CH 2 COOM
  • the number of metals will decrease to 2 or even 1 per cyclohexane skeleton.
  • the cyclohexane functionality admits of varying stereochemistry, and the aforementioned formula is not intended to limit the molecule to any specific stereochemistry.
  • both amino functionalities may be either cis or trans to each other.
  • a preferred cyclohexane ring-containing chelator is of the formula:
  • R 3 is (C r C 4 )alkyl or CH 2 CO 2 " and M is one equivalent of a cationic metallic radioisotope, such as ln In or 99m Tc.
  • the cyclohexane may be unsubstituted (except for the two nitrogen functionalities) or may be substituted, especially at the 4-position, with a hydroxy or acylated hydroxy group, such as with a lower acyl substitution.
  • a hydroxy or acylated hydroxy group such as with a lower acyl substitution.
  • other cyclohexane-based analogs such as alkyl derivatives (e.g., lower alkyl) or substitution products, wherein the derivatization or substitution do not interfere with the linking of the cyclohexane skeleton to N, with the chelating ability (affinity, geometry, etc.) of the individual chelating moieties, are equivalent to those actually shown.
  • Substitutions which are equivalent for the purposes of this invention are hydroxy, acyl, halogen, amino, and the like.
  • any metal capable of being detected in a diagnostic procedure in vivo or in vitro can be employed as M in the Det moieties.
  • any radioactive metal ion capable of producing a diagnostic result in a human or animal body or in an in vitro diagnostic assay may be used in the practice of the present invention.
  • Suitable ions include the following: Antimony-124, Antimony-125, Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, Cadmium-109, Cadmium- 115m, Calcium-45, Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60, Erbium-169, Europium-152, Gadolinium- 153, Gold-195, Gold-199, Hafnium-175, Hafnium-175-181, Indium-I l l, Iridium-192, Iron-55, Iron-59, Krypton-85, Lead-210, Manganese-54, Mercury-197, Mercury-203, Molybdenum-99, Neodymium- 147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185 + 191, Palladium
  • Autologous human lymphocytes can be labeled for in vivo tracking and for imaging lymph nodes in normal humans, for imaging lymphatic malignancies, for imaging tumor-involved lymph nodes and staging Hodgkin's disease, for imaging sites of chronic inflammation, and for the diagnosis of acute kidney-graft rejection.
  • Cultured lymphocytes labeled with the present compounds including interleukin-2 (IL-2) activated autologous peripheral blood lymphocytes (PBLs), tumor-activated killer lymphocytes (TAKs), lymphokine- activated killer cells (LAKs), and tumor infiltrating lymphocytes (TTLs) can be used to image tumors.
  • IL-2 interleukin-2
  • TTLs tumor infiltrating lymphocytes
  • Lymphocytes are extremely sensitive to radiation damage, and radiotoxicity resulting from nuclear accumulation of m In results in decreased lymphocyte proliferative capacity and severe chromosomal abe ⁇ ation. It is well documented that radiotoxicity generally decreases as the distance of a nuclide from the cell nucleus increases. According to various studies, radiation damage from Auger-electron emitters such as m In can be reduced 85-fold if the nuclide is confined to the cytoplasm rather than the nucleus, and reduced 120-fold if the nuclide is restricted to the cell membrane. Thus, the present compounds are expected to be particularly useful to label lymphocytes, as they are membrane- restricted.
  • Labeling of mixed leukocytes (harvested from 20 mL canine blood) with 4a-d was performed using five different procedures: 1) labeling in Diluent C (a commercial non-ionic cell labeling media developed by Zynaxis Co. that is not approved for human use) after two saline washes of the cell pellet; 2) labeling in saline after two saline washes of the cell pellet; 3) labeling in saline with no saline washes of the cell pellet; 4 and 5) labeling in ether 10% or 100% platelet poor plasma (PPP).
  • Diluent C a commercial non-ionic cell labeling media developed by Zynaxis Co. that is not approved for human use
  • Peripheral blood lymphocytes from dogs and rats as well as rat splenic lymphocytes were labeled in high yields with either 4a or 4b (Table 2). Viability of labeled lymphocytes was >90% both before and after radiolabeling (Trypan Blue exclusion test).
  • the effects of a reduced volume of plasma on the cell labeling yield in saline were determined by evaluating the labeling with 125 I-4b, (in 1.5 ml saline) of mixed leukocytes, whose prelabeling cell pellets were washed with either 2 x 10 mL saline or 1 x 10 mL saline or not washed. Unwashed mixed leukocytes in 1.5 mL 10% PPP in saline were also tested. Labeling yields were 82%, 84%o, 80%), and 83%, respectively. No evidence was found that residual plasma in unwashed cells reduced the labeling yield. Although 10% PPP reduced the yield somewhat, labeling yields are high enough to make this method practical. This ability to label leukocytes in the presence of as little as 10% plasma protects cells from premature activation and damage.
  • the present compounds can label both lymphocytes and mixed leukocytes in high yields, and that the latter can be used to image sites of focal inflammation.
  • the higher localization of leukocytes labeled with 131 I-4b compared to m In-oxine in induced inflammatory sites is significant, since " 'In-oxine gives the highest absolute inflammatory lesion uptake of all agents yet tested.
  • the increased localization observed with the present compounds may be due to an increase in cell viability. Since it has been suggested that the lymphocyte component of labeled mixed leukocytes increases the sensitivity of these mixtures for detecting chronic infections over the sensitivity obtained with pure granulocytes, this improved localization may also be due to an increased contribution by labeled lymphocytes spared the radiation damage they would receive from m In-oxine. Thus, the present compounds may be appropriate for tagging lymphocytes in in vivo tracking studies. These results also suggest that mixed leukocytes labeled with the present compounds may image chronic infectious foci with low neutrophilic exudation better than other agents.
  • NCS Triester 12 shown in Figure 3, was made by two routes. In the first, ethylenediamine was converted to the mono-t-Boc derivative and reacted with CDTAMA to give JJ . ( Figure 3). Methyl iodide esterification gave 14 and was followed by selective hydrolysis to 12 (overall yield 15%). Compound JJ , was also converted to J2 by refluxing the ammonium salt of JJ in a solution of methanol, chloroform, and sulfuric acid over 3A molecular sieves (50% yield). Compound 12 reacted with the NHS ester of several ⁇ -haloacetic acids to give 15.
  • Di-8-ASP H a 58.48 (d, 2H); H b 57.27 (d, 2H); H, 56.58 (d, 2H); H d 7.37 (d, 2H); H, or H f 57.18 (d, IH); H e or H f 56.72 (d, IH); H g 53.28 (app.t, 4H); H h 51.58 (br s, 4H); H* 50.88 (app.t, 6H); H j 51.27 (br s, 20H).
  • Di-6-ASP H a 58.48 (d, 2H); Hsorge 57.27 (d, 2H); H, 56.58 (d, 2H); H d 57.37 (d, 2H); H e or H f 57.18 (d, IH); H e or H f 56.72 (d, IH); E ⁇ 53.28 (app.t, 4H); H h 51.58 (br s, 4H); H- 50.88 (app.t, 6H); H j 51.27 (br s, 12H).
  • Di-4-ASP H a 58.48 (d, 2H); H b 57.27 (d, 2H); H, 56.58 (d, 2H); H d 57.37 (d, 2H); H, or H f 57.18 (d, IH); H e or H f 56.72 (d, IH); H g 53.26 (app.t, 4H); H h 51.55 (br m, 4H); H* 50.95 (app.t, 6H); H j 51.33 (br m, 4H).
  • Example 2 Example 2
  • Bu3Sn-Di-8-ASP Ha 58.53 (d, 2H); Hb 57.75 (d, 2H); He 56.60 (d, 2H); Hd 57.45 (d, 2H); He or Hf 57.56 (d, IH); He or Hf 56.76 (d, IH); Hg 53.32 (app.t, 4H); Hh,Hi,Hj, butyl-Sn 51.58, 51.43, 51.26, 5.0.84 (br m integrates higher than theoretical 57H); Hk 56.28 (m, IH); HI 56.03 (m, IH); Hm 55.09 (d,
  • Bu3Sn-Di-6-ASP Ha 58.60 (d, 2H); Hb 57.77 (d, 2H); He 56.60 (d, 2H); Hd 57.45 (d, 2H); He or Hf 57.55 (d, IH); He or Hf 56.75 (d, IH); Hg 53.30 (app.t, 4H); Hh,Hi,Hj, butyl-Sn 51.58, 51.43, 51.26, 5.0.84 (br m integrates higher than theoretical 57H); Hk 56.30 (m, IH); HI 56.06 (m, IH); Hm 55.15 (d, 2H).
  • Bu3Sn-Di-4-ASP Ha 58.50 (d, 2H); Hb 57.76 (d, 2H); He 56.60 (d, 2H); Hd 57.44 (d, 2H); He or Hf 57.55 (d, IH); He or Hf 56.75 (d, IH); Hg 53.32 (app.t, 4H); Hh,Hi,Hj, butyl-Sn 51.58, 51.43, 51.26, 5.0.84 (br m integrates higher than theoretical 41H); Hk 56.31 (m, IH); HI 56.05 (m, IH); Hm 55.04 (d, 2H).
  • Nonradioactive I-Di-10-ASP was prepared directly from Di-10-ASP by an alternative procedure and used as a standard against which to compare the physical properties of radioiodinated I-Di-10-ASP prepared from Bu 3 Sn-Di-10-ASP.
  • Nonradioactive I-Di-10-ASP was characterized by NMR and the identification was confirmed by accurate mass mass spectral analysis.
  • N-Succinimidyl-iodoacetate was prepared by the method previously used to make N-succinimdyl-bromoacetate (R.C. Mease et al, U.S. Patent No. 5,089,663).
  • Iodoacetic acid 2.0 g, 10.8 mmol
  • 100 mL dry CH 2 C1 2 was added to this.
  • 1.2 g (10.8 mmol) N-hydroxysuccinimide, and 2.2 g (10.8 mmol) dicyclohexylcarbodiimide was stirred for three days as room temperature under a nitrogen atmosphere.
  • the by-product, dicyclohexylurea was filtered and the filtrate concentrated to give a yellow solid.
  • Example 8 Radioiodination and cell labeling.
  • the reaction was diluted with 200 ul 5 mM sodium acetate in methanol and injected on to another HPLC system equipped with an inline UV detector, a radioactive detector and a 10 micron Spherisorb column.
  • the column was eluted with 5 mM sodium acetate in methanol (1 mL/min.).
  • the radioactive peak that eluted at 22 minutes was collected and this fraction was counted to give 15.9 uCi (53%). No UV peak was observed at this retention time so the specific activity was estimated to be 2170 Ci/mmol.
  • the column was eluted with 5 mM sodium acetate in methanol (1 mL/min.). The radioactive and UV absorbing fraction that eluted at 16 minutes was collected and this fraction was counted to give 858 uCi (73%). From the size of the UV peak, the specific activity was estimated to be 300 Ci/mmol.
  • Radiolabeling mixed leukocytes with 3-[4-[2-(N,N- dihexylamino)phenyl]ethyenyI]pyridino-E-l-[ 131 I]-iodo-propene A 1 mL microcentrifuge tube was charged with 500 ul (600 uCi) of the HLPC eluate of 3- [4-[2-(N,N-dihexylamino)phenyl]ethyenyl]pyridino-E-l-[ 131 I]-iodo-propene. This was evaporated to dryness under vacuum in a speed vac and reconstituted into 30 ul ethanol.
  • canine blood Forty milliliters of canine blood was drawn into a 60 mL syringe containing 6.5 mL acetate-citrate-dextrose solution. To this was added 5.5 mL 2% methyl cellulose in saline. The contents of the syringe were gently mixed by several inversions of the syringe and the syringe placed vertically, needle end up, for 45 minutes to allow the red blood cells to settle. The plasma was drawn off and placed into a 50 mL Falcon tube. The tube was centrifuged at 1100 rpm for 15 minutes to pellet the leukocytes. The plasma was removed and the cells resuspended in 2 mL saline.
  • Sprague-Dawley rat was sacrificed to remove the spleen (1.133 g).
  • the spleen was perfused with phosphate buffered saline to remove the lymphocytes.
  • the lymphocytes in 10 mL PBS were split into two equal portions and each portion was layered onto 5 mL of Lympholyte-Rat (Cedar Lane Laboratories Limited, Ontario, Canada) in a separate 15 mL centrifuge tube. The tubes were centrifuged ar 2700 rpm for 20 minutes at room temperature.
  • the lymphoctyes appeared as a layer at the interface of the saline and Lympholyte-Rat layers.
  • the saline layer was removed and discarded.
  • the lymphocyte containing layer from each tube was removed and transferred to a single 50 mL Falcon tube. To this tube was added 10 mL saline and the tube centrifuged for 7 minutes at 1600 ⁇ m. The supematant was removed, the cells resuspended in 10 mL saline, and centrifuged at 1600 rpm for 7 minutes. The supematant was removed and the cells suspended in 1 mL saline. Using a hemocytometer, a total of 5 million lymphocytes were counted. Trypan blue exclusion test showed that 12% of the cells were dead.
  • the supernatant, cells, and labeling tube were counted for radioactivity.
  • the cells were centrifuged again at 1600 rpm for 7 minutes, the supematant removed, and the cells resuspended. After the second centrifugation, 11.9uCi (77%) of 131 I was bound to the lymphocytes. After labeling, 12% of the cells were dead by the trypan blue exclusion test. Therefore, the labeling did not affect cell survival.

Abstract

L'invention se rapporte à des composés du type représenté par la formule (I). Dans ladite formule, n est compris entre 4 et 16, Det est un groupe organique comprenant un radioisotope ou capable de soumettre un radioisotope à une chélation, et Z- est un équivalent d'un anion biologiquement acceptable. Les composés de ce type sont utiles pour le radiomarquage des membranes cellulaires, par exemple celles des cellules hématopoïétiques.
PCT/US1995/008460 1995-07-06 1995-07-06 Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires WO1997002246A1 (fr)

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PCT/US1995/008460 WO1997002246A1 (fr) 1995-07-06 1995-07-06 Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires
CA002225861A CA2225861A1 (fr) 1995-07-06 1995-07-06 Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires
US08/673,798 US5840859A (en) 1995-07-06 1996-06-27 (Aminostyryl)pyridinium compounds for radiolabelling cell membranes

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CA002225861A CA2225861A1 (fr) 1995-07-06 1995-07-06 Composes a (aminostyryl)pyridinium pour le radiomarquage des membranes cellulaires
US08/673,798 US5840859A (en) 1995-07-06 1996-06-27 (Aminostyryl)pyridinium compounds for radiolabelling cell membranes

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