WO1994022446A1 - Pharmaceutical composition for the prevention of neuronal cell death - Google Patents

Pharmaceutical composition for the prevention of neuronal cell death Download PDF

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Publication number
WO1994022446A1
WO1994022446A1 PCT/DK1994/000101 DK9400101W WO9422446A1 WO 1994022446 A1 WO1994022446 A1 WO 1994022446A1 DK 9400101 W DK9400101 W DK 9400101W WO 9422446 A1 WO9422446 A1 WO 9422446A1
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WO
WIPO (PCT)
Prior art keywords
furosemide
cell death
neuronal cell
dihydroxy
nitro
Prior art date
Application number
PCT/DK1994/000101
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English (en)
French (fr)
Inventor
Lars Dalgaard
Original Assignee
Novo Nordisk A/S
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU64234/94A priority Critical patent/AU6423494A/en
Publication of WO1994022446A1 publication Critical patent/WO1994022446A1/en

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Classifications

    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines

Definitions

  • the present invention relates to pharmaceutical preparations having the ability to protect against neuronal cell death associated with neuronal focal or global ischemia, oedema, or traumatic events.
  • the invention also relates to a method of protection against neuronal cell death associated with neuronal focal or global ischemia, oedema, or traumatic events. Such conditions occur in subjects suffering from stroke, cardiac arrest, suffoca ⁇ tion or traumatic injury of the brain or spinal cord.
  • NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxali- ne) is useful in the treatment of indications caused by hyperactivity of the exitatory neurotransmitters, US Pat. No. 4,889,855.
  • NBQX has proved to be effective in animal models of cerebral global ischemia (Sheardown et al., Science, 247:571 , 1990; Buchan et al. Neurosci. Lett., 132: 255, 1991) and focal ischemia (Buchan et al. NeuroReport, 2:473, 1991).
  • the neuropro- tecting effect is due to blockade of especially the AMPA receptor, which is a subtype of the glutamate receptor.
  • Even 8 hours after the ischemic insult treatment with NBQX has been shown to be reduce the injury in the hippocampus of rat brain (Pulsinelli et al., Neurology 42: Suppl. 3:532S, 1992 (Abstract)).
  • NBQX also reduces glutamate mediated brain edema in the rat (Westergren et al., Brain Res. 573: 324, 1992) and has an anticon- vulsa ⁇ t effect in mice (Chapman et al. Epilepsy Res. 9:92, 1991 and Smith et al., Eur. J. Pharmacol., 201 :179, 1991).
  • NBQX as an antiischemic agent has been materially improved by administration simultaneously, separately or sequentially with a diuretic, more specific a loop-diuretic such as furosemide.
  • Furosemide is a highly potent diuretic agent which act on the lumi ⁇ al surface of the acsending loop of Henle by inhibiting the active reabsorbtion of chloride. Along with chloride, there is an enhanced excretion of sodium, potassium, protons, calcium, magnesium, ammonium, bicarbonate and possibly phosphate. The resulting low osmolality in the medulla, decreases the ability of the kidney to reabsorb water. The effect on pH is low, although temporary increases have been observed.
  • the average peak diuretic response to furosemide in rats and humans are about 30 times the control values of 70 ⁇ l/min/kg and 15 ⁇ l/min/kg, respect ⁇ ively (Andreasen, 1991).
  • the present invention relates to a method of protection against neuronal cell death associated with neuronal focal or global ischemia, oedema, or traumatic events. Such conditions occur in subjects suffering from stroke, cardiac arrest, suffocation or traumatic injury of the brain or spinal cord. Further, the present invention relates to pharmaceutical compositions useful in the treatment of such conditions.
  • the invention relates to a method of treating ischemia and related conditions in mammals, including humans, which comprises simulta ⁇ neous, separate or sequential administration of NBQX and of an effective loop-diuretic, such as furosemide, to a patient suffering from the noted conditions, and to pharmaceutical compositions containing effective doses of both of these compounds.
  • loop-diuretics which are potent in improving the antiischemic effect of NBQX, are bumetanid, 3- (aminosulfonyl)-5-(butylamino)-4-phenoxybenzoic acid or ethacrynic acid, [2,3-dichloro-4-(2-methylene-1 -oxobutyl)phenoxy] acetic acid.
  • diuretics which are carbonic anhydrase inhibitors such as chlorothiazide, hydrochloride or be ⁇ zthiazide.
  • This invention is based on the discovery that simultaneous, separate or sequential administration of NBQX and a diuretic agent, such as furosemi ⁇ de, preferably in two separate compositions containing effective amounts of the two compounds, significantly increases the safety margin between the dose causing a potential risk of precipitation of NBQX in the renal tubuli and the dose required for the neuroprotective effect.
  • a diuretic agent such as furosemi ⁇ de
  • the safe dose of NBQX which can be administered is 5-30 times higher than without furosemide, resulting in an increased plasma and brain con ⁇ centration of NBQX.
  • the increased diuresis causes loss of water and salts, in particular sodium and potassium which may be replaced by i.v. infusion of physiological saline or Ringer solution.
  • MAP Mean arterial pressure
  • the rats Four days after the ischemic challenge, the rats were anaesthetized with pentobarbital and perfusion fixed with 4% buffered paraformaldehyde. After embedding, the brains were coronally sectioned and stained with haema- toxylin and eosin. Damage was assessed in a "blind" manner in hippocam- pus CA1 region, and other regions by counting the number of dead neuro ⁇ nes (eosinophilic) as well as normal neurones. Results were compared using two-tailed Student's t-test. (Kaiser et al., Mol. Neuropharm, 2:219-220 1991.
  • mice Male Sprague-Dawley rats weighing between 180 - 300 g were anesthetized with an intraperitoneal injection of tribromoethanol in 0.9 % saline (also containing 8 % ethanol) and then cannulated in the right jugular vein. The cannula were transferred subcutaneously, externalised in the neck and connected to a swirvel. The animals were placed in a metabolism cage and allowed to recover for one day before the infusion experiments were started. Dosing was performed as described below and then after 24 hours the cannula was removed and the animals put in an ordinary cage with free acces to food and water for 4 days. Then the rats were killed, the kidneys removed and put in formalin until fixation and histopathologic examination.
  • the rats were initially given a solution of 3.8 mg/ml KCI + 6 mg/ml NaCI (at a rate of 25 ml/kg/h) for one hour in order to increase the water load. Then, a solution of 10 mg/ml furosemide was given for 5 min at a rate of 24 ml/kg/h corresponding to a dose of 20 mg/kg. Immediately afterwards was given a formulation containing NBQX ( 1.0 mg/ml) and furosemide (0.67 mg/ml) in a vehicle consisting of PVP 12 (5 %) and glucose (4%) adjusted to pH 7.4.
  • the rate of infusion was 30 + 60 + 2 ml/kg/h correponding to 30 + 60 + 2 mg/kg/h for Vz + Va +23 hours, respectively (Fig. 1). In all, a volume of 72 ml/kg was given for the first two hours (one hour pre dosing + the first hour of dosing).
  • the infusion solutions were introduced through the vein cannula using a gastight glass syringe fitted into a Harvard 22 infusion pump.
  • Urine was collected before (-1 - 0 hour) furosemide was given and then at intervals between 0 - 1 , 1 - 3, 3 - 24 hours.
  • Blood samples were taken from treated animals. After one hour, by the end of the NBQX infusion at highest rate (60 mg/kg/h), the animals were put in light CO 2 anesthesia and blood was collected by heart puncture.
  • the HPLC system consisted of a Kontron 420 pump, a Kontron 425 gradient former, a Kontron 460 autosampler, a LiChrospher RP 18, endcapped 5 ⁇ m particle analytical column (250 x 4 mm i.d.), a precolumn (4 x 4 mm i.d.) containing the same material.
  • the column was thermostatted in a Kontron 480 oven at 40 °C. Detection was performed with a Kontron 432 UV- detector at 294 nm using Range 0.1 and response time 0.5.
  • a Kontron 450 MT1 data system was used to control the HPLC units and for data aquisiti- on.
  • the mobile phase consisted of tetrahydrofuran:13 mM phosphoric acid adjusted to pH 2.35 (with NaOH) in a 15:85 v/v mixture. The flow rate was 1.0 ml/min.
  • Bond-Elut C 8 extraction columns 500 mg, 3 ml were conditioned and eluted using a gentle suction on the outlet. Each column was rinsed twice with 1 ml of methanol and twice with 1 ml of water, followed by 200 ⁇ l of 13 mM phosphoric acid (pH 1.85). A 500 ⁇ l sample of plasma, 50 ⁇ l of internal standard (1 ⁇ g/ml) and 500 ⁇ l of 8M urea in 13 mM phosphoric acid were added and the columns were submitted to gentle suction. A volume of 1 ml of 13 mM phosphoric acid was then added and suction applied again.
  • Collecting tubes (10 ml glass tubes with conically shaped bottoms) were placed under the extraction columns and 2 ml of methanol was used for eluation. The eluate was evaporated under nitrogen at 60 °C. The residue was redissolved in 100 ⁇ l of the mobile phase and centrifuged at 2000 x g for 5 min. The supernatant was transferred to 200 ⁇ l vials and capped. A volume of 25 ⁇ l of the sample was injected on the HPLC system.
  • Urine was diluted 1 :1 with the mobile phase used for HPLC and 10 ⁇ l was injected on the HPLC system using the same chromatographic conditions as used with plasma.
  • Aqueous NBQX solutions were used as standards while controls consisted of urine spiked with NBQX.
  • the results of the NBQX urine concentration, excreted dose, urine volume and pH are sum ⁇ marized in table 1.
  • the urine concentration versus the pH is plotted in fig 2. The concentration is below approx. 200 ⁇ g/ml in all fractions including the 0 - 1 hours and the 1 - 3 hours fractions where high concentrations of NBQX could be expected, if not furosemide was administered.
  • Similar concentra ⁇ tions were observed in another study where NBQX was given alone, but at much lower dose rate (1.3 mg/kg/h). In that same study, NBQX related toxicity was not observed after 1 month of dosage.
  • NNC 07-9202 URINE CONCENTRATION AND EXCRETED DOSE, URINE VOLUME AND PH FROM RATS GIVEN AN IN. INFUSION OF FUROSEMIDE AND NNC 07-9202 IN A NEUROPROTECTIVE DOSE (30 + 60 +2 MG/KG/H FOR Vz + Vz +23 H) ⁇
  • Urine collected one hour before dosing i.e. -1 - 0 hours amounted to 4.3 - 4.6 ml.
  • the pharmaceutical preparations or compositions comprising the com ⁇ pounds of the invention may be administered to humans or animals by oral and preferably by intravenous administration.
  • the loop diuretic agent e.g. furosemide can be given either by the oral or intravenous administration while NBQX is given' by intraveneous administration alone.
  • An effective amount of the active compounds may be determined in accord ⁇ ance with the usual factors, such as the nature and severity of the condition and the weight of the mammal requiring treatment.
  • Conventional excipients are such pharmaceutically acceptable organic or inorganic carrier substances which do not deleteriously react with the active compounds.
  • Such carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, glucose and other carbohybra- tes, gelatine, lactose, amylose, magnesium stearate, talc, silicic acid, fatty acid monoglycerides and diglycerides, pentaerythhtol fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.
  • the pharmaceutical preparations can be sterilized and mixed, if desired, with auxiliary agents, such as preservatives, stabilizers, wetting agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring substances and the like, which do not deleteriously react with the active compounds.
  • auxiliary agents such as preservatives, stabilizers, wetting agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring substances and the like, which do not deleteriously react with the active compounds.
  • the dosage of the compounds according to this invention is 0.1 mg-4 g/day of NBQX and 0.1 mg-4 g/day of the diuretic agent.
  • the compounds of the invention may be placed into the form of phamaceutical composi ⁇ tions and unit dosages thereof.
  • a preferred dosage form for intravenous infusion consists of NBQX (3 ⁇ g/ml-3 mg/ml) and furosemide (25 ⁇ g/ml-25 mg/ml) and the carriers Polyvidone Ph.Eur. (5%) and glucose (4%) in an aqueous solution made basic with a small excess of sodium hydroxide and then adjusted to pH 7 - 8 with hydrochloric acid.
  • the content of PVP and glucose can be varied in order to obtain an isotonic solution.
  • a loading dose of furos ⁇ emide alone can be given in the same vehicle as described above or by oral administration using a conventional dosage form.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
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PCT/DK1994/000101 1993-03-30 1994-03-09 Pharmaceutical composition for the prevention of neuronal cell death WO1994022446A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64234/94A AU6423494A (en) 1993-03-30 1994-03-09 Pharmaceutical composition for the prevention of neuronal cell death

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DK0373/93 1993-03-30
DK37393A DK37393D0 (cs) 1993-03-30 1993-03-30

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101879B2 (en) * 2000-11-03 2006-09-05 Massachusetts Institute Of Technology Treatments for neurotoxicity in Alzheimer's Disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EUROPEAN JOURNAL OF PHARMACOLOGY - MOLECULAR PHARMACOLOGY SECTION, Volume 244, 1993, AOENDREW J. PALMER et al., "Cyclothiazide Reverses AMPA Receptor Antagonism of the 2,3-Benzodiazepine, GYKI 53655", page 193 - page 194. *
MOLECULAR PHARMACOLOGY, Volume 41, 1992. JUAN LERMA et al., "Chloride Transport Blockers Prevent N-Methyl-D-Aspartate Receptor-Channel Complex Activation", page 217 - page 222. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101879B2 (en) * 2000-11-03 2006-09-05 Massachusetts Institute Of Technology Treatments for neurotoxicity in Alzheimer's Disease

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DK37393D0 (cs) 1993-03-30

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