Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-05-23
2002-01-15
Jarvis, William R. A. (Department: 1714)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
Reexamination Certificate
active
06339095
ABSTRACT:
The present invention relates to the use of 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline in the prevention and/or treatment of motor neuron diseases.
1,6-Dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxy-ergoline or 1-methyl-10&agr;-methoxy-9,10-dihydrolysergol is one of the metabolites of nicergoline (F. ARCAMONE et al., Biochem. Pharmacol., 21 (16), 2205-2013 (1972)). Like nicergoline, but to a lesser degree, this compound exhibits &agr;1-adrenergic and 5HT1a-serotonergic properties. It is also useful as an intermediate for preparing nicergoline (French Patent No. 2,616,788).
It has now been found that 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline increases the survival of motor neurons and can therefore be used in preventing and/or treating motor neuron diseases.
Motor neuron diseases include, in particular, amyotrophic lateral sclerosis, progressive spinal muscular atrophy, infantile muscular atrophy and primary lateral sclerosis.
In the presence of trophic support supplied by the neurotrophic factors BDNF or GDNF, motor neuron cultures are composed of broad and homogeneous neurons with long branched axons. However, the motor neurons die by apoptosis if the culture is performed in the absence of trophic support.
The effect of 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline has therefore been determined in a degeneration model which is induced by depriving cultured motor neurons of neurotrophic factor.
Furthermore, astrocytes play a major role in controlling and maintaining an environment which is suitable for motor neuron survival.
1,6-Dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline has therefore also been tested on a co-culture of motor neurons and astrocytes.
The following protocols were employed:
MOTOR-NEURON-ENRICHED CULTURES:
The motor-neuron-enriched cultures are prepared using the centrifugation method which was described by R. L. SCHNAAR and A. E. SCHAFFNER, J. Neurosci., 1, 204-217 (1981) and modified by W. CAMU and C. E. HENDERSON, J. Neurosci. Methods, 44, 59-70 (1992). The motor neurons are spread, at a density of 2500 cells per plate, on 35 mm culture plates which have previously been coated with laminin/ornithine in accordance with the method of A. G. ESTEVEZ et al., J. Neurosci., 18 (3), 923-931 (1998). The cultures are then maintained in L15 medium (GIBCO BRL) containing sodium bicarbonate (22 mM), conalbumin (0.1 mg/ml), putrescine (0.1 mM), insulin (5 &mgr;g/ml), sodium selenite (31 mM), glucose (20 mM), progesterone (21 nM), penicillin (100 IU/ml) and streptomycin (100 &mgr;/ml).
The motor neurons which are thus obtained consist of large (25-30 &mgr;m) and homogeneous neurons which possess long branched axons. More than 70% of the cells are immunoreactive for the neurotrophin p75 receptor and the Islet ½ markers for spinal motor neurons. Approximately 70% of the motor neurons die by apoptosis 24 hours after the spreading if the culture is performed in the absence of a trophic factor.
CULTURES OF SPINAL CORD ASTROCYTES:
The astrocytes are obtained from young, one-day-old rats using the slightly modified method of R. P. SANETO and J. DE VELLIS as described in Neurochemistry a practical approach (A. J. TURNER and H. A. St JOHN) IRL Press, Oxford-Washington DC, pp. 27-63. The spinal cords are dissected out under sterile conditions and freed of meninges and dorsal ganglia. Five to ten spinal cords are transferred into PBS (phosphate buffered saline) and cut before being incubated at 37° C. for 25 minutes in PBS to which 0.25% trypsin has been added. The enzymatic treatment is stopped by adding 10 ml of Dulbeccols modified Eagle medium (DMEM), to which 10% fetal calf serum (FCS) has been added, and the cells are collected by centrifugation. Another step of mechanical dissociation is performed using the end of a 1 ml pipette. The cells are spread, at a density of 1.5-2×10
6
cells per 25 cm
2
of culture medium, in DMEM containing 10% FCS. After 2 days in vitro, the cultures are fed every day. When a visible monolayer of cells has been completed, the cultures are stirred at 250 rpm for 48 hours and, on the following day, the monolayers are treated with cytosine arabinoside (10
−5
M) for 48 hours. The astrocyte monolayers are then amplified to a density of five on 35 mm culture plates for initially 25 cm
2
culture flasks.
The spinal astrocyte cultures consist, to an extent of more than 98%, of flat, polygonal cells which are immunoreactive for the glial fibrillar acid protein (GFAP). The monolayers are exposed to the product to be tested and then incubated with the motor neuron medium in order to obtain a conditioned culture medium. This medium is transferred and tested at different dilutions in order to determine its effects on neuronal survival.
IMMUNOCHEMISTRY
The cells are fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (pH 7.4 and at 4° C. for 15 minutes) and in a cold methanolic solution. The cultures are then washed and the nonspecific sites are blocked with 10% goat serum and 2% bovine serum albumin (BSA) in PBS and treated for immunochemistry using antibodies against the p75 weak affinity neurotrophin receptor or a 200 kD neurofilament protein (Amersham) using the manufacturer's instructions and applying the avidin-biotin-DAB/hydrogen peroxide enhancement reaction.
TREATING THE ASTROCYTES WITH 1,6-DIMETHYL-8&bgr;-HYDROXYMETHYL-10&agr;-METHOXYERGOLINE
The astrocytes are treated with 1,6-dimethyl-80&bgr;-hydroxymethyl-10&agr;-methoxyergoline in the following manner: the product is dissolved in methanol, sterilized by filtration and used immediately after preparation. The treatment which is applied to the enriched motor neuron cultures is effected by adding aliquots of solutions of the products to be tested to the L15 medium by spreading. The astrocyte monolayers are exposed to the vehicle or to the solutions of the compound to be tested for 24 hours and at different concentrations. The astrocyte monolayers are washed 3 times with DMEM and incubated with complete L15 medium. he astrocyte-conditioned medium is recovered 24 hours later and centrifuged at 1800 g for 15 minutes and used immediately or stored at−70° C. for a maximum of 2 weeks without loss of trophic activity.
COUNTING THE CELLS AND STATISTICAL ANALYSIS
The cells which are immunoreactive for neurofilaments and which exhibit axons which are longer than the diameters of the cells are regarded as being viable motor neurons. The number of motor neurons is estimated by counting the labelled cells in an area of 0.4-1 cm
2
under a microscope which enlarges 200 times. In all cases, the values are expressed as a percentage of the number of motor neurons which are present in cultures maintained using trophic factors. The experiments are carried out at least 3 times.
The statistical analyses are performed using the Student's test (t test).
The following results were obtained:
1—Effect of 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline on neuronal survival in astrocyte/motor neuron co-cultures:
% of labelled motor neurons
All the motor
Very large motor
neurons
neurons
Vehicle alone
100 ± 9.4
100 ± 2.8
1,6-Dimethyl-8&bgr;-
hydroxymethyl-10&agr;-
methoxyergoline
0.1 &mgr;M
123 ± 9.3**
122.6 ± 8.2*
1 &mgr;M
158.9 ± 32*
117 ± 5.6*
10 &mgr;M
137.5 ± 19***
ND
*significantly different from the vehicle (p < 0.05)
**significantly different from the vehicle (p < 0.01)
ND — not determined
These results demonstrate that 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline (0.1-1 &mgr;M) prevents the neuronal death of the co-cultures and increases the number of large motor neurons.
2—Effect of 1,6-dimethyl-8&bgr;-hydroxymethyl-10&agr;-methoxyergoline on the neurotrophic activity of the motor neurons which is produced by the spinal astrocytes:
Motor neuron survival in %
Dilutions of the astrocytes in the
conditioned medium
1:50
1:10
1:4
Vehicle alone
36.9 ± 3.2
45.4 ± 4.2
59.9 ± 8&
Aventis Pharma S.A.
Gupta Balaram
Jarvis William R. A.
Kim Vickie
LandOfFree
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