European Journal of Clinical Investigation (1992) 22,630-634
Colchicine analogues: Effect on amyloidogenesis in a murine model and, in vitro, on polymorphonuclear leukocytes B. WOLACH*, M. GOTFRIED?, A. JEDEIKINt, M. LISHNERt, A. BROSSIS & M. RAVIDt, Departments of *Pediatrics and ?Medicine, Sackler Faculty of Medicine Tel-Aviv University and Meir Hospital, Kfar-Saba, Israel, and $Laboratory of Medicinal Chemistry NIDDK, NIH Bethesda MD, USA
Received 14 November 1991 and in revised form 21 April 1992; accepted 5 May 1992
Abstract. Colchicine has been used in diverse clinical settings such as gout, familial Mediterranean fever, liver cirrhosis, Behcet's disease and pericarditis. It also has an antimitotic potential hitherto unexplored due to its narrow therapeutic toxic ratio. The aim of the present study was to compare the effectiveness and the toxicity of colchicine and three analogues: thiocolchicine, 2,3 dimethyl-colchicine and 3-dimethylthiocolchicine in the blockage of amyloid synthesis in a murine model. 3-demethylthiocolchicine was equipotent to colchicine in the blockage of casein induced amyloidogenesis. However, it was markedly less toxic (LD50 11.3 mg kg-' vs. 1-6mg kg-I). Thiocolchicine was toxic (LD50 1.0 mg kg-I) and 2,3 didemethyl-colchicine was far less effective. The effect of 3-dimethylthiocolchicineon polymorphonuclear leukocytes was then compared to colchicine. The effect of this analogue on inhibition of chemotaxis was equivalent to that of colchicine whereas the latter was superior to the analogue in the suppression of phagocytosis (by a ratio of 2: 1) and in the inhibition of bactericidal activity (by a ratio of 10: I). Since in therapeutic concentrations the only detectable effect of colchicine on PMNs is inhibition of chemotaxis, our data may point to 3-demethylthiocolchicine as an optional, perhaps superior alternative to colchicine for some of its therapeutic indications. Keywords. Amyloidosis, colchicine, 3-demethylthiocolchicine. Introduction Colchicine is one of the oldest alkaloides in medical use. Extracts of colchicum autumnale were used in acute gouty arthritis over 200 years ago. At present, colchicine is employed in diverse clinical settings such as gout, familial Mediterranean fever [ 11, and prevention of systemic AA amyloidosis [2]. There is also preliminary evidence of its therapeutic potential in Correspondence: M. Ravid MD, Department of Medicine, Meir Hospital, Kfar-Saba 44281, Israel.
630
slowing liver fibrosis [3,4], in Behcet's disease [5] and in recurrent idiopathic pericarditis [6]. The antimitotic potential of this preparation, suggested by some interesting animal experiments [7] could not be explored in man due to its very narrow therapeutic toxic ratio. Natural and synthetic colchicine analogues were therefore tested with the hope to identify preparations with reduced toxicity and to explore their therapeutic potential. The present report describes the comparative evaluation of three synthetic analogues, thiocolchicine, 2,3 didemethyl-colchicine, 3-demethylthiocolchicine and colchicine in a known biological model of colchicine effect on the blockage of amyloid synthesis in mice [8]. 3-demethylthiocolchicine which was found to be equipotent but considerably less toxic than colchicine was compared to colchicine also by its inhibitory effect on random migration, chemotaxis, phagocytosis and bactericidal activity of polymorphonuclear leukocytes. Materials and methods Colchicine was purchased from Eli Lilly and Co. (Indianapolis, USA). The methods of preparation of the analogues were previously described [7]. Acute toxicity experiments, by a single intraperitoneal injection to mice showed the LD50 after 7 days to be 1.6 mg kg-' for colchicine, 1 1-3mg kg-' for 3-methylthiocolchicine, 20 mg kg- for 2,3 didemethyl-colchicine and 1.0 mg kg-I for thiocolchicine [9]. There was no correlation between the degree of tubuline binding of these analogues and their antiinflammatory or antimitotic activity as tested in the carrageenin induced footpad oedema model in rats [ 101 and in P338 lymphocytic leukemia model in mice [ 1 13. The tubuline binding of 3-demethylthiocolchicine and its anti-inflammatory effect were equivalent to those of colchicine [7]. The compounds were obtained as lyophylized powder. For the animal experiments they were dissolved in normal saline to a concentration of 0.1 mg ml-I.
'
The animals White Swiss mice, 10 weeks old and weighing approximately 30 g each were obtained from the Veterinary
COLCHICINE ANALOGUES AND AMY LOIDOGENESIS
63 1
Table 1. Blockage of murine amyloid synthesis by colchicine and by three analogues: 3-demethylthiocolchicine, thiocolchicine and 2,3 didemethyl-colchicine
Daily dose mg animal-
Experiment No. Agent ~
I
No. of animals Amyloidosis
~
I 2 3
4 5 6 1
8 9 10
I1 12 13 14 15
0.2 ml NaCl Colchicine 0.02 Colchicine 0.04 3-dimethylthiocolchicine 0-02 3-demethylthiocolchicine 0.04 3-demethylthiocolchicine 0.06 3-demethylthiocolchicine 0.08 3-demethylthiocolchicine 0.I 3-demethylthiocolchicine 0.2 thiocolchicine 0.01 thiocolchicine 0.02 2,3-didemethylcolchicine 0.02 2,3-didemethylcolchicine 0.04 2,3-didemethylcolchicine 0.06 2,3-didemethylcolchicine 0.1
Institute (Beth Dagan, Israel). They were fed a standard Purina diet ad libitum. Induction of amyloidosis
The amyloidogenic stimulus consisted of 14 daily subcutaneous injections of 0.5 ml 13% vitamin free casein (N.B.C., Cleveland, Ohio, USA) in 0.05N NaOH [8]. Colchicine and the analogue compounds were administered intraperitoneally, parallel to the casein injections. The experiments were conducted on 15 groups of animals. The first was a control group which received NaCl to test the amyloidogenic effect of the casein batch. The second and third groups received colchicine in daily doses of 0.02 and 0.04 mg animal-' respectively. Groups 4-9 received 3-demethylthiocolchicine in rising doses from 0.02 to 0.2 mg animal-'. Groups 10 and I 1 received thiocolchicine 0.01 and 0.02 mg animal-'. Groups 12-1 5 received 2,3 didemethylcolchicine in doses of 0.02, 0.04, 0.06 and 0.1 mg animal-' (Table I). On the 15th day the animals were sacrificed and amyloid deposits were sought in their spleens by polarization microscopy of Congo red stained slides. Isolation of polymorphonuclear leukocytes
Human purified PMNs (98% PMNs, with less than 1 platelet per 1000 cells) were isolated from heparinized venous blood. The blood samples were obtained from healthy volunteers who had given consent in accord with the Helsinki declaration. After sedimentation in 6% dextran, the leukocyte-enriched plasma was layered on a ficoll-hypaque gradient, centrifuged at 400 g for 30 min at 4°C as previously described [12]. The supernatant was discarded and the pellet was subjected to hypotonic lysis for 20 s to free the PMNs
40 40 40 40 20 20
20 20
39/39
0138 011 0139 0119
1/18
20 20 20
0119 016 010 417 010
40
18/38
20 20 20
6/11 5/19 4/19
of contaminating red cells. The PMNs were resuspended in PCM-G-A (phosphate-calcium-magnesium buffer-glucose-albumin) for phagocytosis, in PBS-GA (phosphate-buffered saline-glucose-albumin) for bactericidal activity and in MI99 medium (Earle's salt with L-glutamine; Biological Industries, Kibbutz Beth Haemek, Israel) for chemotaxis. For the assays the cells were incubated with the drugs at different concentrations, (or the suspension solutions for control), for 1 h at 37°C in a shaking water bath. The cell pellets were washed twice with the suspension solutions. Phagocytosis
Phagocytosis of 14C-labelled Staphylococcus aureus by PMNs was studied as previously described [13]. Incubation mixtures containing 10 x lo6 cells ml-I, 0.5% human albumin AB serum and buffer solution (PCM) with 5-5 mp glucose, were preincubated at 37°C for 10 min. Phagocytosis was initiated by the addition of 14C-labelled staphylococcus aureus (1 0 x I O7 bacteria ml-'--Staph. aureus 502A-ATCC, Rockville, MD, USA). This resulted in a PMN: bacteria ratio of 1 : 10. The uptake of bacteria was stopped by the addition of 1 ml ice-cold PBS containing 20 nM NaF and 10% human plasma solution, at zero and 60 min time-interval. Thereafter, 3 U ml- lysostaphin was added and the samples were incubated for 15 min at 37°C to lyse the non-phagocytosed bacteria. After centrifugation the supernatants were discarded and the cell pellets were washed twice with PBS containing NaF. Cell pellets were dissolved in Soluene-100 (Packard), 10 ml of scintillation fluid and counted in a liquid scintillation counter. All incubations were performed in duplicate. The percent of uptake at 1 : 10 cell to bacteria ratio was calculated.
'
632
B. WOLACH et al.
Bactericidal activity
The quantitation of maximal bactericidal capability was conducted in triplicate. A mixture of 1 x lo7ml-' of PMN's, 5 x lo7 ml-' bacteria (Staph. aureus 502AATCC, Rockville, MD, USA) and AB serum, were incubated at 37°C at zero and 60 min. This resulted in a PMN/bacteria ratio of 1 :5. Bacteria were grown fresh before each experiment and allowed to enter an early stationary phase (I8 h at 37°C). The final concentration of bacteria was calculated by spectrophotometry ~41. After the incubation period, cells were lysed with distilled water, diluted, and plated in triplicates in broth agar plates for 24 h at 37°C. After counting the colonies the log decrease was calculated by the determination of the 100% value (incubation of bacteria without cells) and the number of colonies after 60 min of incubation.
Results Blockage of amyloidogenesis As expected, all 39 surviving mice of the saline treated control group showed splenic amyloidosis. The blockage of amyloid synthesis by colchicine, 0.02 mg animal-', was complete. None of the surviving 38 animals showed any visible amyloid. Doubling the colchicine dose resulted in massive mortality and only seven animals survived, they were all amyloid free. 3-demethylthiocolchicine effectively blocked amyloidogenesis. Animal mortality was marginal in the first three dose schedules (0.02, 0.04 and 0.06 mg animal-'), further increase of the dose was associated with considerable mortality. Thiocolchicine was very toxic. Only seven animals survived from the low dose group, four of them with splenic amyloidosis. 0.02 mg Table 2. Bactericidal and phagocytic activity of PMNs treated with colchicine and 3-demethylthiocolchicine
Chemotaxis assay
A 48 well chemotaxic microchamber (Neuro Probe, Inc., Bethesda, MD, USA) was used to determine random migration and chemotaxis [15]. The chemoatractant FMLP (N-formylmethionyl-leucyl-phenylalanine) or the suspension medium were added to the bottom wells at a concentration of lop7M. A polycarbonate filter sheet, without PVP coating, containing 3 um holes (Nucleopore Corp., Pleasanton, CA, USA) was placed on top of the wells in the bottom plate. The gasket and top plate were fixed in place and lo6 PMNs ml-' were added to the upper wells. The assembly was incubated for 60 min at 37°C in humidified air. After incubation, the filter was wiped off and stained with May-Grunwald-Giemsa. The number of cells in five fields was counted under light microscopy with a 40 x objective, and an optical grid at 10 x magnification. The chemotactic index was calculated by subtracting the random migration from the chemotactic activity. Experiments were performed in quadruplicate. Statistical analysis was performed by the Student t-test.
*Bactericidal activity (Mean f SD) Control group Colchicine (IO-2M) (5 x 1 0 - 3 ~ )
1.31 f 0 . 3 ~ 3 ) 0.02 +0.0I $(12) 0.52 f 0.3
(IO-IM)
1.37f0.37 $(7) 0.2 f 0.2 H9) 0.73 f0.32
3-demethylthiocolchicine (10-*M) (5 x IO-IM) (10-3~)
1.14f 0.3 $(7)
*Log decrease of colonies/hr ?Percent of phagocytosis at I : 10 PMN to bacteria ratio $ Number of samples tested
Table 3. Chemotacticand random migration of PMNs treated with colchicineand 3demethylthiocolchicine Chemotactic index (Mean f SD)
Chemotaxis Random migration (Mean f SD) (Mean f SD) ~
64.7f3.7 *(W 28.7 f 2.9 *(9) 38.8 f 2.4 30.3 f 3.3 *(9) 40.5 f 2.8 *(8)
Control group Colchicine (10-4~) (10-6M)
104.3f 7.9 *(W 65.2 f 6.5 *(9) 75.8 f 7.9 *(7) 3-demethylthiocolchicine 62.9f 6.3 (10-4~) *(9) (10-6M) 75. I f 6.2
Number of samples tested
TPhagocytic activity (Meanf SD)
*(a)
39.7 5.4 '(12) 36.4 f 5. I *(9) 37.0 f 6.7 *(7) 32.5 f 3.4 *(9) 34.6 f 4.8 *(8)
~~
COLCHICINE ANALOGUES AND AMYLOIDOGENESIS animal-’ was lethal to all the animals. 2,3 didemethylcolchicine was more toxic in this model, and less effective than either colchicine or 3-demethylthiocolchicine. These results are summarized in Table 1. Leukocyte experiments
A significant depression of bactericidal and phagocytic activity of PMNs was demonstrated by both drugs (P< 0.001 as compared to control). However colchicine was evidently more potent than its analogue. The difference at lo-’ M concentration was 10-fold for suppression of bactericidal activity and 2-fold for suppression of phagocytosis. These data are shown in Table 2. Chemotaxis was significantly and equally suppressed by both colchicine and 3-demethylthiocolchicine (P
Colchicine analogues: Effect on amyloidogenesis in a murine model and, in vitro, on polymorphonuclear leukocytes B. WOLACH*, M. GOTFRIED?, A. JEDEIKINt, M. LISHNERt, A. BROSSIS & M. RAVIDt, Departments of *Pediatrics and ?Medicine, Sackler Faculty of Medicine Tel-Aviv University and Meir Hospital, Kfar-Saba, Israel, and $Laboratory of Medicinal Chemistry NIDDK, NIH Bethesda MD, USA
Received 14 November 1991 and in revised form 21 April 1992; accepted 5 May 1992
Abstract. Colchicine has been used in diverse clinical settings such as gout, familial Mediterranean fever, liver cirrhosis, Behcet's disease and pericarditis. It also has an antimitotic potential hitherto unexplored due to its narrow therapeutic toxic ratio. The aim of the present study was to compare the effectiveness and the toxicity of colchicine and three analogues: thiocolchicine, 2,3 dimethyl-colchicine and 3-dimethylthiocolchicine in the blockage of amyloid synthesis in a murine model. 3-demethylthiocolchicine was equipotent to colchicine in the blockage of casein induced amyloidogenesis. However, it was markedly less toxic (LD50 11.3 mg kg-' vs. 1-6mg kg-I). Thiocolchicine was toxic (LD50 1.0 mg kg-I) and 2,3 didemethyl-colchicine was far less effective. The effect of 3-dimethylthiocolchicineon polymorphonuclear leukocytes was then compared to colchicine. The effect of this analogue on inhibition of chemotaxis was equivalent to that of colchicine whereas the latter was superior to the analogue in the suppression of phagocytosis (by a ratio of 2: 1) and in the inhibition of bactericidal activity (by a ratio of 10: I). Since in therapeutic concentrations the only detectable effect of colchicine on PMNs is inhibition of chemotaxis, our data may point to 3-demethylthiocolchicine as an optional, perhaps superior alternative to colchicine for some of its therapeutic indications. Keywords. Amyloidosis, colchicine, 3-demethylthiocolchicine. Introduction Colchicine is one of the oldest alkaloides in medical use. Extracts of colchicum autumnale were used in acute gouty arthritis over 200 years ago. At present, colchicine is employed in diverse clinical settings such as gout, familial Mediterranean fever [ 11, and prevention of systemic AA amyloidosis [2]. There is also preliminary evidence of its therapeutic potential in Correspondence: M. Ravid MD, Department of Medicine, Meir Hospital, Kfar-Saba 44281, Israel.
630
slowing liver fibrosis [3,4], in Behcet's disease [5] and in recurrent idiopathic pericarditis [6]. The antimitotic potential of this preparation, suggested by some interesting animal experiments [7] could not be explored in man due to its very narrow therapeutic toxic ratio. Natural and synthetic colchicine analogues were therefore tested with the hope to identify preparations with reduced toxicity and to explore their therapeutic potential. The present report describes the comparative evaluation of three synthetic analogues, thiocolchicine, 2,3 didemethyl-colchicine, 3-demethylthiocolchicine and colchicine in a known biological model of colchicine effect on the blockage of amyloid synthesis in mice [8]. 3-demethylthiocolchicine which was found to be equipotent but considerably less toxic than colchicine was compared to colchicine also by its inhibitory effect on random migration, chemotaxis, phagocytosis and bactericidal activity of polymorphonuclear leukocytes. Materials and methods Colchicine was purchased from Eli Lilly and Co. (Indianapolis, USA). The methods of preparation of the analogues were previously described [7]. Acute toxicity experiments, by a single intraperitoneal injection to mice showed the LD50 after 7 days to be 1.6 mg kg-' for colchicine, 1 1-3mg kg-' for 3-methylthiocolchicine, 20 mg kg- for 2,3 didemethyl-colchicine and 1.0 mg kg-I for thiocolchicine [9]. There was no correlation between the degree of tubuline binding of these analogues and their antiinflammatory or antimitotic activity as tested in the carrageenin induced footpad oedema model in rats [ 101 and in P338 lymphocytic leukemia model in mice [ 1 13. The tubuline binding of 3-demethylthiocolchicine and its anti-inflammatory effect were equivalent to those of colchicine [7]. The compounds were obtained as lyophylized powder. For the animal experiments they were dissolved in normal saline to a concentration of 0.1 mg ml-I.
'
The animals White Swiss mice, 10 weeks old and weighing approximately 30 g each were obtained from the Veterinary
COLCHICINE ANALOGUES AND AMY LOIDOGENESIS
63 1
Table 1. Blockage of murine amyloid synthesis by colchicine and by three analogues: 3-demethylthiocolchicine, thiocolchicine and 2,3 didemethyl-colchicine
Daily dose mg animal-
Experiment No. Agent ~
I
No. of animals Amyloidosis
~
I 2 3
4 5 6 1
8 9 10
I1 12 13 14 15
0.2 ml NaCl Colchicine 0.02 Colchicine 0.04 3-dimethylthiocolchicine 0-02 3-demethylthiocolchicine 0.04 3-demethylthiocolchicine 0.06 3-demethylthiocolchicine 0.08 3-demethylthiocolchicine 0.I 3-demethylthiocolchicine 0.2 thiocolchicine 0.01 thiocolchicine 0.02 2,3-didemethylcolchicine 0.02 2,3-didemethylcolchicine 0.04 2,3-didemethylcolchicine 0.06 2,3-didemethylcolchicine 0.1
Institute (Beth Dagan, Israel). They were fed a standard Purina diet ad libitum. Induction of amyloidosis
The amyloidogenic stimulus consisted of 14 daily subcutaneous injections of 0.5 ml 13% vitamin free casein (N.B.C., Cleveland, Ohio, USA) in 0.05N NaOH [8]. Colchicine and the analogue compounds were administered intraperitoneally, parallel to the casein injections. The experiments were conducted on 15 groups of animals. The first was a control group which received NaCl to test the amyloidogenic effect of the casein batch. The second and third groups received colchicine in daily doses of 0.02 and 0.04 mg animal-' respectively. Groups 4-9 received 3-demethylthiocolchicine in rising doses from 0.02 to 0.2 mg animal-'. Groups 10 and I 1 received thiocolchicine 0.01 and 0.02 mg animal-'. Groups 12-1 5 received 2,3 didemethylcolchicine in doses of 0.02, 0.04, 0.06 and 0.1 mg animal-' (Table I). On the 15th day the animals were sacrificed and amyloid deposits were sought in their spleens by polarization microscopy of Congo red stained slides. Isolation of polymorphonuclear leukocytes
Human purified PMNs (98% PMNs, with less than 1 platelet per 1000 cells) were isolated from heparinized venous blood. The blood samples were obtained from healthy volunteers who had given consent in accord with the Helsinki declaration. After sedimentation in 6% dextran, the leukocyte-enriched plasma was layered on a ficoll-hypaque gradient, centrifuged at 400 g for 30 min at 4°C as previously described [12]. The supernatant was discarded and the pellet was subjected to hypotonic lysis for 20 s to free the PMNs
40 40 40 40 20 20
20 20
39/39
0138 011 0139 0119
1/18
20 20 20
0119 016 010 417 010
40
18/38
20 20 20
6/11 5/19 4/19
of contaminating red cells. The PMNs were resuspended in PCM-G-A (phosphate-calcium-magnesium buffer-glucose-albumin) for phagocytosis, in PBS-GA (phosphate-buffered saline-glucose-albumin) for bactericidal activity and in MI99 medium (Earle's salt with L-glutamine; Biological Industries, Kibbutz Beth Haemek, Israel) for chemotaxis. For the assays the cells were incubated with the drugs at different concentrations, (or the suspension solutions for control), for 1 h at 37°C in a shaking water bath. The cell pellets were washed twice with the suspension solutions. Phagocytosis
Phagocytosis of 14C-labelled Staphylococcus aureus by PMNs was studied as previously described [13]. Incubation mixtures containing 10 x lo6 cells ml-I, 0.5% human albumin AB serum and buffer solution (PCM) with 5-5 mp glucose, were preincubated at 37°C for 10 min. Phagocytosis was initiated by the addition of 14C-labelled staphylococcus aureus (1 0 x I O7 bacteria ml-'--Staph. aureus 502A-ATCC, Rockville, MD, USA). This resulted in a PMN: bacteria ratio of 1 : 10. The uptake of bacteria was stopped by the addition of 1 ml ice-cold PBS containing 20 nM NaF and 10% human plasma solution, at zero and 60 min time-interval. Thereafter, 3 U ml- lysostaphin was added and the samples were incubated for 15 min at 37°C to lyse the non-phagocytosed bacteria. After centrifugation the supernatants were discarded and the cell pellets were washed twice with PBS containing NaF. Cell pellets were dissolved in Soluene-100 (Packard), 10 ml of scintillation fluid and counted in a liquid scintillation counter. All incubations were performed in duplicate. The percent of uptake at 1 : 10 cell to bacteria ratio was calculated.
'
632
B. WOLACH et al.
Bactericidal activity
The quantitation of maximal bactericidal capability was conducted in triplicate. A mixture of 1 x lo7ml-' of PMN's, 5 x lo7 ml-' bacteria (Staph. aureus 502AATCC, Rockville, MD, USA) and AB serum, were incubated at 37°C at zero and 60 min. This resulted in a PMN/bacteria ratio of 1 :5. Bacteria were grown fresh before each experiment and allowed to enter an early stationary phase (I8 h at 37°C). The final concentration of bacteria was calculated by spectrophotometry ~41. After the incubation period, cells were lysed with distilled water, diluted, and plated in triplicates in broth agar plates for 24 h at 37°C. After counting the colonies the log decrease was calculated by the determination of the 100% value (incubation of bacteria without cells) and the number of colonies after 60 min of incubation.
Results Blockage of amyloidogenesis As expected, all 39 surviving mice of the saline treated control group showed splenic amyloidosis. The blockage of amyloid synthesis by colchicine, 0.02 mg animal-', was complete. None of the surviving 38 animals showed any visible amyloid. Doubling the colchicine dose resulted in massive mortality and only seven animals survived, they were all amyloid free. 3-demethylthiocolchicine effectively blocked amyloidogenesis. Animal mortality was marginal in the first three dose schedules (0.02, 0.04 and 0.06 mg animal-'), further increase of the dose was associated with considerable mortality. Thiocolchicine was very toxic. Only seven animals survived from the low dose group, four of them with splenic amyloidosis. 0.02 mg Table 2. Bactericidal and phagocytic activity of PMNs treated with colchicine and 3-demethylthiocolchicine
Chemotaxis assay
A 48 well chemotaxic microchamber (Neuro Probe, Inc., Bethesda, MD, USA) was used to determine random migration and chemotaxis [15]. The chemoatractant FMLP (N-formylmethionyl-leucyl-phenylalanine) or the suspension medium were added to the bottom wells at a concentration of lop7M. A polycarbonate filter sheet, without PVP coating, containing 3 um holes (Nucleopore Corp., Pleasanton, CA, USA) was placed on top of the wells in the bottom plate. The gasket and top plate were fixed in place and lo6 PMNs ml-' were added to the upper wells. The assembly was incubated for 60 min at 37°C in humidified air. After incubation, the filter was wiped off and stained with May-Grunwald-Giemsa. The number of cells in five fields was counted under light microscopy with a 40 x objective, and an optical grid at 10 x magnification. The chemotactic index was calculated by subtracting the random migration from the chemotactic activity. Experiments were performed in quadruplicate. Statistical analysis was performed by the Student t-test.
*Bactericidal activity (Mean f SD) Control group Colchicine (IO-2M) (5 x 1 0 - 3 ~ )
1.31 f 0 . 3 ~ 3 ) 0.02 +0.0I $(12) 0.52 f 0.3
(IO-IM)
1.37f0.37 $(7) 0.2 f 0.2 H9) 0.73 f0.32
3-demethylthiocolchicine (10-*M) (5 x IO-IM) (10-3~)
1.14f 0.3 $(7)
*Log decrease of colonies/hr ?Percent of phagocytosis at I : 10 PMN to bacteria ratio $ Number of samples tested
Table 3. Chemotacticand random migration of PMNs treated with colchicineand 3demethylthiocolchicine Chemotactic index (Mean f SD)
Chemotaxis Random migration (Mean f SD) (Mean f SD) ~
64.7f3.7 *(W 28.7 f 2.9 *(9) 38.8 f 2.4 30.3 f 3.3 *(9) 40.5 f 2.8 *(8)
Control group Colchicine (10-4~) (10-6M)
104.3f 7.9 *(W 65.2 f 6.5 *(9) 75.8 f 7.9 *(7) 3-demethylthiocolchicine 62.9f 6.3 (10-4~) *(9) (10-6M) 75. I f 6.2
Number of samples tested
TPhagocytic activity (Meanf SD)
*(a)
39.7 5.4 '(12) 36.4 f 5. I *(9) 37.0 f 6.7 *(7) 32.5 f 3.4 *(9) 34.6 f 4.8 *(8)
~~
COLCHICINE ANALOGUES AND AMYLOIDOGENESIS animal-’ was lethal to all the animals. 2,3 didemethylcolchicine was more toxic in this model, and less effective than either colchicine or 3-demethylthiocolchicine. These results are summarized in Table 1. Leukocyte experiments
A significant depression of bactericidal and phagocytic activity of PMNs was demonstrated by both drugs (P< 0.001 as compared to control). However colchicine was evidently more potent than its analogue. The difference at lo-’ M concentration was 10-fold for suppression of bactericidal activity and 2-fold for suppression of phagocytosis. These data are shown in Table 2. Chemotaxis was significantly and equally suppressed by both colchicine and 3-demethylthiocolchicine (P
