Br. J. clin. Pharmac. (1977), 4, 357-366
THE ABSORPTION AND ELIMINATION OF ICI 74,917 IN MAN M.E. PICKUP1, A.J. JOHNSON, C.S. MAY,2 J.W. PATERSON3 & M.P. HARRISON4 Asthma Research Council Clinical Pharmacology Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, London SW3 6HP
I The pharmacokinetics of ICI 74,917 were studied in both asthmatic patients and normal volunteers. 2 The tritiated compound was administered to the lungs by inhalation from an aerosol and a bronchoscope, and by intravenous, oral and buccal routes. Radioactivity was measured in plasma, urine, faeces, sputum and exhaled air. 3 After bronchoscopic administration 63% of the available dose was absorbed; after aerosol administration 8% was absorbed from the lung and more than 50% swallowed. 4 Intravenous studies indicated that the drug is excreted in the bile and urine in the ratio 2:1. 5 Minimal oral and no buccal absorption occurred. 6 There was no evidence of tritium exchange or drug metabolism. 7 The mean terminal half-life following administration by all routes was 16.1 hours. However, the majority of the dose was rapidly excreted. 8 Aerosol administration is the method of choice for the clinical use of ICI 74,917.
Introduction
The introduction of disodium cromoglycate (DSCG) has been a significant advance in the treatment of bronchial asthma (Brompton Hospital/MRC collaborative trial). Subsequently other compounds have been shown to possess similar inhibitory effects on immediate hypersensitivity responses. Of these, 6 n-butyl- 2,8-dicarboxy-4, 10-dioxo- 1,4,7,10-tetrahydro- 1,7-phenanthroline (ICI 74,917) as the disodium salt (Figure 1) has been shown to be 300 times more potent than DSCG in inhibiting dermal blueing reactions provoked by Compound 48/80 in rats (Evans & Thomson, 1975). This novel compound also inhibits allergic bronchospasm in the guinea-pig in a dose dependent manner. ICI 74,917 given intranasally by pressurized aerosol to man prevents allergic rhinitis produced by -
nasal provocation testing (Mygind & Thomsen, 1975); it is undergoing clinical trials in asthma administered by dry pressurized aerosol. The absorption, metabolism and excretion of DSCG has been studied (Walker, Evans, Richards, Paterson, 1972b) and the finding of poor gastro-intestinal absorption but rapid absorption of the drug from the lung confirms that inhalation is the optimal route of administration in bronchial asthma. The present study examines the absorption and excretion of ICI 74,917 in order to confirm the results of animal studies which show that aerosol administration is the route of choice. Methods Materials
Present addresses: Rheumatism Research Clinical Pharmacology Unit, Royal Bath Hospital, Cornwall Road, Harrogate, Yorkshire HG1 2PS; 2 Department of Medicine, Aberdeen University, Foresterhill, Aberdeen AB9 2ZD, Scotland; I Department of Pharmacology, University of Western Australia, The Medical School, Perth Medical Centre, Western Australia 6008; 4Imperial Chemical Industries Limited, Pharmaceutical Division, Alderley Park, Macclesfield, Cheshire SK 10 4TG
ICI 74,917 was tritiated by an exchange process and equilibrated to remove labile isotope. The following formulations were prepared by Imperial Chemical Industries Ltd, (a) an aerosol, identical to that being used in clinical trials, capable of delivering at least 80 metered doses each of 500,g (100 gCi) of the free acid equivalent as a suspension in a surfactantpropellant mixture (b) ampoules containing 5.0,g (13.4 gCi) of the salt in 1.0 ml isotonic saline for
358
M.E. PICKUP, A.J. JOHNSON, C.S. MAY, J.W. PATERSON & M.P. HARRISON
O
COONa
~~NH
H
NaOOC
(CH2)3CH3 0
Figure 1- Structural formula of ICI 74,917, disodium salt.
bronchoscopic studies (c) ampoules containing 2.5 jig (6.7 jiCi) of the salt in 10 ml sterile pyrogen free water for intravenous use (d) ampoules containing 1.0 mg (200 gCi) of the salt for buccal and oral studies. Drug administration All patients and volunteers gave written consent to the administration of the drug, and to the collection of specimens, after the procedures had been fully explained.
(a) Aerosol study. Seven hospital in-patients (Table 1) who were in the convalescent phase of acute asthma participated while continuing their usual drug therapy. Prior to administration of the drug each person inhaled two doses from a salbutamol aerosol (100 jig/dose) to ensure adequate bronchodilatation, and that their technique of inhalation was satisfactory. A short cannula was inserted into a forearm vein and a blood sample was taken in order to determine the baseline level of plasma radioactivity. A sample of urine was collected for the same purpose.
Table 1
One dose of the study aerosol was inhaled by each person and the breath held for approximately 10 seconds. Blood samples were drawn into lithium heparin tubes at approximately 1, 2, 4, 6, 10, 15, 30, 45 min, 1, 2, 3, 4, 6 and 8 h after inhalation. Sufficient blood was obtained to allow separation of duplicate 1 ml aliquots of plasma. Urine was collected for 72 hours. Sputum, if produced, was collected until levels of radioactivity decreased to twice background (3 to 4 days). Two patients, in addition, expired the first 10 breaths after inhalation of the drug into a Douglas bag which was fitted with a one-way valve and contained sodium bicarbonate solution (0.1 M, 500 ml). This solution was recovered following vigorous shaking of the bag. Subsequent rinses (2-3) of the bag and valve were performed until levels of radioactivity recovered decreased to twice background. After collecting expired gas, the mouth was immediately rinsed with sodium bicarbonate solution (30 ml) and the fluid recovered. During administration and subsequent manoeuvres, one of the two patients used a noseclip. All faecal samples from both patients were collected up to 7 days. In these two studies, and on two other occasions, the recovery of drug from the aerosol mouthpiece was estimated.
(b) Bronchoscopic administration. Direct administration to the lung was achieved by use of a bronchoscopic technique. Three patients with suspected bronchogenic carcinoma of the left lung, one of whom also had emphysema, and another quiescent pulmonary tuberculosis, were undergoing fibreoptic bronchoscopy for diagnostic purposes under local anesthetic (premedication: atropine and omnopon; local anesthetic, lignocaine hydrochloride). After diagnostic examination the tip of the bronchoscope was placed in a visually normal segmental bronchus of the right lower lobe. The drug solution (1.0 ml) was instilled via the suction/instru-
Patient data: Aerosol study Patient
Asthma
EB
CP
DB
JC
FC
MO
PC
Intrinsic
Extrinsic
Intrinsic
Intrinsic Bronchiectasis
Extrinsic Aortic
Extrinsic
Extrinsic
Other disease
incompetance
Sex
Age (years) Weight (kg) Height (cm) PEFR (I min-1) FEV1 (I) FVC (I)
F
64 53 158 145 ND ND
F
M
39 95 170 480 ND ND
43 60 160 150 ND ND
* Prior to commencement; N D = Not determined.
M
63 69 170 ND 2.1 3.5
F 70 45 154 ND 1.4 2.0
F
24 55 164 325 2.7 3.0
F
37 64 173 435 2.8 3.6
ICI 74,917 IN MAN
ment channel of the bronchoscope and flushed through with normal saline (10 ml). The bronchoscope was then withdrawn, and blood and urine samples were collected as in the aerosol study. Additional blood samples were taken at approximately 20 and 24hours. All sputum produced was collected until levels of radioactivity decreased to twice background (2 days). The ampoule, syringe and bronchoscope rinsings were estimated for residual drug.
(c) Intravenous study. Four healthy volunteers were each given an intravenous infusion of the drug solution (10 ml) over an 8 min period. After completion of the infusion blood samples were withdrawn via a cannula in a vein of the opposite forearm in a similar manner to, and at approximately the same times as, the aerosol study. Urine samples were collected for 72 h in all subjects. The recovery of drug from syringe, cannula, and ampoule was estimated. In two subjects faeces were collected up to 7 days.
(d) Buccal studies. For each of four healthy volunteers, the drug (1 mg) was dissolved in distilled water (50 ml) and an aliquot (25 ml) swilled round the mouth for 3 min before expulsion (Beckett & Pickup, 1975). Blood samples were collected up to 4 h at times comparable with the aerosol study. Urine samples were collected up to 36 hours. (e) Oral studies. For each of four healthy volunteers, the drug (1 mg) was dissolved in distilled water (50 ml) and an aliquot (25 ml) swallowed. This was washed down with a further quantity of distilled water (10 ml). Blood samples were collected up to 6.5 h and urine up to 48 h after administration. Physiological assessments
359
bronchoscope was rinsed with normal saline, and aliquots were counted. Sputum was homogenized with a known volume of distilled water and an aliquot counted. Faeces were homogenized with distilled water and the pH adjusted to 10-11 using sodium hydroxide solution to release all bound drug (modified from Walker et al., 1 972b). Following centrifugation an aliquot (0.1 ml) of supematant was counted in scintillant (9 ml) after allowing at least 12 h at 4 IC for any chemiluminescence to disappear. Urine samples from subjects following intravenous or aerosol administration were examined for the presence of tritiated water by a freeze-drying technique. Aliquots were placed in glass scintillation vials and freeze dried. The dry residues were reconstituted with distilled water and the radioactivity compared with that of the equivalent whole urine samples to assess
the amount of free tritiated water present. Samples of urine from different subjects were examined by thin-layer chromatography (t.l.c.). Where necessary the radioactivity was concentrated by partial freeze-drying. Duplicate aliquots of each sample (up to 50 gl) were applied to silica gel on glass t.l.c. plates (Anachem Uniplates Silica gel GF 0.25 mm) as 2 cm bands, and in each case one band was overspotted with [3H]-ICI 74,917. The chromatograms were developed with (a) ethanolchloroform-ammonia-water (55:30:15:10 v/v), (b) isopropanol-ammonia-water (70:30: 10) and (c) isopropanol-water (80:60) solvent systems. Quantitation and location of radioactivity was obtained by scraping 0.5 cm segments in sequence from the plates into scintillation vials. Each segment was suspended in triton toluene scintillant (10 ml) containing 3.5 ml of water to form a stable gel and radioactivity was measured. Control urine specimens containing [3H]-ICI 74,917 were compared to the
study specimens. Before and during the aerosol and intravenous studies, periodic assessments of pulse, blood pressure, forced expiratory volume (FEVy), forced vital capacity (FVC) and peak expiratory flow rate (PEFR) were carried out. Pulse and blood pressure were measured during the bronchoscopic study. Quantitative estimation ofPHJ-ICI 74,917 The total 3H activity of each sample was measured in a Tri-Carb Liquid Scintillation Spectrometer (Model 3375 Packard). All counting was performed at 40C, and the counting efficiency was determined from a standard curve by the automatic external standard method. Aliquots (1 ml) of each of the sample fluids were counted in Ne260 Liquid Scintillant (9 ml, Nuclear Enterprises Ltd). Aliquots of plasma, urine and recoveries from mouthrinse and Douglas bag were counted undiluted. Ampoules, syrings, aerosol mouthpieces and one-way valves were rinsed with a measured volume of distilled water, while the
Estimation of half l(fefrom urine data
Linear least squares regression analysis was used to obtain the line of closest fit to the logarithms of the calculated values (Um,x-Ut) at various times t of the terminal exponential phases, where Um,x is the maximum urinary recovery of drug and U, the cumulative recovery at time t (Cummings, Martin & Park, 1967; Lin, Magat, Calesnick & Symchowicz, 1972). The drug half-life for the terminal elimination phase was derived from the gradient of this regression line (Pickup, May, Ssendagire, Paterson, 1976) (Figure 3). Results
Drug adninistration
Aerosol study. The drug appeared rapidly in the plasma of all patients and the mean maximum con-
360
M.E. PICKUP, A.J. JOHNSON, C.S. MAY, J.W. PATERSON & M.P. HARRISON
0
C1)
COCV
-v
0
*
co
. .r..
. L C.
O
C)o r0)
o
0
_ It
CE)
CN
*
0 0
*
r o CO0)' O co0
()
._
2U) Q, N
CL
oN o 0 cl, Z Z -( C')~~~~~~~V
coLE
vtec;
aiz
0._
0U) 20
.
.
LU
a.
L
C 0
NOD IOZ
ON''-NO
9
00
00N
-
0
o
o
C.
Z
.7
E
* *
0
.
Z
Z
L
i oe L)v
Et~ 0.
0.
*i: ~ ~ ~
~
~~L 10
.Ec
~._
Z z ._
0
_ -- E Z
Ln
z
O :l
0.
Z =0
L
ct
0
:.2
0>.
0
0w2 .E
0
C .
Z
ooz
E
-C co
n *X
0~~
V0
C4
..
O.CL-C '
e c~ °
E
u' E
0~~~
_.0 4-.
NOQ CE
40 0
0
0
>~~ co ~~0
.wa)
0E E
THE ABSORPTION AND ELIMINATION OF ICI 74,917 IN MAN M.E. PICKUP1, A.J. JOHNSON, C.S. MAY,2 J.W. PATERSON3 & M.P. HARRISON4 Asthma Research Council Clinical Pharmacology Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, London SW3 6HP
I The pharmacokinetics of ICI 74,917 were studied in both asthmatic patients and normal volunteers. 2 The tritiated compound was administered to the lungs by inhalation from an aerosol and a bronchoscope, and by intravenous, oral and buccal routes. Radioactivity was measured in plasma, urine, faeces, sputum and exhaled air. 3 After bronchoscopic administration 63% of the available dose was absorbed; after aerosol administration 8% was absorbed from the lung and more than 50% swallowed. 4 Intravenous studies indicated that the drug is excreted in the bile and urine in the ratio 2:1. 5 Minimal oral and no buccal absorption occurred. 6 There was no evidence of tritium exchange or drug metabolism. 7 The mean terminal half-life following administration by all routes was 16.1 hours. However, the majority of the dose was rapidly excreted. 8 Aerosol administration is the method of choice for the clinical use of ICI 74,917.
Introduction
The introduction of disodium cromoglycate (DSCG) has been a significant advance in the treatment of bronchial asthma (Brompton Hospital/MRC collaborative trial). Subsequently other compounds have been shown to possess similar inhibitory effects on immediate hypersensitivity responses. Of these, 6 n-butyl- 2,8-dicarboxy-4, 10-dioxo- 1,4,7,10-tetrahydro- 1,7-phenanthroline (ICI 74,917) as the disodium salt (Figure 1) has been shown to be 300 times more potent than DSCG in inhibiting dermal blueing reactions provoked by Compound 48/80 in rats (Evans & Thomson, 1975). This novel compound also inhibits allergic bronchospasm in the guinea-pig in a dose dependent manner. ICI 74,917 given intranasally by pressurized aerosol to man prevents allergic rhinitis produced by -
nasal provocation testing (Mygind & Thomsen, 1975); it is undergoing clinical trials in asthma administered by dry pressurized aerosol. The absorption, metabolism and excretion of DSCG has been studied (Walker, Evans, Richards, Paterson, 1972b) and the finding of poor gastro-intestinal absorption but rapid absorption of the drug from the lung confirms that inhalation is the optimal route of administration in bronchial asthma. The present study examines the absorption and excretion of ICI 74,917 in order to confirm the results of animal studies which show that aerosol administration is the route of choice. Methods Materials
Present addresses: Rheumatism Research Clinical Pharmacology Unit, Royal Bath Hospital, Cornwall Road, Harrogate, Yorkshire HG1 2PS; 2 Department of Medicine, Aberdeen University, Foresterhill, Aberdeen AB9 2ZD, Scotland; I Department of Pharmacology, University of Western Australia, The Medical School, Perth Medical Centre, Western Australia 6008; 4Imperial Chemical Industries Limited, Pharmaceutical Division, Alderley Park, Macclesfield, Cheshire SK 10 4TG
ICI 74,917 was tritiated by an exchange process and equilibrated to remove labile isotope. The following formulations were prepared by Imperial Chemical Industries Ltd, (a) an aerosol, identical to that being used in clinical trials, capable of delivering at least 80 metered doses each of 500,g (100 gCi) of the free acid equivalent as a suspension in a surfactantpropellant mixture (b) ampoules containing 5.0,g (13.4 gCi) of the salt in 1.0 ml isotonic saline for
358
M.E. PICKUP, A.J. JOHNSON, C.S. MAY, J.W. PATERSON & M.P. HARRISON
O
COONa
~~NH
H
NaOOC
(CH2)3CH3 0
Figure 1- Structural formula of ICI 74,917, disodium salt.
bronchoscopic studies (c) ampoules containing 2.5 jig (6.7 jiCi) of the salt in 10 ml sterile pyrogen free water for intravenous use (d) ampoules containing 1.0 mg (200 gCi) of the salt for buccal and oral studies. Drug administration All patients and volunteers gave written consent to the administration of the drug, and to the collection of specimens, after the procedures had been fully explained.
(a) Aerosol study. Seven hospital in-patients (Table 1) who were in the convalescent phase of acute asthma participated while continuing their usual drug therapy. Prior to administration of the drug each person inhaled two doses from a salbutamol aerosol (100 jig/dose) to ensure adequate bronchodilatation, and that their technique of inhalation was satisfactory. A short cannula was inserted into a forearm vein and a blood sample was taken in order to determine the baseline level of plasma radioactivity. A sample of urine was collected for the same purpose.
Table 1
One dose of the study aerosol was inhaled by each person and the breath held for approximately 10 seconds. Blood samples were drawn into lithium heparin tubes at approximately 1, 2, 4, 6, 10, 15, 30, 45 min, 1, 2, 3, 4, 6 and 8 h after inhalation. Sufficient blood was obtained to allow separation of duplicate 1 ml aliquots of plasma. Urine was collected for 72 hours. Sputum, if produced, was collected until levels of radioactivity decreased to twice background (3 to 4 days). Two patients, in addition, expired the first 10 breaths after inhalation of the drug into a Douglas bag which was fitted with a one-way valve and contained sodium bicarbonate solution (0.1 M, 500 ml). This solution was recovered following vigorous shaking of the bag. Subsequent rinses (2-3) of the bag and valve were performed until levels of radioactivity recovered decreased to twice background. After collecting expired gas, the mouth was immediately rinsed with sodium bicarbonate solution (30 ml) and the fluid recovered. During administration and subsequent manoeuvres, one of the two patients used a noseclip. All faecal samples from both patients were collected up to 7 days. In these two studies, and on two other occasions, the recovery of drug from the aerosol mouthpiece was estimated.
(b) Bronchoscopic administration. Direct administration to the lung was achieved by use of a bronchoscopic technique. Three patients with suspected bronchogenic carcinoma of the left lung, one of whom also had emphysema, and another quiescent pulmonary tuberculosis, were undergoing fibreoptic bronchoscopy for diagnostic purposes under local anesthetic (premedication: atropine and omnopon; local anesthetic, lignocaine hydrochloride). After diagnostic examination the tip of the bronchoscope was placed in a visually normal segmental bronchus of the right lower lobe. The drug solution (1.0 ml) was instilled via the suction/instru-
Patient data: Aerosol study Patient
Asthma
EB
CP
DB
JC
FC
MO
PC
Intrinsic
Extrinsic
Intrinsic
Intrinsic Bronchiectasis
Extrinsic Aortic
Extrinsic
Extrinsic
Other disease
incompetance
Sex
Age (years) Weight (kg) Height (cm) PEFR (I min-1) FEV1 (I) FVC (I)
F
64 53 158 145 ND ND
F
M
39 95 170 480 ND ND
43 60 160 150 ND ND
* Prior to commencement; N D = Not determined.
M
63 69 170 ND 2.1 3.5
F 70 45 154 ND 1.4 2.0
F
24 55 164 325 2.7 3.0
F
37 64 173 435 2.8 3.6
ICI 74,917 IN MAN
ment channel of the bronchoscope and flushed through with normal saline (10 ml). The bronchoscope was then withdrawn, and blood and urine samples were collected as in the aerosol study. Additional blood samples were taken at approximately 20 and 24hours. All sputum produced was collected until levels of radioactivity decreased to twice background (2 days). The ampoule, syringe and bronchoscope rinsings were estimated for residual drug.
(c) Intravenous study. Four healthy volunteers were each given an intravenous infusion of the drug solution (10 ml) over an 8 min period. After completion of the infusion blood samples were withdrawn via a cannula in a vein of the opposite forearm in a similar manner to, and at approximately the same times as, the aerosol study. Urine samples were collected for 72 h in all subjects. The recovery of drug from syringe, cannula, and ampoule was estimated. In two subjects faeces were collected up to 7 days.
(d) Buccal studies. For each of four healthy volunteers, the drug (1 mg) was dissolved in distilled water (50 ml) and an aliquot (25 ml) swilled round the mouth for 3 min before expulsion (Beckett & Pickup, 1975). Blood samples were collected up to 4 h at times comparable with the aerosol study. Urine samples were collected up to 36 hours. (e) Oral studies. For each of four healthy volunteers, the drug (1 mg) was dissolved in distilled water (50 ml) and an aliquot (25 ml) swallowed. This was washed down with a further quantity of distilled water (10 ml). Blood samples were collected up to 6.5 h and urine up to 48 h after administration. Physiological assessments
359
bronchoscope was rinsed with normal saline, and aliquots were counted. Sputum was homogenized with a known volume of distilled water and an aliquot counted. Faeces were homogenized with distilled water and the pH adjusted to 10-11 using sodium hydroxide solution to release all bound drug (modified from Walker et al., 1 972b). Following centrifugation an aliquot (0.1 ml) of supematant was counted in scintillant (9 ml) after allowing at least 12 h at 4 IC for any chemiluminescence to disappear. Urine samples from subjects following intravenous or aerosol administration were examined for the presence of tritiated water by a freeze-drying technique. Aliquots were placed in glass scintillation vials and freeze dried. The dry residues were reconstituted with distilled water and the radioactivity compared with that of the equivalent whole urine samples to assess
the amount of free tritiated water present. Samples of urine from different subjects were examined by thin-layer chromatography (t.l.c.). Where necessary the radioactivity was concentrated by partial freeze-drying. Duplicate aliquots of each sample (up to 50 gl) were applied to silica gel on glass t.l.c. plates (Anachem Uniplates Silica gel GF 0.25 mm) as 2 cm bands, and in each case one band was overspotted with [3H]-ICI 74,917. The chromatograms were developed with (a) ethanolchloroform-ammonia-water (55:30:15:10 v/v), (b) isopropanol-ammonia-water (70:30: 10) and (c) isopropanol-water (80:60) solvent systems. Quantitation and location of radioactivity was obtained by scraping 0.5 cm segments in sequence from the plates into scintillation vials. Each segment was suspended in triton toluene scintillant (10 ml) containing 3.5 ml of water to form a stable gel and radioactivity was measured. Control urine specimens containing [3H]-ICI 74,917 were compared to the
study specimens. Before and during the aerosol and intravenous studies, periodic assessments of pulse, blood pressure, forced expiratory volume (FEVy), forced vital capacity (FVC) and peak expiratory flow rate (PEFR) were carried out. Pulse and blood pressure were measured during the bronchoscopic study. Quantitative estimation ofPHJ-ICI 74,917 The total 3H activity of each sample was measured in a Tri-Carb Liquid Scintillation Spectrometer (Model 3375 Packard). All counting was performed at 40C, and the counting efficiency was determined from a standard curve by the automatic external standard method. Aliquots (1 ml) of each of the sample fluids were counted in Ne260 Liquid Scintillant (9 ml, Nuclear Enterprises Ltd). Aliquots of plasma, urine and recoveries from mouthrinse and Douglas bag were counted undiluted. Ampoules, syrings, aerosol mouthpieces and one-way valves were rinsed with a measured volume of distilled water, while the
Estimation of half l(fefrom urine data
Linear least squares regression analysis was used to obtain the line of closest fit to the logarithms of the calculated values (Um,x-Ut) at various times t of the terminal exponential phases, where Um,x is the maximum urinary recovery of drug and U, the cumulative recovery at time t (Cummings, Martin & Park, 1967; Lin, Magat, Calesnick & Symchowicz, 1972). The drug half-life for the terminal elimination phase was derived from the gradient of this regression line (Pickup, May, Ssendagire, Paterson, 1976) (Figure 3). Results
Drug adninistration
Aerosol study. The drug appeared rapidly in the plasma of all patients and the mean maximum con-
360
M.E. PICKUP, A.J. JOHNSON, C.S. MAY, J.W. PATERSON & M.P. HARRISON
0
C1)
COCV
-v
0
*
co
. .r..
. L C.
O
C)o r0)
o
0
_ It
CE)
CN
*
0 0
*
r o CO0)' O co0
()
._
2U) Q, N
CL
oN o 0 cl, Z Z -( C')~~~~~~~V
coLE
vtec;
aiz
0._
0U) 20
.
.
LU
a.
L
C 0
NOD IOZ
ON''-NO
9
00
00N
-
0
o
o
C.
Z
.7
E
* *
0
.
Z
Z
L
i oe L)v
Et~ 0.
0.
*i: ~ ~ ~
~
~~L 10
.Ec
~._
Z z ._
0
_ -- E Z
Ln
z
O :l
0.
Z =0
L
ct
0
:.2
0>.
0
0w2 .E
0
C .
Z
ooz
E
-C co
n *X
0~~
V0
C4
..
O.CL-C '
e c~ °
E
u' E
0~~~
_.0 4-.
NOQ CE
40 0
0
0
>~~ co ~~0
.wa)
0E E
