ACTA 0 P H T H A L M 0 L O G I CA
70 (1992)570-577
Long-term kinetic vitreous fluorophotometry in normal and diabetic subjects Lars L. Knudsen’, Thomas Olsen’ and Folmer Nielsen-Kudsk‘ Department of Ophthalmology’, Arhus University Hospital, and Institute of Pharmacology*, University of Aarhus, hhus, Denmark
Abstract. Nine normal and 24 diabetic subjects were examined by long-term vitreous and plasma fluorescein fluorophotometry and the observed concentration profiles were described by biexponential time courses. The rate constant of elimination of fluorescein from the body (Klo) was significantly decreased in diabetics with background and proliferative retinopathy, presumably caused by affection of the liver and possibly representing alterations in membranes of liver cells. Increased kidney albumin excretion was observed with increasing degree of retinopathy. The apparent rate constant of fluorescein penetration into the eye (Ki,) was found significantlydecreased in background as well as in proliferative retinopathy; while the permeability index, calculated as areas under vitreous and plasma fluorescein curves, was significantly increased. In the normal subjects Kin was significantly higher than the rate constant of fluorescein transfer (K12) from the apparent central to the peripheral tissue compartment, whereas in the diabetics this difference was only found in the group with background retinopathy. The findings seem compatible with the concept that the breakdown of the blood-ocular barrier could be caused at least partly by affection of an active transport system for fluorescein, but thickening and compositional changes of the basement membranes in the eye might also be of importance. Key words: vitreous fluorophotometry - diabetes - fluorescein - blood-ocular barrier - ocular pharmacokinetics permeability index.
The vitreous is surrounded by a tight barrier, which controls drug exchange between blood and eye. In order to understand the properties of this 570
barrier, several vitreous fluorophotometric studies have been performed (Chahal et al. 1985; CunhaVaz & Zeimer 1985). These investigations have all been carried out in a way which estimates net flux of fluorescein into the eye (after 1 h). In this model the presence of a simultaneous active elimination of fluorescein from the eye cannot be taken into accout (Zeimer et al. 1983; Larsen et al. 1983), even though experimental animal models suggest this kind of transport (Cunha-Vaz & Maurice 1967). Recently, a new method based on an open twocompartment pharmacokinetic model was introduced (Knudsenet al. 1991a),in which a long-term observation of fluorescein in plasma and vitreous was made. The penetration into, as well as the elimination of fluorescein from the eye was studied in a group of normal volunteers and compared to penetration and elimination of fluorescein from the apparent peripheral compartment. An active elimination of fluorescein from the eye was observed in a group of normal volunteers (Knudsen et al. 1991a). In diabetics with severe eye disease a breakdown of the blood-retinal barrier has been reported (Cunha-Vaz et al. 1975; Krogsaa et al. 1981).These findings have been interpreted as evidence of an inceased penetration of fluorescein into the eye. Studies from human diabetics and animals with experimental diabetes have also reported cellular dysfunction from various parts of the organism (Reddi 1978; Caldwell et al. 1986; Shimorura &
Spiro 1987;Rasmussen & Ledet 1988).If this cellular dysfunction includes those parts of the eye which build up the blood-retinal barrier (pigment epithelium and vascular endothelium), one might suspect an affection of the active elimination of fluorescein from the eye. The present study explores the possible affection of an active elimination system for fluorescein in diabetics in conjunction with a detailed pharmacokinetic analysis of fluorescein parameters obtained from plasma and eye.
Materialsand Methods 1. Subjects Sodium fluorescein (14 mg/kg body weight) was administered in a cubital vein to 9 normal subjects and 24 early onset insulin treated diabetics, all aged between 19 and 46 years. The 24 diabetics were subdivided into 3 groups: 7 diabetics without retinopathy, 11 diabetics with background retinopathy and 6 diabetics with proliferative retinopathy. Blood sampleswere drawn from an opposite cubital vein at selected time intervals within the experimental period up to 30 h. Before the start of vitreous fluorophotometry each patient was examined by slit-lamp investigation, ophthalmoscopy,measurements of intraocular tension, axial length of the eye, corneal curvature, refraction, visual acuity, blood pressure and weight. In the diabetic group the examination also included measurement of fasting blood glucose, Hb-Alc and kindey albumin excretion (Christensen & 0rskov 1984). 2. Vitreous and plasma fluorophotometry
Vitreous and plasma fluorescein fluorophotometxy were performed as described by Knudsen et al. (1991a,b). 3. Theory a. Model description
The distribution of fluorescein in the organism was described in accordance with an open twocompartment model, in which plasma was considered part of the apparent central compartment and vitreous was assumed to be a special part of the apparent peripheral compartment, as described by Knudsen et al. (1991a,b).A brief review is given below.
b. Pharmacokineticsof free plasma fluorescein
The time course of free plasma fluorescein after a single intravenous dose was described by a biexponential disposition curve (Chahal et al. 1985; Blair et al. 1986; Knudsen et al. 1991b): CF= A*e-at + B*e-Bt, where A and B are zero time concentration coefficients, t the time (h) and a and p are the disposition rate constants of the initial and terminal phase, respectively. The concentration coefficients and the rate constants were found by iterative non-linear regression analysis of the experimental plasma concentration/time data (Nielsen-Kudsk 1983). In the assumed open two-compartment model representing the above stated function, K21 was defined as the rate constant of drug transfer from the apparent peripheral to the central compartment, and it was calculated as: K21= (a*B + p *A)/ (A+B). The elimination rate constant of fluorescein from the central compartment was designated Klo and determined as: Klo= a*p/K21. K12 was defined as the rate constant of transfer from the apparent central to the apparent peripheral compartment and calculated as: K12 = a + p-0(21+ Klo). The apparent volume of distribution of fluorescein in the organism was determined as: Vdp=Klo*Vc/p, where Vc is the apparent volume of the central compartment calculated as: Vc= Do/(A+B). c. Fluorescein in the vitreous body
The vitreous fluorescein concentration profile could be described by the folowing beexponential function (Knudsen et al. 1991a): CF"= C(e-Koutte--Kint), where C is a concentration coefficient determined by extrapolation of the KO,, phase to zero time. Kin is the apparent rate constant of permeation of fluorescein and KO,, is the apparent elimination rate constant from the vitreous. The rate constants and C were found by iterative nonh e a r regression analysis of the experimental concentratiodtime data (Nielsen-Kudsk 1983). d. Permeability index
The permeability index was defined as described by Knudsen et al. (1991a). P index=(C/KOut(C/Kh))/(A/a+ (B/P)), which is the ratio of total areas under the vitreous and plasma concentration curves for fluorescein, respectively, as defined from zero to infinity. 571
4. Statistical methods Based on probit analysis the distribution of the pharmacokinetic parameters represented a normal distribution after logarithmic transformation. For comparison of two kinetic parameters determined from the same group of patients, a paired Student t-test was used. For comparison of two kinetic parameters determined from different groups of patients, an unpaired Student t-test was brought to use. Calculated mean values, 95% confidence limits, coefficientsof correlationand statistical tests were based on the logarithmically transformed parameter values.
N
Results The disposition of fluorescein from plasma could be described by a biexponential decay curve, which was assumed to represent an open two-compartment model. From the determined pharmacokinetic parameters the volume of the apparent central compartment (Vc) was found to be 0.68 llkg body weight in the normal group. Similar results were found in all 3 diabetic groups (Table 1). The apparent total volume of distribution (Vdp) was found to be 5.72 llkg body weight in the normal group. The corresponding values found in all diabetic groups decreased slightly but not si@icantly (Table 1).In all normal and diabetic subjects the major part of fluoresceinwas located in the apparent peripheral tissue compartment. The elimination rate constant of fluorescein from the central compartment (Klo) was similar to
Table 2. Mean values (mean) and 95% confidence limits (confl for determined vitreous kinetic fluorescein parameters after a single intravenous dose (14 mg/kg body weight) in 9 normal subjects (N), 7 diabetics without retinopathy (-R), 11 diabetics with background retinopathy (BR) and 6 diabetics with proliferative retinopathy (PR).
-R
BR
PR
a (h-1) mean 2.03 cod 1.44-2.87
2.63 1.88-3.68
1.54 1.20-1.97
1.57 1.13-2.20
P (h-) mean 0.21 c o d 0.17-0.26
0.29 0.19-0.45
0.2 1 0.16-0.28
0.17 0.1 1-0.28
t 'h6 (h) mean 3.36 c o d 2.68-4.22
2.40 1.53-3.76
3.22 2.37-4.37
3.97 2.50-6.30
C
Vc (1 k g l ) mean 0.68 conf 0.59-0.78
0.63 0.54-0.74
0.66 0.55-0.79
0.70 0.60-0.82
mean 0.101 0.065" 0.075" 0.074'# conf 0.084-0.122 0.050-0.085 0.066-0.085 0.063-0.086
Vdp (1 k g l ) mean 5.72 conf 4.80-6.82
4.39 3.26-5.92
4.44 3.51-5.62
5.27 3.36-8.26
Kl,lll/ B mean 0.38 conf 0.29-0.50
0.22 0.14-0-36
0.36 0.27-0.49
0.42 0.26-0.09
Kio (h-l) mean 1.74 cod 1.36-2.23
2.00 1.58-2.53
1.40" 1.16-1.69
1.31" 1.02-1.69
AUC,I (pmol 1-1 h) 31.4 28.9 mean conf 25.9-38.0 23.3-35.9
40.2 32.8-49.3
40.4 28.5-57.2
(pmol 1-1 h) 0.85 0.98 0.67-1.08 0.80-1.20
1.97"
7.88""
1.36-2.86
6.27-9.90
I
Kin (h-l) 0.67 mean ~0.48-0.93
O
Kout
0-1)
N
I
-
R
R
I
P
R
0.55
0.33"
0.15""
0.23-0.48
0.10-0.22
(h-1)
AUCvitreous
mean conf
KIZ(h-1) mean 0.149 0.339 0.097 0.170 conf 0.060-0.367 0.124-0.927 0.053-0.178 0.080-0.361
Permeability index ("10) 3.4 mean 2.7 cod 2.1-3.5 2.8-4.1
" Indicates significant deviation from diabetics without
*, ** Indicates
572
B
0.45-0.67
mean 0.241 0.385 0.232 0.210 conf 0.175-0.332 0.214-0.692 0.167-0.322 0.123-0.356
retinopathy at 5%level.
I
4.9" 3.1-7.7
19.5~: 16.4-23.3
significant deviations from normal subjects af 5%and 1%levels.
50.00 40.00 30.00
50.00 40.00 30.00
20.00
20.00
fluorescein
Plasma A
I = 10.00 :
5 10.00
-5
-d
zW
t:
0
W
8
op
r 5
2
1.00
: Z
.-8
-E L
a
t
c
m
0
s
0
1.00
8
c
0
?
0.10
0.0 1
O.1°
0.01 0
2
4 6
8 10 12 14 16 18 2 0 2 2 24 26 28 30
Time (hours)
Fig.la Free plasma fluorescein and vitreous fluorescein time course cuvers in a representative non diabetic subjects.
the initial rate constant of distributive disposition (a)in all groups (Table 2). Klo decreased significantly in diabetics with background (p
70 (1992)570-577
Long-term kinetic vitreous fluorophotometry in normal and diabetic subjects Lars L. Knudsen’, Thomas Olsen’ and Folmer Nielsen-Kudsk‘ Department of Ophthalmology’, Arhus University Hospital, and Institute of Pharmacology*, University of Aarhus, hhus, Denmark
Abstract. Nine normal and 24 diabetic subjects were examined by long-term vitreous and plasma fluorescein fluorophotometry and the observed concentration profiles were described by biexponential time courses. The rate constant of elimination of fluorescein from the body (Klo) was significantly decreased in diabetics with background and proliferative retinopathy, presumably caused by affection of the liver and possibly representing alterations in membranes of liver cells. Increased kidney albumin excretion was observed with increasing degree of retinopathy. The apparent rate constant of fluorescein penetration into the eye (Ki,) was found significantlydecreased in background as well as in proliferative retinopathy; while the permeability index, calculated as areas under vitreous and plasma fluorescein curves, was significantly increased. In the normal subjects Kin was significantly higher than the rate constant of fluorescein transfer (K12) from the apparent central to the peripheral tissue compartment, whereas in the diabetics this difference was only found in the group with background retinopathy. The findings seem compatible with the concept that the breakdown of the blood-ocular barrier could be caused at least partly by affection of an active transport system for fluorescein, but thickening and compositional changes of the basement membranes in the eye might also be of importance. Key words: vitreous fluorophotometry - diabetes - fluorescein - blood-ocular barrier - ocular pharmacokinetics permeability index.
The vitreous is surrounded by a tight barrier, which controls drug exchange between blood and eye. In order to understand the properties of this 570
barrier, several vitreous fluorophotometric studies have been performed (Chahal et al. 1985; CunhaVaz & Zeimer 1985). These investigations have all been carried out in a way which estimates net flux of fluorescein into the eye (after 1 h). In this model the presence of a simultaneous active elimination of fluorescein from the eye cannot be taken into accout (Zeimer et al. 1983; Larsen et al. 1983), even though experimental animal models suggest this kind of transport (Cunha-Vaz & Maurice 1967). Recently, a new method based on an open twocompartment pharmacokinetic model was introduced (Knudsenet al. 1991a),in which a long-term observation of fluorescein in plasma and vitreous was made. The penetration into, as well as the elimination of fluorescein from the eye was studied in a group of normal volunteers and compared to penetration and elimination of fluorescein from the apparent peripheral compartment. An active elimination of fluorescein from the eye was observed in a group of normal volunteers (Knudsen et al. 1991a). In diabetics with severe eye disease a breakdown of the blood-retinal barrier has been reported (Cunha-Vaz et al. 1975; Krogsaa et al. 1981).These findings have been interpreted as evidence of an inceased penetration of fluorescein into the eye. Studies from human diabetics and animals with experimental diabetes have also reported cellular dysfunction from various parts of the organism (Reddi 1978; Caldwell et al. 1986; Shimorura &
Spiro 1987;Rasmussen & Ledet 1988).If this cellular dysfunction includes those parts of the eye which build up the blood-retinal barrier (pigment epithelium and vascular endothelium), one might suspect an affection of the active elimination of fluorescein from the eye. The present study explores the possible affection of an active elimination system for fluorescein in diabetics in conjunction with a detailed pharmacokinetic analysis of fluorescein parameters obtained from plasma and eye.
Materialsand Methods 1. Subjects Sodium fluorescein (14 mg/kg body weight) was administered in a cubital vein to 9 normal subjects and 24 early onset insulin treated diabetics, all aged between 19 and 46 years. The 24 diabetics were subdivided into 3 groups: 7 diabetics without retinopathy, 11 diabetics with background retinopathy and 6 diabetics with proliferative retinopathy. Blood sampleswere drawn from an opposite cubital vein at selected time intervals within the experimental period up to 30 h. Before the start of vitreous fluorophotometry each patient was examined by slit-lamp investigation, ophthalmoscopy,measurements of intraocular tension, axial length of the eye, corneal curvature, refraction, visual acuity, blood pressure and weight. In the diabetic group the examination also included measurement of fasting blood glucose, Hb-Alc and kindey albumin excretion (Christensen & 0rskov 1984). 2. Vitreous and plasma fluorophotometry
Vitreous and plasma fluorescein fluorophotometxy were performed as described by Knudsen et al. (1991a,b). 3. Theory a. Model description
The distribution of fluorescein in the organism was described in accordance with an open twocompartment model, in which plasma was considered part of the apparent central compartment and vitreous was assumed to be a special part of the apparent peripheral compartment, as described by Knudsen et al. (1991a,b).A brief review is given below.
b. Pharmacokineticsof free plasma fluorescein
The time course of free plasma fluorescein after a single intravenous dose was described by a biexponential disposition curve (Chahal et al. 1985; Blair et al. 1986; Knudsen et al. 1991b): CF= A*e-at + B*e-Bt, where A and B are zero time concentration coefficients, t the time (h) and a and p are the disposition rate constants of the initial and terminal phase, respectively. The concentration coefficients and the rate constants were found by iterative non-linear regression analysis of the experimental plasma concentration/time data (Nielsen-Kudsk 1983). In the assumed open two-compartment model representing the above stated function, K21 was defined as the rate constant of drug transfer from the apparent peripheral to the central compartment, and it was calculated as: K21= (a*B + p *A)/ (A+B). The elimination rate constant of fluorescein from the central compartment was designated Klo and determined as: Klo= a*p/K21. K12 was defined as the rate constant of transfer from the apparent central to the apparent peripheral compartment and calculated as: K12 = a + p-0(21+ Klo). The apparent volume of distribution of fluorescein in the organism was determined as: Vdp=Klo*Vc/p, where Vc is the apparent volume of the central compartment calculated as: Vc= Do/(A+B). c. Fluorescein in the vitreous body
The vitreous fluorescein concentration profile could be described by the folowing beexponential function (Knudsen et al. 1991a): CF"= C(e-Koutte--Kint), where C is a concentration coefficient determined by extrapolation of the KO,, phase to zero time. Kin is the apparent rate constant of permeation of fluorescein and KO,, is the apparent elimination rate constant from the vitreous. The rate constants and C were found by iterative nonh e a r regression analysis of the experimental concentratiodtime data (Nielsen-Kudsk 1983). d. Permeability index
The permeability index was defined as described by Knudsen et al. (1991a). P index=(C/KOut(C/Kh))/(A/a+ (B/P)), which is the ratio of total areas under the vitreous and plasma concentration curves for fluorescein, respectively, as defined from zero to infinity. 571
4. Statistical methods Based on probit analysis the distribution of the pharmacokinetic parameters represented a normal distribution after logarithmic transformation. For comparison of two kinetic parameters determined from the same group of patients, a paired Student t-test was used. For comparison of two kinetic parameters determined from different groups of patients, an unpaired Student t-test was brought to use. Calculated mean values, 95% confidence limits, coefficientsof correlationand statistical tests were based on the logarithmically transformed parameter values.
N
Results The disposition of fluorescein from plasma could be described by a biexponential decay curve, which was assumed to represent an open two-compartment model. From the determined pharmacokinetic parameters the volume of the apparent central compartment (Vc) was found to be 0.68 llkg body weight in the normal group. Similar results were found in all 3 diabetic groups (Table 1). The apparent total volume of distribution (Vdp) was found to be 5.72 llkg body weight in the normal group. The corresponding values found in all diabetic groups decreased slightly but not si@icantly (Table 1).In all normal and diabetic subjects the major part of fluoresceinwas located in the apparent peripheral tissue compartment. The elimination rate constant of fluorescein from the central compartment (Klo) was similar to
Table 2. Mean values (mean) and 95% confidence limits (confl for determined vitreous kinetic fluorescein parameters after a single intravenous dose (14 mg/kg body weight) in 9 normal subjects (N), 7 diabetics without retinopathy (-R), 11 diabetics with background retinopathy (BR) and 6 diabetics with proliferative retinopathy (PR).
-R
BR
PR
a (h-1) mean 2.03 cod 1.44-2.87
2.63 1.88-3.68
1.54 1.20-1.97
1.57 1.13-2.20
P (h-) mean 0.21 c o d 0.17-0.26
0.29 0.19-0.45
0.2 1 0.16-0.28
0.17 0.1 1-0.28
t 'h6 (h) mean 3.36 c o d 2.68-4.22
2.40 1.53-3.76
3.22 2.37-4.37
3.97 2.50-6.30
C
Vc (1 k g l ) mean 0.68 conf 0.59-0.78
0.63 0.54-0.74
0.66 0.55-0.79
0.70 0.60-0.82
mean 0.101 0.065" 0.075" 0.074'# conf 0.084-0.122 0.050-0.085 0.066-0.085 0.063-0.086
Vdp (1 k g l ) mean 5.72 conf 4.80-6.82
4.39 3.26-5.92
4.44 3.51-5.62
5.27 3.36-8.26
Kl,lll/ B mean 0.38 conf 0.29-0.50
0.22 0.14-0-36
0.36 0.27-0.49
0.42 0.26-0.09
Kio (h-l) mean 1.74 cod 1.36-2.23
2.00 1.58-2.53
1.40" 1.16-1.69
1.31" 1.02-1.69
AUC,I (pmol 1-1 h) 31.4 28.9 mean conf 25.9-38.0 23.3-35.9
40.2 32.8-49.3
40.4 28.5-57.2
(pmol 1-1 h) 0.85 0.98 0.67-1.08 0.80-1.20
1.97"
7.88""
1.36-2.86
6.27-9.90
I
Kin (h-l) 0.67 mean ~0.48-0.93
O
Kout
0-1)
N
I
-
R
R
I
P
R
0.55
0.33"
0.15""
0.23-0.48
0.10-0.22
(h-1)
AUCvitreous
mean conf
KIZ(h-1) mean 0.149 0.339 0.097 0.170 conf 0.060-0.367 0.124-0.927 0.053-0.178 0.080-0.361
Permeability index ("10) 3.4 mean 2.7 cod 2.1-3.5 2.8-4.1
" Indicates significant deviation from diabetics without
*, ** Indicates
572
B
0.45-0.67
mean 0.241 0.385 0.232 0.210 conf 0.175-0.332 0.214-0.692 0.167-0.322 0.123-0.356
retinopathy at 5%level.
I
4.9" 3.1-7.7
19.5~: 16.4-23.3
significant deviations from normal subjects af 5%and 1%levels.
50.00 40.00 30.00
50.00 40.00 30.00
20.00
20.00
fluorescein
Plasma A
I = 10.00 :
5 10.00
-5
-d
zW
t:
0
W
8
op
r 5
2
1.00
: Z
.-8
-E L
a
t
c
m
0
s
0
1.00
8
c
0
?
0.10
0.0 1
O.1°
0.01 0
2
4 6
8 10 12 14 16 18 2 0 2 2 24 26 28 30
Time (hours)
Fig.la Free plasma fluorescein and vitreous fluorescein time course cuvers in a representative non diabetic subjects.
the initial rate constant of distributive disposition (a)in all groups (Table 2). Klo decreased significantly in diabetics with background (p
