A C T A O P H T H A L M O L O G I C A VOL. 5 6 1 9 7 8
Medical Microbiology (Head: P . Toiwanen), University of Turku and Debartment of Obhthalmology (Head: A . Oksala), University Central Hospital, Turku, Finland
Department
of
CLOXACILLIN DISTRIBUTION IN THE RABBIT EYE AFTER INTRAVENOUS INJECTION BY
LOTTA SALMINEN
Distribution of isotopically labelled and intravenously injected cloxacillin was studied in the rabbit eye. The antibiotic concentration determined by liquid scintillation counting proved to be a reliable measure of the total antibiotic concentration when controlled by microbiological assay. In the rabbit eye after an intravenous injection of 50 mg/kg of cloxacillin sodium, longlasting antibiotic concentration regarded as therapeutic against penicillinase producing staphylococci was obtained in all vascularized ocular structures and in the cornea. The antibiotic present in the iris and ciliary body, and in the retina and choroid preparations, proved to be partly intravascular, whereas it penetrated better into the extravascular tissue compartment of the sclera and limbal area. Cloxacillin failed to achieve a therapeutic antibiotic concentration in the vitreous body and in the lens. Administration of probenecid had an enhancing effect on ocular cloxacillin concentration allowing improved drug diffusion into the eye by means of an elevated plasma concentration. No specific ocular effect of probenecid was noticed. Therapeutic concentration of cloxacillin in the aqueous humour, otherwise barely achieved, was more satisfactorily obtained with a previous injection of probenecid.
Key words: cloxacillin - Ekvacillin@ - ocular drug concentration radioassay - bioassay - probenecid - ProbecidB - plasma concentration. Received June 20, 1 9 i i .
11
Lotta Salminen
Cloxacillin, with oxacillin and dicloxacillin, is a member of the isoxazolyl family of semisynthetic penicillins. The isoxazolyl penicillins are acid stable and strongly resistant to destruction by bacterial penicillinase and thus indicated primarily in the treatment of infections due to penicillinase producing staphylococci. These organisms have been implicated as the pathogens responsible f o i many cases of postoperative bacterial endophthalmitis (Allen & Mangiaracine 1964; Locatcher-Khorazo & Gutierrez 1972). Reports on ocular penetration of the isoxazolyl penicillins are conflicting and defective. Oxacillin failed to penetrate into the aqueous humour of the rabbit when administered systemically in doses ranging from 1 1 to 22 mg/kg (Furgiuele 1964) or from 25 to 75 mg/kg (Records & Ellis 1967). Also samples of the secondary aqueous humour showed no antibiotic activity after a single intravenous injection of 75 mg/kg of oxacillin. Therapeutic antibiotic activity in the human aqueous humour after a n intravenous injection of about 50 mg/kg of oxacillin was reported by Meyer et al. (1973) as opposed to Records (1967) who found no detectable antibiotic activity in the aqueous humour of noninflamed human eyes after systemic administration of oxacillin. After a single intravenous injection of 25 mg/kg of dicloxacillin, no antibiotic activity was observed in the aqueous humour of the normal rabbit eye. Concentrations in the secondary aqueous humour were nearly as high as those present in plasma (Records 1968). Cloxacillin penetrated into the aqueous humour of the rabbit in therapeutic amount when administered systemically in doses of 25 to 50 mg/kg (Faris & Uwaydah 1974), but no cloxacillin was detectable in human aqueous humour (Uwaydah et al. 1976). This study was undertaken to determine whether systemically administered cloxacillin, bacterial to penicillinase producing staphylocicci at concentrations of about 1.5 pglg (Nayler et al. 1962), would reach therapeutically useful concentrations in the structures of the rabbit eye. T o produce higher and more sustained plasma levels of cloxacillin, some rabbits received probenecid prior to the antibiotic injection.
Material and Methods Animals
The material in the present study consisted of 30 rabbits of both sexes weighing from 1.9 to 2.5 kg. Inspection of external ocular structures and ophthalmoscopy revealed no abnormalities in their eyes.
12
Cloxacillin in the Rabbit Eye Injection of antibiotic 3%-cloxacillin sodium (specific activity 37.6 pCilmg) and non-radioactive cloxacillin sodium (EkvacillinB) (both kindly supplied by Astra, Sodertalje, Sweden) were used. Each rabbit received intravascularly 1 ml/kg of an aqueous solution containing 50 mg/ml of cloxacillin sodium and a n amount of 25 pCi/ml of the labelled material. Injection of probenecid
Nine rabbits received 100 mg/kg of probenecid (ProbecidB) intraperitoneally 20 rnin prior to the antibiotic injection. Preparation and radioassay of plasma and ocular samples T o follow the antibiotic concentration in plasma, several blood samples were taken from all rabbits until the animal was killed by a rapid injection of pentobarbital sodium (NembutalB) at intervals of 5 to 240 min from the injection of antibiotic. Both eyes were promptly enucleated and frozen in liquid nitrogen. The frozen eyes were dissected on a salt-ice mixture. The cornea was cut 1 mm from the limbus. The frozen aqueous humour was separated into pieces. Then the eye was cut longitudinally into two equal halves which were dissected separately. The lens was easily separated from the frozen vitreous body. The aqueous humour, possibly contaminating the lens, was separated. Several specimens were taken from the vitreous body. The frozen iris and ciliary body were taken together as well as the retina and choroid. If low radioactivity was expected, the two halves of the i n s and ciliary body, as well as those of the retina and choroid, were united. The limbal area, 2 mm in width, was separated from the rest of the sclera. Possible blood contamination was observed, and the contaminated tissues were mechanically cleaned. If this failed, the specimen was rejected. The specimens were weighed in pre-weighed counting vials and digested with 0.1 ml of 6Oo/o perchloric acid and 0.2 ml of 3Oo/o hydrogen peroxide at 80°C for two h. Thereafter a counting solution containing 4.6 ml of ethylene glycerol monoethyl ether and 10.4 ml of toluol with 6 g/l 2,5-diphenyloxazole was added. Plasma samples were treated in the same way. Counting was performed with a Packard Tri-Card model 3375 liquid scintillation spectrometer. External standardisation was used for calculating the results in disintergrations per min (DPM). The reduction in radioactivity of sulphur-35 was observed. Mlcrobiologlcal assay of plasma In addition to radioassay, parallel microbiological assay of total antibiotic concentration in plasma of five rabbits killed at 180 to 240 min after the cloxacillin injection was undertaken as described by Jalling et al. (1972). Plasma samples of 10 ,ul were pipetted on a Ford 428 Mill filter paper and dried. Standard discs were prepared from appropriate dilutions of cloxacillin sodium in pooled rabbit plasma in the same way. The test organism, Sarcina lutea ATCC 9341, and the plates were prepared according to Grove & Randall (1955). The plates were prepared for one day’s use a t a time. They were allowed to dry for 30 min, after which the pre-pipetted discs were placed on the agar surface. At least three series of standard discs for each inoculated agar were used each time the assay was run. After a pre-diffusion time of 30 min, the plates were placed at +35OC for 12 h. The diameter of the inhibition zone was read
13
Lotta Salminen twice, and the mean of the two readings taken. Antibiotic concentration of the sample was obtained from the standard curve, which was linear to semilogarithmic plot within the range of concentration for standard discs. Statistics
The number of normal eyes studied for their cloxacillin concentration was 31. This number includes both eyes, with one exception, of 11 rabbits killed at 30, 60, 90, 120, 150, 180, 210 and 240 min respectively following the injection of antibiotic, as well as contralateral eyes of 10 rabbits which underwent an anterior chamber puncture before the antibiotic injection. The results of the punctured eyes will be presented in a later paper (Salminen 1977). The number of eyes obtained from rabbits with probenecid injection before cloxacillin injection was 18. They comprised both eyes of 9 rabbits killed at 30, 60, 90, 120, 180 and 210 min respectively following the antibiotic injection. From the normal material and from the eyes of the animals with probenecid injection, the mean values and their standard errors were calculated. Student's t-test was used in comparing the two materials. The correlation coefficient between plasma liquid scintillation counting method and antibiotic concentrations obtained by bioassay was determined (Hald 1952), and the significance of the correlation was tested.
)OO
100
,a0
,o 0
100 I00
100
TOO
'0 0
100
100
300
2:
508 40 a 30
;
30
20
20 I0
I3
05
05
821
i
0 3
I
,
02
O'
5 k 30 6c
&
Ic
140
I00
2:o
2:
MIULIrES
Fig. I B. Aqueous humour.
Fig. I A . Cornea. Fig. - 1.
Ocular (0,0 ) and plasma (curves) radioactivity and corresponding drug concentration after an intravenous injection of 3~S-cloxacillinsodium. Open circles (n = 2 to 6, mean 3.2) and lower curve are samples obtained from rabbits without, and black circles ( n = 2 to 6, mean 3.0) and upper curve are samples obtained from rabbits with a previous injection of probenecid (100 mg/kg) before the antibiotic injection. Vertical lines equal k SEM. The amount of 3~S-cloxacillinsodium injected was 25.0 pCilkg with 50 mg/kg of unlabelled drug. 14
Cloxacillin in the Rabbit Eye
500 40.0 30.0 20 0
200
200 10 0
10.0
10.0
* \ 30L
2.0
05
0.2
MINUTES
MINUTES
Fig. 1 D . Retina-choroid.
Fig. 1 C . Iris-ciliary body.
Results Radioactivity obtained by liquid scintillation method and cloxacillin concentration obtained by bioassay in plasma corresponded well with each other (correlation coefficient 0.94 1 ; P < 0.001). Radioactivity (disintegrations per min) found in a microbiologically determined concentration of 1 pg of cloxacillin sodium in plasma samples taken 180 to 240 min after the antibiotic injection was 1252 f 279. Radioactivity (DPM) added to 1 pg of unlabelled cloxa-
200 0
-1
1
1004: 400. 500: 300:
1200
105
0 5: 04. 0 3.
0"
0 I530
60
w
I20 I50 MINUTES
I I00
2;o
02O I Lo
24;
b,;
60
&
o1;
160
, I I
180 210
MINUTES
Fig. 1 F. Sclera.
Fig. 1 E. Limbal area.
15
240
Lotta Salminen
cillin sodium to make the injected solution was 1110. It seems probable, therefore, that the measured radioactivity gives reliable information about the ocular concentration of the studied antibiotic. Ocular and plasma radioactivity and corresponding drug concentration, with the exception of the lens, are summarized in Figs. 1-2. Radioactivity measured from the lens did not differ from that of the background. Cloxacillin concentration in plasma, after equal values at 5 and 15 rnin after the antibiotic injection, diminished more rapidly thereafter in animals with no previous injection of probenecid (P< 0.01). In animals with a probenecid injection before the antibiotic injection, statistically higher (P < 0.001 or P < 0.01) cloxacillin levels were measured in the cornea (30, 60, 90, 150 and 210 min), aqueous humour (30 and 60 min), iris-ciliary body and retina-choroid (30, 60 and 120 min) and in the sclera and limbal area (120 and 180 min).
Discussion In the present investigation the therapeutic level of 1.5 pg/g of cloxacillin was achieved in all vascularized ocular structures and in the cornea of the rabbit eye after an intravenous injection of 50 mg/kg of cloxacillin sodium. In the aqueous humour this level was only achieved in animals which had had a previous injection of probenecid prior to the antibiotic injection. Cloxacillin failed to penetrate into the lens and vitreous body to any remarkable degree.
- 0.10
f 0
0.05 ao4 0.03
o
\
o
cm
0
0.020.01
a
f 0
I
'
0
'
4
1
I
Radioactivity and corresponding drug concentration in rabbit vitreous body after an intravenous injection of 3%-cloxacillin sodium. Determinations were made from the same rabbits as presented in Fig. 1. See the legend to Fig. 1 for explanation. 16
Cloxacillin in the Rabbit Eye
The intravenous injection of 50 mg/kg of cloxacillin resulted in an extremely high plasma antibiotic concentration immediately after the antibiotic injection. In the course of the next 30 min, cloxacillin plasma concentration greatly diminished. This was due to the rapid diffusion of the drug into the extravascular tissue compartment (Dittert et al. 1969). Moreover the elimination began immediately after the intravenous injection. The huge intravascular cloxacillin concentration led to a rapid diffusion into the extravascular tissue compartment in the sclera and limbal area. As a sign of extravascular diffusion of cloxacillin into the sclera and limbal area, their antibiotic concentration reached plasma level at 60 min and was equal or superior to it during the next 60 min. In the iris-ciliary body and in the retina-choroid, circulating cloxacillin met capillary walls which appeared to have lower permeability. Unfortunately, retina and choroid were not examined separately and any difference in their capillary permeability cannot be assessed. Plasma cloxacillin concentration was never reached in the iris and ciliary body. In the aqueous humour cloxacillin reached its greatest concentration after 30 min. It is obvious that the high cloxacillin plasma concentration immediately after injection led to a rapid diffusion of the drug through the iris and ciliary body into the aqueous humour. Cloxacillin concentration in the aqueous humour was too low to cause any remarkable diffusion into the lens. There was a marked difference in cloxacillin concentration between the limbal area and the cornea during the first hour after the antibiotic injection. Drug diffusion from the limbal area into the cornea took place gradually. As a sign of the resistance of the vitreous barrier to drug penetration (Bleeker et al. 1968), cloxacillin concentration in the vitreous body was far below that in the aqueous humour. Administration of probenecid resulted in a significant increase of cloxacillin concentration in all ocular structures with the exception of the lens and vitreous body. Therapeutic concentration of cloxacillin in the aqueous humour, otherwise barely achieved, was more satisfactorily obtained by means of a previous injection of probenecid. Administration of probenecid caused a significant increase in cloxacillin plasma concentration. Drug diffusion into ocular tissues was enhanced by a greater concentration gradient and by a longer contact of the antibiotic with capillary walls. No increase in the ratio of ocular tissue cloxacillin concentration to that of plasma was noticed. This agrees well with the observation of Forbes & Becker (1960) that systemic administration of probenecid to nephrectomized animals failed to increase penetration of organic anions into the vitreous or aqueous humour. The total cloxacillin concentration obtained by radioactive tracer method, 17 Acta ophthal. 56, 1
2
Lotta Salminen
however, gives incomplete information on the biological activity of the samples studied. The antibiotic activity is smaller than the total antibiotic activity would indicate (Bloome et al. 1970). This reduction of biological activity is caused by binding of the antibiotic to plasma and tissue proteins. T h e inactivation is for the great part reversible, being influenced by local circumstances. and not measurable in vitro.
References Allen M. F. & Mangiaracine A. B. (1964) Bacterial endophthalmitis after cataract extraction. Arch. Ophthal. (Chicago) 72, 454-462. Bleeker G. M., van Haeringen N. J., Maas E. R. & Glasius E. (1968) Selective properties of the vitreous barrier. Exp. Eye Res. 7, 37-46. Bloome M. A., Golden B. & MacKee A. D. (1970) Antibiotic concentration in ocular tissues. Arch. Ophthal. (Chicago) 83, 78-83. Dittert L. W., Griffen W . O., LaPiana J. C., Shainfeld F. J. & Doluisio J. T. (1969) Pharmacokinetic interpretation of penicillin levels in serum and urine after intravenous administration. In: Hobby G. L., Ed. Antimicrobial agents in chemotherapy, pp. 42-48. American Society for Microbiology, Bethesda, Maryland. Faris B. M. & Uwaydah M. M. (1974) Intraocular penetration of semisynthetic penicillins. Methicillin, cloxacillin, ampicillin and carbenicillin studies in experimental animals with a review of the literature. Arch. Ophthal. (Chicago) 92, 501-505. Forber M. & Becker B. (1960) The transport of organic anions by the rabbit eye. 11. In vivo transport of iodopyracet (Diodrast). Amer. /. Ophthal. 50, 197-203. Furgiuele F. P. (1964) New antibiotics: Their intraocular penetration. Amer. /. Ophthal. 58, 443-446. Grove D. C . & Randall W . A. (1955) Assay methods of antibiotics. A laboratory manual. Medical Encyclopedia, Inc., New York. Hald A. (1952) Statistical theory witfa engineering applications. John Wiley and Sons, London. Jalling B., Malmborg A,-S., Lindman A. & Bortus L. 0. (1972) Evaluation of a micromethod for determination of antibiotic concentration in plasma. EZlro/J. /. d i n . Pharmacol. 4 , 150-158. Locatcher-Khorazo D. & Gutierrez E. (1972) Postoperative infection of the eye. In: Locatcher-Khorazo D. and Seegal B. C., Eds. Microbiology of the Eye, pp. 77-85, Mosby, St. Louis. Meyer K., Matz D., Uterman D. & Fedder J. (1973) Ober antibakterielle Wirkstoffkonzentrationen im Kammerwasser des Menschen nach parenteraler Applikation verschiedener Antibiotica. Albrecftt v . Graeies Arch. klin. exp. Ophthal. 185, 55-70. Nayler H. C., Long A. A. W., Brown D. M., Acred P., Rolinson G. N., Batchelor F. R., Stevens S. Sutherland R. (1962) Chemistry, toxicology, pharmacology and microbiology of a new acid-stable penicillin, resistant to penicillinase (BRL. 1621). Nature (Lond.) 195, 1264-1267. Records R. E. (1967) Human intraocular penetration of sodium oxacillin. Arch. Ophthal. (Chicago) 77, 693-695.
18
Cloxacillin in the Rabbit Eye Records R. E. (196s) Intraocular penetration of dicloxacillin. Invest. Olhthal. 7, 663-667. Records R. E. & Ellis P. P. (1967) T h e intraocular penetration of ampicillin, methicillin, and oxacillin. Amer. /. Ophtlzal. 64, 135-143. Salminen L. (1978) Effect of paracentesis on ocular cloxacillin concentration. Acta ophthal. (Kbh.) 56, 20-26. Uwaydah M. M., Faris B. M., Smara I. N., Shammas H. F. & To’Mey K. F. (1976) Cloxacillin penetration. Amer. /. O/?ldzal. 82, 114-1 16.
Atittior’s address:
Lotta Salminen, M. D., Turku University Central Hospita I, Department of Ophthalmology, SF-20520 Turku 52, Finland.
19
Medical Microbiology (Head: P . Toiwanen), University of Turku and Debartment of Obhthalmology (Head: A . Oksala), University Central Hospital, Turku, Finland
Department
of
CLOXACILLIN DISTRIBUTION IN THE RABBIT EYE AFTER INTRAVENOUS INJECTION BY
LOTTA SALMINEN
Distribution of isotopically labelled and intravenously injected cloxacillin was studied in the rabbit eye. The antibiotic concentration determined by liquid scintillation counting proved to be a reliable measure of the total antibiotic concentration when controlled by microbiological assay. In the rabbit eye after an intravenous injection of 50 mg/kg of cloxacillin sodium, longlasting antibiotic concentration regarded as therapeutic against penicillinase producing staphylococci was obtained in all vascularized ocular structures and in the cornea. The antibiotic present in the iris and ciliary body, and in the retina and choroid preparations, proved to be partly intravascular, whereas it penetrated better into the extravascular tissue compartment of the sclera and limbal area. Cloxacillin failed to achieve a therapeutic antibiotic concentration in the vitreous body and in the lens. Administration of probenecid had an enhancing effect on ocular cloxacillin concentration allowing improved drug diffusion into the eye by means of an elevated plasma concentration. No specific ocular effect of probenecid was noticed. Therapeutic concentration of cloxacillin in the aqueous humour, otherwise barely achieved, was more satisfactorily obtained with a previous injection of probenecid.
Key words: cloxacillin - Ekvacillin@ - ocular drug concentration radioassay - bioassay - probenecid - ProbecidB - plasma concentration. Received June 20, 1 9 i i .
11
Lotta Salminen
Cloxacillin, with oxacillin and dicloxacillin, is a member of the isoxazolyl family of semisynthetic penicillins. The isoxazolyl penicillins are acid stable and strongly resistant to destruction by bacterial penicillinase and thus indicated primarily in the treatment of infections due to penicillinase producing staphylococci. These organisms have been implicated as the pathogens responsible f o i many cases of postoperative bacterial endophthalmitis (Allen & Mangiaracine 1964; Locatcher-Khorazo & Gutierrez 1972). Reports on ocular penetration of the isoxazolyl penicillins are conflicting and defective. Oxacillin failed to penetrate into the aqueous humour of the rabbit when administered systemically in doses ranging from 1 1 to 22 mg/kg (Furgiuele 1964) or from 25 to 75 mg/kg (Records & Ellis 1967). Also samples of the secondary aqueous humour showed no antibiotic activity after a single intravenous injection of 75 mg/kg of oxacillin. Therapeutic antibiotic activity in the human aqueous humour after a n intravenous injection of about 50 mg/kg of oxacillin was reported by Meyer et al. (1973) as opposed to Records (1967) who found no detectable antibiotic activity in the aqueous humour of noninflamed human eyes after systemic administration of oxacillin. After a single intravenous injection of 25 mg/kg of dicloxacillin, no antibiotic activity was observed in the aqueous humour of the normal rabbit eye. Concentrations in the secondary aqueous humour were nearly as high as those present in plasma (Records 1968). Cloxacillin penetrated into the aqueous humour of the rabbit in therapeutic amount when administered systemically in doses of 25 to 50 mg/kg (Faris & Uwaydah 1974), but no cloxacillin was detectable in human aqueous humour (Uwaydah et al. 1976). This study was undertaken to determine whether systemically administered cloxacillin, bacterial to penicillinase producing staphylocicci at concentrations of about 1.5 pglg (Nayler et al. 1962), would reach therapeutically useful concentrations in the structures of the rabbit eye. T o produce higher and more sustained plasma levels of cloxacillin, some rabbits received probenecid prior to the antibiotic injection.
Material and Methods Animals
The material in the present study consisted of 30 rabbits of both sexes weighing from 1.9 to 2.5 kg. Inspection of external ocular structures and ophthalmoscopy revealed no abnormalities in their eyes.
12
Cloxacillin in the Rabbit Eye Injection of antibiotic 3%-cloxacillin sodium (specific activity 37.6 pCilmg) and non-radioactive cloxacillin sodium (EkvacillinB) (both kindly supplied by Astra, Sodertalje, Sweden) were used. Each rabbit received intravascularly 1 ml/kg of an aqueous solution containing 50 mg/ml of cloxacillin sodium and a n amount of 25 pCi/ml of the labelled material. Injection of probenecid
Nine rabbits received 100 mg/kg of probenecid (ProbecidB) intraperitoneally 20 rnin prior to the antibiotic injection. Preparation and radioassay of plasma and ocular samples T o follow the antibiotic concentration in plasma, several blood samples were taken from all rabbits until the animal was killed by a rapid injection of pentobarbital sodium (NembutalB) at intervals of 5 to 240 min from the injection of antibiotic. Both eyes were promptly enucleated and frozen in liquid nitrogen. The frozen eyes were dissected on a salt-ice mixture. The cornea was cut 1 mm from the limbus. The frozen aqueous humour was separated into pieces. Then the eye was cut longitudinally into two equal halves which were dissected separately. The lens was easily separated from the frozen vitreous body. The aqueous humour, possibly contaminating the lens, was separated. Several specimens were taken from the vitreous body. The frozen iris and ciliary body were taken together as well as the retina and choroid. If low radioactivity was expected, the two halves of the i n s and ciliary body, as well as those of the retina and choroid, were united. The limbal area, 2 mm in width, was separated from the rest of the sclera. Possible blood contamination was observed, and the contaminated tissues were mechanically cleaned. If this failed, the specimen was rejected. The specimens were weighed in pre-weighed counting vials and digested with 0.1 ml of 6Oo/o perchloric acid and 0.2 ml of 3Oo/o hydrogen peroxide at 80°C for two h. Thereafter a counting solution containing 4.6 ml of ethylene glycerol monoethyl ether and 10.4 ml of toluol with 6 g/l 2,5-diphenyloxazole was added. Plasma samples were treated in the same way. Counting was performed with a Packard Tri-Card model 3375 liquid scintillation spectrometer. External standardisation was used for calculating the results in disintergrations per min (DPM). The reduction in radioactivity of sulphur-35 was observed. Mlcrobiologlcal assay of plasma In addition to radioassay, parallel microbiological assay of total antibiotic concentration in plasma of five rabbits killed at 180 to 240 min after the cloxacillin injection was undertaken as described by Jalling et al. (1972). Plasma samples of 10 ,ul were pipetted on a Ford 428 Mill filter paper and dried. Standard discs were prepared from appropriate dilutions of cloxacillin sodium in pooled rabbit plasma in the same way. The test organism, Sarcina lutea ATCC 9341, and the plates were prepared according to Grove & Randall (1955). The plates were prepared for one day’s use a t a time. They were allowed to dry for 30 min, after which the pre-pipetted discs were placed on the agar surface. At least three series of standard discs for each inoculated agar were used each time the assay was run. After a pre-diffusion time of 30 min, the plates were placed at +35OC for 12 h. The diameter of the inhibition zone was read
13
Lotta Salminen twice, and the mean of the two readings taken. Antibiotic concentration of the sample was obtained from the standard curve, which was linear to semilogarithmic plot within the range of concentration for standard discs. Statistics
The number of normal eyes studied for their cloxacillin concentration was 31. This number includes both eyes, with one exception, of 11 rabbits killed at 30, 60, 90, 120, 150, 180, 210 and 240 min respectively following the injection of antibiotic, as well as contralateral eyes of 10 rabbits which underwent an anterior chamber puncture before the antibiotic injection. The results of the punctured eyes will be presented in a later paper (Salminen 1977). The number of eyes obtained from rabbits with probenecid injection before cloxacillin injection was 18. They comprised both eyes of 9 rabbits killed at 30, 60, 90, 120, 180 and 210 min respectively following the antibiotic injection. From the normal material and from the eyes of the animals with probenecid injection, the mean values and their standard errors were calculated. Student's t-test was used in comparing the two materials. The correlation coefficient between plasma liquid scintillation counting method and antibiotic concentrations obtained by bioassay was determined (Hald 1952), and the significance of the correlation was tested.
)OO
100
,a0
,o 0
100 I00
100
TOO
'0 0
100
100
300
2:
508 40 a 30
;
30
20
20 I0
I3
05
05
821
i
0 3
I
,
02
O'
5 k 30 6c
&
Ic
140
I00
2:o
2:
MIULIrES
Fig. I B. Aqueous humour.
Fig. I A . Cornea. Fig. - 1.
Ocular (0,0 ) and plasma (curves) radioactivity and corresponding drug concentration after an intravenous injection of 3~S-cloxacillinsodium. Open circles (n = 2 to 6, mean 3.2) and lower curve are samples obtained from rabbits without, and black circles ( n = 2 to 6, mean 3.0) and upper curve are samples obtained from rabbits with a previous injection of probenecid (100 mg/kg) before the antibiotic injection. Vertical lines equal k SEM. The amount of 3~S-cloxacillinsodium injected was 25.0 pCilkg with 50 mg/kg of unlabelled drug. 14
Cloxacillin in the Rabbit Eye
500 40.0 30.0 20 0
200
200 10 0
10.0
10.0
* \ 30L
2.0
05
0.2
MINUTES
MINUTES
Fig. 1 D . Retina-choroid.
Fig. 1 C . Iris-ciliary body.
Results Radioactivity obtained by liquid scintillation method and cloxacillin concentration obtained by bioassay in plasma corresponded well with each other (correlation coefficient 0.94 1 ; P < 0.001). Radioactivity (disintegrations per min) found in a microbiologically determined concentration of 1 pg of cloxacillin sodium in plasma samples taken 180 to 240 min after the antibiotic injection was 1252 f 279. Radioactivity (DPM) added to 1 pg of unlabelled cloxa-
200 0
-1
1
1004: 400. 500: 300:
1200
105
0 5: 04. 0 3.
0"
0 I530
60
w
I20 I50 MINUTES
I I00
2;o
02O I Lo
24;
b,;
60
&
o1;
160
, I I
180 210
MINUTES
Fig. 1 F. Sclera.
Fig. 1 E. Limbal area.
15
240
Lotta Salminen
cillin sodium to make the injected solution was 1110. It seems probable, therefore, that the measured radioactivity gives reliable information about the ocular concentration of the studied antibiotic. Ocular and plasma radioactivity and corresponding drug concentration, with the exception of the lens, are summarized in Figs. 1-2. Radioactivity measured from the lens did not differ from that of the background. Cloxacillin concentration in plasma, after equal values at 5 and 15 rnin after the antibiotic injection, diminished more rapidly thereafter in animals with no previous injection of probenecid (P< 0.01). In animals with a probenecid injection before the antibiotic injection, statistically higher (P < 0.001 or P < 0.01) cloxacillin levels were measured in the cornea (30, 60, 90, 150 and 210 min), aqueous humour (30 and 60 min), iris-ciliary body and retina-choroid (30, 60 and 120 min) and in the sclera and limbal area (120 and 180 min).
Discussion In the present investigation the therapeutic level of 1.5 pg/g of cloxacillin was achieved in all vascularized ocular structures and in the cornea of the rabbit eye after an intravenous injection of 50 mg/kg of cloxacillin sodium. In the aqueous humour this level was only achieved in animals which had had a previous injection of probenecid prior to the antibiotic injection. Cloxacillin failed to penetrate into the lens and vitreous body to any remarkable degree.
- 0.10
f 0
0.05 ao4 0.03
o
\
o
cm
0
0.020.01
a
f 0
I
'
0
'
4
1
I
Radioactivity and corresponding drug concentration in rabbit vitreous body after an intravenous injection of 3%-cloxacillin sodium. Determinations were made from the same rabbits as presented in Fig. 1. See the legend to Fig. 1 for explanation. 16
Cloxacillin in the Rabbit Eye
The intravenous injection of 50 mg/kg of cloxacillin resulted in an extremely high plasma antibiotic concentration immediately after the antibiotic injection. In the course of the next 30 min, cloxacillin plasma concentration greatly diminished. This was due to the rapid diffusion of the drug into the extravascular tissue compartment (Dittert et al. 1969). Moreover the elimination began immediately after the intravenous injection. The huge intravascular cloxacillin concentration led to a rapid diffusion into the extravascular tissue compartment in the sclera and limbal area. As a sign of extravascular diffusion of cloxacillin into the sclera and limbal area, their antibiotic concentration reached plasma level at 60 min and was equal or superior to it during the next 60 min. In the iris-ciliary body and in the retina-choroid, circulating cloxacillin met capillary walls which appeared to have lower permeability. Unfortunately, retina and choroid were not examined separately and any difference in their capillary permeability cannot be assessed. Plasma cloxacillin concentration was never reached in the iris and ciliary body. In the aqueous humour cloxacillin reached its greatest concentration after 30 min. It is obvious that the high cloxacillin plasma concentration immediately after injection led to a rapid diffusion of the drug through the iris and ciliary body into the aqueous humour. Cloxacillin concentration in the aqueous humour was too low to cause any remarkable diffusion into the lens. There was a marked difference in cloxacillin concentration between the limbal area and the cornea during the first hour after the antibiotic injection. Drug diffusion from the limbal area into the cornea took place gradually. As a sign of the resistance of the vitreous barrier to drug penetration (Bleeker et al. 1968), cloxacillin concentration in the vitreous body was far below that in the aqueous humour. Administration of probenecid resulted in a significant increase of cloxacillin concentration in all ocular structures with the exception of the lens and vitreous body. Therapeutic concentration of cloxacillin in the aqueous humour, otherwise barely achieved, was more satisfactorily obtained by means of a previous injection of probenecid. Administration of probenecid caused a significant increase in cloxacillin plasma concentration. Drug diffusion into ocular tissues was enhanced by a greater concentration gradient and by a longer contact of the antibiotic with capillary walls. No increase in the ratio of ocular tissue cloxacillin concentration to that of plasma was noticed. This agrees well with the observation of Forbes & Becker (1960) that systemic administration of probenecid to nephrectomized animals failed to increase penetration of organic anions into the vitreous or aqueous humour. The total cloxacillin concentration obtained by radioactive tracer method, 17 Acta ophthal. 56, 1
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Lotta Salminen
however, gives incomplete information on the biological activity of the samples studied. The antibiotic activity is smaller than the total antibiotic activity would indicate (Bloome et al. 1970). This reduction of biological activity is caused by binding of the antibiotic to plasma and tissue proteins. T h e inactivation is for the great part reversible, being influenced by local circumstances. and not measurable in vitro.
References Allen M. F. & Mangiaracine A. B. (1964) Bacterial endophthalmitis after cataract extraction. Arch. Ophthal. (Chicago) 72, 454-462. Bleeker G. M., van Haeringen N. J., Maas E. R. & Glasius E. (1968) Selective properties of the vitreous barrier. Exp. Eye Res. 7, 37-46. Bloome M. A., Golden B. & MacKee A. D. (1970) Antibiotic concentration in ocular tissues. Arch. Ophthal. (Chicago) 83, 78-83. Dittert L. W., Griffen W . O., LaPiana J. C., Shainfeld F. J. & Doluisio J. T. (1969) Pharmacokinetic interpretation of penicillin levels in serum and urine after intravenous administration. In: Hobby G. L., Ed. Antimicrobial agents in chemotherapy, pp. 42-48. American Society for Microbiology, Bethesda, Maryland. Faris B. M. & Uwaydah M. M. (1974) Intraocular penetration of semisynthetic penicillins. Methicillin, cloxacillin, ampicillin and carbenicillin studies in experimental animals with a review of the literature. Arch. Ophthal. (Chicago) 92, 501-505. Forber M. & Becker B. (1960) The transport of organic anions by the rabbit eye. 11. In vivo transport of iodopyracet (Diodrast). Amer. /. Ophthal. 50, 197-203. Furgiuele F. P. (1964) New antibiotics: Their intraocular penetration. Amer. /. Ophthal. 58, 443-446. Grove D. C . & Randall W . A. (1955) Assay methods of antibiotics. A laboratory manual. Medical Encyclopedia, Inc., New York. Hald A. (1952) Statistical theory witfa engineering applications. John Wiley and Sons, London. Jalling B., Malmborg A,-S., Lindman A. & Bortus L. 0. (1972) Evaluation of a micromethod for determination of antibiotic concentration in plasma. EZlro/J. /. d i n . Pharmacol. 4 , 150-158. Locatcher-Khorazo D. & Gutierrez E. (1972) Postoperative infection of the eye. In: Locatcher-Khorazo D. and Seegal B. C., Eds. Microbiology of the Eye, pp. 77-85, Mosby, St. Louis. Meyer K., Matz D., Uterman D. & Fedder J. (1973) Ober antibakterielle Wirkstoffkonzentrationen im Kammerwasser des Menschen nach parenteraler Applikation verschiedener Antibiotica. Albrecftt v . Graeies Arch. klin. exp. Ophthal. 185, 55-70. Nayler H. C., Long A. A. W., Brown D. M., Acred P., Rolinson G. N., Batchelor F. R., Stevens S. Sutherland R. (1962) Chemistry, toxicology, pharmacology and microbiology of a new acid-stable penicillin, resistant to penicillinase (BRL. 1621). Nature (Lond.) 195, 1264-1267. Records R. E. (1967) Human intraocular penetration of sodium oxacillin. Arch. Ophthal. (Chicago) 77, 693-695.
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Cloxacillin in the Rabbit Eye Records R. E. (196s) Intraocular penetration of dicloxacillin. Invest. Olhthal. 7, 663-667. Records R. E. & Ellis P. P. (1967) T h e intraocular penetration of ampicillin, methicillin, and oxacillin. Amer. /. Ophtlzal. 64, 135-143. Salminen L. (1978) Effect of paracentesis on ocular cloxacillin concentration. Acta ophthal. (Kbh.) 56, 20-26. Uwaydah M. M., Faris B. M., Smara I. N., Shammas H. F. & To’Mey K. F. (1976) Cloxacillin penetration. Amer. /. O/?ldzal. 82, 114-1 16.
Atittior’s address:
Lotta Salminen, M. D., Turku University Central Hospita I, Department of Ophthalmology, SF-20520 Turku 52, Finland.
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