AQUEOUS HUMOR PENETRATION OF ALPHA-DEOXYOXYTETRA-CYCLINE (DOXYCYCLINE) IN MAN

U L F KRAUSE, M.D., VEIJO RAUNIO, M.D., AND EILA MUSTONEN, M.D. Oulu, Finland

Doxycycline, or α-deoxyoxytetracycline, is a modified tetracycline which was introduced in 1966.1 It is synthesized by hydrogénation of methacycline. The spectrum of activity of doxycycline is similar to that of the tetracy-clines.2 Doxycycline differs from the other tetracyclines in that it has an augmented in­testinal absorbtion and a prolonged serum half-life, reaching 18.5 to 22.2 hours after ingestion of 200 mg of doxycycline.1 The maximum serum concentration of 3.06 μg/ ml (average) occurs between 2.25 and nine hours. The protein binding of doxycycline in the serum varies between 25 and ΖΙψο? which is similar in magnitude to that of the other tetracyclines. On the whole, the pro­tein binding is greater when the serum levels of doxycycline are higher.1 A serum protein-binding of 82 and even 92% has been re­ported.3·4

The distribution of the antibiotics between tissue depots and interstitial fluid correlates well with the partition coefficient between chloroform and water,5 a measure of the lipid solubilities of compounds. Doxycycline has a distribution coefficient between chloroform and aqueous buffer6: 475 X 103. Because it is lipophilic, doxycycline reaches a high con­centration in the tissues where it acts.

The purpose of our work is to investigate whether therapeutic levels of doxycycline can be achieved in aqueous humor in man, and whether doxycycline concentration is as­sociated with the aqueous protein concentra­tion in these eyes.

MATERIALS AND METHODS

A single oral dose of 200 mg of doxycy­cline (Vibramycin) was given to 10 patients

From the University Eye Hospital (Drs. Krause and Mustonen) and the State Public Health Labora­tory (Dr. Raunio), Oulu, Finland.

Reprint requests to Ulf Krause, M.D., the Uni­versity Eye Hospital, Uusikatu 50, Oulu, Finland.

on the first day of the study. On the succeed­ing day, a 100 mg dose of doxycycline was given to these same patients and about two hours later a sample of the aqueous humor was withdrawn from the anterior chamber. Because this series was only a part of an­other investigation, the sample was collected in a teflon tube some 2.5 m long with an inte­rior diameter of 0.5 mm, attached to the puncture needle of a paracentesis gun.7 A sample of blood from the cubital vein of the patients was withdrawn at the same time. The samples of both the aqueous humor and the serum were stored at a temperature of —40° C so that future investigations could be performed at the same time.

Two patients were without any sign of definite ocular inflammation, but had intra­ocular melanomas. Eight patients had signs of severe acute iridocyclitis. These eight pa­tients were also given the usual local therapy for iridocyclitis (i.e., instillation of cortico-steroids and mydriatics), as the inflammatory process was so advanced that the postpone­ment of therapy until after paracentesis could not be considered. Some of these pa­tients were also given systemic corticoste-roids, but no other antibiotics or medicines were given.

ASSAY PROCEDURE

All serum samples were first studied by the serial dilution method in tubes to get a rough estimation of doxycycline concentra­tions. The final assay was made by the plate method. In the case of aqueous humor, the screening method was omitted because of the scarcity of samples and assays were per­formed directly by the plate method.

A purified doxycycline preparation was used as a standard. The assay procedure was performed according to the instructions given by the Pfizer Laboratories in Sandwich, En­gland, as follows :

77

78 AMERICAN JOURNAL OF OPHTHALMOLOGY JULY, 1972

TABLE 1 CONCENTRATION OF DOXYCYCLINE IN SERUM AND AQUEOUS HUMOR AND TOTAL

PROTEIN CONTENT OF AQUEOUS HUMOR IN 1 0 PATIENTS STUDIED

Case No., Sex, and Age (in years)

1. F-51 2. F-65 3. M-57 4. M-36 S. M-56 6. M-45 7. M-36 8. M-30 9. M-48

10. M-36

Diagnosis

Choroidal melanoma Choroidal melanoma Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis Acute iridocyclitis

Average in patients with melanoma in patients with iridocyclitis

Hours after Last

Dose

2.3 2.6 2.2 2.6 2 .4 2.3 2.1 2.5 2 .3 2.4

Doxycycline Concen­tration

Serum

5.40 7.68 3.36 3.60 5.40 2.70 2.04 1.71 1.74 1.35 6.54 2.74

G*g/ml)

Aqueous Humor

0.27 0.51 0.92 0.74 1.44 1.08 0.51 0.68 0.47 0.69 0.39 0.82

Protein Content of

Aqueous Humor

(mg/100 ml)

73 101 231 676

1,072 1,163 1,193 1,316 1,438 1,508

Each 0.15 ml sample of aqueous humor was serially diluted, 1:3 (ΤΊ) and 1:9 (T 2 ) , in a phosphate buffer of pH 4.5. Similarly, the doxycycline standards were made up in a phosphate buffer of pH 4.5 to give two stan­dards of 0.33 (Si) and 0.11 (S2) μg/ml. The test dilutions Ti and T2 from two specimens, and the standards Si and S2 were replicated in a random manner on a large plate having 6x6 punch-holes (6 mm in diameter). Hence, providing there was sufficient volume of ΤΊ and T2, they, along with Si and S2 were filled with 120 ml of antibiotic medium No. 1 (Difco) seeded with bacillus cereus NCIB 8849. After overnight incubation at 28° C, the zone of inhibition was measured and the concentration of doxycycline in the test sam­ples was computed.

The total protein concentration of the samples of aqueous humor was determined by precision spectrophotometry using Folin-Ciocalteau's reagent.8-9 Table 1 shows the concentration of doxycycline in both the se­rum and the aqueous humor, and the total protein content of the aqueous humor of 10 patients.

DISCUSSION

Our work confirms others' observations10

concerning doxycycline penetration in the aqueous humor in man. Several factors affect the penetration of antibiotics into the aqueous humor, such as liposolubility of the drug,5

the state of the blood-aqueous barrier,4 and plasma concentration. We adjusted the dosage so that the antibiotic concentration in the sera of all patients reached therapeutic levels. The average minimum inhibitory concentration (MIC) of doxycycline is 0.5 μg/ml, deter­mined for clinical pathogens such as pneu-mococci, group A streptococci, Haemofilus influenzae, susceptible staphylococci, and neisseria species. Doxycycline concentrations of 0.5 to 1.5 μg/ml in vivo are considered therapeutic.11 The two patients with choroidal melanoma had a definitely higher serum con­centration than patients in the iridocyclitis group. We are not yet able to offer any ex­planation for this finding.

On the other hand, doxycycline concentra­tion in the aqueous humor of the patients with melanoma was in one case well below the MIC and in the other case at the level of MIC, whereas in the iridocyclitis group doxy­cycline concentration exceeded the MIC except in Case 9, when it was a little below that level. Thus, when all 10 patients are considered, an association between serum

VOL. 74, NO. 1 DOXYCYCLINE PENETRATION 79

and aqueous humor concentration is ob­served. It is known that the blood-aqueous barrier is not the same in these two patient groups and this perhaps explains the differ­ence in passage of doxycycline into the ante­rior chamber. Because there were only two patients with melanoma these findings can only be suggestive. If the doxycycline concentration is examined only in patients with iridocyclitis, here, too, the data suggest a rough correlation between serum and aque­ous humor antibiotic level. These findings are compatible with what is known about in­creased permeability of the blood-aqueous barrier in inflammation: the antibiotics reach a higher concentration in inflamed than in normal eyes. This has been shown in animal experiments,12"16 as well as in man.16"19

Since doxycycline has a marked affinity for the plasma proteins, a high concentration of plasma protein in the anterior chamber is likely to parallel high doxycycline concentra­tion. However, it appears that there is no linear relation between protein and doxy­cycline concentrations in the aqueous humor of patients with iridocyclitis (Table 1). Con­versely, comparing patients with choroidal melanomas and those with iridocyclitis, the data indicate an association in protein and doxycycline concentrations.

One possible explanation for this is that a relationship between the protein content and doxycycline concentration may exist at protein levels lower than those found in the eyes with iridocyclitis. The other factor which perhaps plays a role in the iridocycli­tis group is the administration of corticoste-roids. The capillary permeability in the nor­mal eye is not influenced by corticosteroids, but they do reduce the increased capillary permeability associated with inflammation.20

We must consider this type of interference in our patients, too, as they received intensive corticosteroid therapy. The protein and doxycycline concentrations in aqueous humor could have been higher and less scattered without corticosteroid therapy. The few cases studied does not allow a meaningful

statistical analysis and thus these results and conclusions are presented only as suggestions.

SUMMARY

Patients with iridocyclitis and two with choroidal melanoma each received a total of 300 mg of doxycycline divided into two doses (200 and 100 mg). About two hours after the last dose, the doxycycline concen­tration in the aqueous humor was deter­mined. The iridocyclitis group had an aver­age level of 0.82 μg/ml, which is well within the therapeutic range. Below this range, an average of 0.39 μg/ml was found in the two patients with melanoma. The results also in­dicate an association between protein and doxycycline concentration in the aqueous hu­mor. However, it was not possible to demon­strate the expected linear relationship, since this may occur at lower protein levels than those found in the eyes with iridocyclitis.

ACKNOWLEDGMENT We thank Mrs. Leena Marjamaa and Mrs. Ritva

Savilaakso for their technical assistance in the labo­ratory work. We also thank Pfizer Laboratories, who supplied the doxycycline used in this study as Vibramycin.

REFERENCES 1. Fabre, J., Pitton, J. S., and Kunz, J. P. : Dis­

tribution and excretion of doxycycline in man. Che-motherapia 11:73, 1966.

2. Dimmling, T. : Bakteriologische Untersuchun­gen über Resorption und Excretion sowie über das Wirkungsspectrum von Alpha-6-Desoxyoxytetra-cyclin (Doxycyclin). Med. Klin. 62:1269, 1967.

3. Schach von Wittenau, M. : Some pharmacoki-netic aspects of doxycycline metabolism in man. Chemotherapy (Suppl.) 13:41, 1968.

4. Williamson, G. M. : The implications of the protein binding; of the tetracyclines. Proc. Int. Cong. Chemotherap. 4:207, 1967.

5. Langham, M. : Factors affecting the penetra­tion of antibiotics into the aqueous humour. Brit. J. Ophth. 35:614, 1951.

6. Schach von Wittenau, M., and Yeary, R. : The excretion and distribution in body fluids of tetracy­clines after intravenous administration to dogs. J. Pharmacol. Exp. Therap. 140:258, 1963.

7. Krause, U., and Raunio, V.: Protein content of normal human aqueous humour in vivo. Acta Ophth. 47:215,1969.

8. Ewing, G. : Instrumental Methods of Chemical Analysis. New York, McGraw-Hill, 1960, p. 57.

9. Krause, U., and Raunio, V. : Proteins of nor-

80 AMERICAN JOURNAL OF OPHTHALMOLOGY JULY, 1972

mal human aqueous humour. Ophthalmologica 159: 178, 1969.

10. Tsacopoulos, M. : The penetration of Vibra-maycin (doxycycline) in human aqueous humor. Ophthalmologica 159:418, 1969.

11. Walter, A., and Heilmeyer, L. : Antibiotika-Fibel. Stuttgart, Thieme, 1969, pp. 294, 270, 264.

12. Records, R. E.: Intraocular penetration of cephalotin. Am. J. Ophth. 66:436, 1968.

13. Green, W. R., and Leopold, I. H. : Intraocular penetration of methicillin. Am. J. Ophth. 60:800, 1965.

14. Furgiuele, F. P. : Ocular penetration and tol-erans of gentamicin. Am. J. Ophth. 64:421, 1967.

15. Leopold, I. H., Nichols, A. C, and Vogel, S. W. : Penetration of chloramphenicol USP

(Chloromycetin) into the eye. Arch. Ophth. 44:22, 1950.

16. Furgiuele, F. P., Sery, T. W., and Leopold, I. H. : Newer antibiotics : Their intraocular penetra­tion. Am. J. Ophth. 50:614, 1960.

17. Kurose, Y., and Leopold, I. H. : Intraocular penetration of ampicillin. Arch. Ophth. 73:361, 1965.

18. Green, W. R., Bennet, J. E., and Goos, R. D. : Ocular penetration of amphotericin B. Arch. Ophth. 73:769,1965.

19. Records, R. E. : The human intraocular pene­tration of methicillin. Arch. Ophth. 76:720, 1966.

20. Cook, C, and MacDonald, R. K. : Effect of cortisone on the permeability of the blood-aqueous barrier to fluorescein. Brit. J. Ophth. 35 :730, 1951.

OPHTHALMIC MINIATURE

Fallacies which beg the question (petitio principii)—circulus in pro-bando (circular reasoning) : In a sense, all of science and logic involve reasoning which begins with basic assumptions which cannot be proven except by proceeding to other conclusions which are based on those as­sumed in the beginning. Thus, we accept the reality of the external world, that the universe is orderly, etc. Science and logic, especially mathematics, have built bodies of knowledge which show internal con­sistency. Ordinary circularity begins with assumptions which are not self-evident. For example, "Swedish instruments are the finest in the world." "How do you know this ?" "Because they are made of Swedish steel."

"Well, this turn goes up the hill, I suppose—no, it doesn't ! This goes straight back to the house! Well then, I'll try it the other way." And so she did, wandering up and down, and taking turn after turn, but always coming back to the house, do what she would.

Dwight J. Ingle Fallacies and errors in the Wonderlands of Biology, Medicine, and Lewis Carroll

Perspectives in Biology and Medicine 15:273, 1972