- Email: [email protected]
Ocular Penetration of Hetrazan in Rabbits
V O L . 66. N O . 2
RETICULUM
CELL SARCOMA
Mikroglia. Arch. Psychiat. Nervenkr. 206 r662, 1965. 15. Miller, A . A . and Ramsden, F. : Primary reticulosis of the central nervous system. Acta Neurochir. 11:439,1963. 16. Yuile, C. L. : Case of primary reticulum cell sarcoma of the brain. Arch. Path. 26:1036, 1938. 17. Del Rio-Hortega, P. : The Microscopic Anatomy of Tumors of the Central and Peripheral Nervous System. Springfield, Thomas, 1962, p. 27. 18. Kinney, T. D. and Adams, R D. : Reticulum cell sarcoma of the brain. Arch. Neurol. Psychiat. 50:552, 1943. 19. Abbot, K. H . and Kernohan, J. W . : Primary sarcomas of the brain. Arch. Neurol. Psychiat 50:43, 1943.
215
20. Dougherty, T. F. : Studies on the cytogenesis of microglia and their relation to cells of the reticulo-endothelial system. Am. J. Anat 74:61, 1944. 21. Russell, D. S. and Rubinstein, L. J. : Pathology of Tumors of the Nervous System. Baltimore, Williams and Wilkins, 1963, p. 66. 22. Albites, V . E. : Primary reticulum-cell sarcoma of the brain. J. Neurosurg. 20:777, 1963. 23. Onofrio, B. M , Kernohan, J. W . and Uihlein, A : Primary meningeal Sarcomatosis. Cancer 15:1197,1962. 24. Hogan, M . J , Spencer, W . H . and Hoyt, W . F. : Primary reticuloendothelial sarcomas of the orbital and cranial meninges : Ophthalmologic aspects. Am. J. Ophth. 61:1146, 1966.
O C U L A R P E N E T R A T I O N O F H E T R A Z A N IN R A B B I T S MOSHE LAZAR, M . D , * THEODORE W . LIEBERMAN, M . D . , MOSES F U R M A N , M . S . AND IRVING H .
LEOPOLD, M . D .
New York Hetrazan ( l-diethylcarbamyl-4-methylpip- t i o n ' ' ' may make initial therapy intolerable erazine) (fig. 1) is an antifilarial agent intro- and subsequent treatment undesirable to the duced by Hewitt and co-workers in 1947. patient.* The drug is used in the treatment of the Since the greatest morbidity of the disease round-worm infection, onchocerciasis, a prev- is due to ocular anterior segment infestation alent cause of ocular morbidity in Africa, with microfilariae, * and since the major Mexico, Guatemala and Venezuela. As an oral route of the microfilariae into the eye appreparation, Hetrazan has proven to be highly pears to be through the conjunctiva,*' it was effective in transiently reducing the number considered that an ophthalmic preparation of of microfilariae found in skin biopsies or seen Hetrazan might be a useful adjunct to the in the aqueous humor of individuals harbor- present therapeutic armamentarium against ing the worm, Onchocerca volvulus. ' The onchocerciasis. This study, a first step in the drug is customarily administered daily over a development of such a preparation, was unseveral-week period. After a course of treat- dertaken to determine the penetration of Hement the microfilariae are practically nonex- trazan into the eye after its ocular adminisistent in skin-snip biopsies, but after several tration. months begin to increase in number due to MATERIALS AND METHODS the ineffectiveness of Hetrazan in eradicatHetrazan dihydrogen citrate (HetDC) ing the adult worms. Consequently, repeated powder}" was used in all experiments. Solutreatments over many years are required to tions were prepared on a weight-to-volume control the disease. Transient pruritis, basis of HetDC to distilled water. The pH edema, dermatitis, arthralgias, conjunctiviwas adjusted to 7.0 with concentrated soties and fever during Hetrazan administradium hydroxide. One hundred and twenty albino and pigFrom the Department of Ophthalmology, Mount mented rabbits weighting from 3 to 4 kg Sinai School of Medicine, the City University of 2
4
1
5
6
7
z z
New York. This investigation was supported by U S P H S Grant N B 06579 from the National Institute of Neurological Diseases and Blindness. * O n leave from Tel-Hashomer Hospital, Israel.
t Contributed by Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York.
216
AMERICAN JOURNAL OF OPHTHALMOLOGY
C H — 2
Fig. 1 (Lazar, Lieberman, Furman and Leopold). Molecular structure of Hetrazan base, 1-diethylcarbamyl-4-methylpiperazine.
A U G U S T , 1968
0 II
- C - N
CH -N 3
C H — 2
were used to study the aqueous humor, vitreous humor and serum concentrations of Hetrazan after topical, subconjunctival and intravenous administration of the drug. Each rabbit was used for a single aqueous and vitreous Hetrazan concentration determination. Topical instillations were made to both eyes of alert animals without anesthesia. The lids were held to prevent blinking for 30 seconds after administration. Subconjunctival injections were given to both eyes of animals anesthetized with intravenous pentobarbital. Intravenous injections of Hetrazan were given in the marginal ear vein after pentobarbital anesthesia. Anterior chamber taps were performed on anesthetized animals. The eyes were washed thoroughly with distilled water before the anterior chambers were entered. Aqueous humor was aspirated with a 26-gauge needle attached to a tuberculin syringe. The aqueous humor from both eyes of a single animal was pooled for Hetrazan determinations. Vitreous humor was aspirated with a 20-gauge needle attached to a 6.0 cc syringe immediately after the aqueous humor tap. Blood samples were obtained by heart puncture. All samples were analyzed for drug concentration within two hours after collection. Hetrazan in the aqueous, vitreous and serum was determined by a modification of the method of Lubran. In the technique used, Hetrazan forms a colored salt with the dye bromthymol blue. The colored salt is extracted into benzene in which bromthymol blue itself is insoluble. The concentration of
/ \
2 5 n
C2 H
5
Hetrazan in the benzene layer is determined spectrophotometrically against a standard curve. The exact technique is as follows : T o 0.5 ml of aqueous or vitreous humor, 0.5 ml of 4.0 N sodium hydroxide and 2.0 ml of benzene* were added and vigorously shaken for 15 seconds with a vortex mixer. The benzene layer was separated by centrifugation. T o an aliquot of the benzene layer 1.0 ml of a 0.05% bromthymol blue solutionf was added. The mixture was shaken for 15 seconds with a vortex mixer and then centrif uged 10 minutes to separate the benzene layer. The optical density of the benzene layer was measured at 400 mu in a Beckman DB spectrophotometer. A standard curve was prepared using the above procedure and known amounts of Hetrazan. Through preliminary determinations of the range of Hetrazan concentrations to be expected during the experiment, the unknown samples were adjusted to read on the straight-line portion of the standard curve. For Hetrazan determinations in the serum 1.0 ml of serum and double the amounts of the reagents given above were used. When blank samples of aqueous humor, vitreous humor and serum were run through the above procedure and compared to distilled water, they gave optical density readings corresponding to concentrations of less than 0.5 u-g of Hetrazan base per ml.
8
•Certified benzene, 99 Mol % pure, thiophenefree (Fisher Scientific Company). t Bromthymol blue (Fisher Scientific Company) was made up in 0.1 M phosphate buffer (pH 7.0) and washed twice with analytical grade benzene.
VOL.
OCULAR PENETRATION O F HETRAZAN
66. N O . 2
217
RESULTS
Each value given in the results is the average of five or more separate determinations.
10-
O--0
serum aqueous humor
A--0
vitreous humor
TOPICAL APPLICATION
After a single topical administration of two drops (approximately 0.12 ml) of 5% HetDC (2.5% Hetrazan base) the ocular penetration of Hetrazan was rapid (9.5 |ig of Hetrazan base/ml aqueous humor at onehalf hour). Within four hours the aqueous humor concentration had fallen to less than 0.5 jig base/ml (fig. 2 ) . No Hetrazan could be detected in the vitreous humor. With a single instillation of two drops of a 1 % solution of HetDC (0.5% base), 2 jig base/ml aqueous humor were found at one hour. This compared to 5 (tg base/ml aqueous humor found at one hour after the instillation of two drops of the 5% HetDC solution. Animals were treated with repeated topical doses of Hetrazan to determine if there was any accumulation of the drug in the aqueous humor. The animals received two drops of 5% HetDC every three hours four times daily. The anterior chambers were tapped in one group of animals four hours after the second dose on the fourth day of treatment and in another group of animals four hours after the first dose on the fifth day of treat-
p
1
1
0
1/2
I
2
t
TINE (hours)
Fig. 3 (Lazar, Lieberman, Furman and Leopold). Changes in serum, aqueous humor and vitreous humor concentrations of Hetrazan base after a single intravenous injection of 20 jig Hetrazan dihydrogen citrateAg body weight The sign f indicates time of injection.
ment. A s in the single dose experiment, no Hetrazan within experimental limits (less than 0.5 |ig Hetrazan base/ml aqueous) could be detected four hours after topical dose. There was no evidence of ocular irritation in any animal receiving topical solutions. SUBCONJUNCTIVAL INJECTIONS
One hour after the subconjunctival administration of 0.1 ml of 5% HetDC, 11 (ig Hetrazan base/ml aqueous humor was detected. After two hours the concentration dropped to 3.5 u,g Hetrazan base/ml aqueous humor. At four hours, less than 1 u,g Hetrazan base/ml aqueous humor was found. No Hetrazan was detected in the vitreous humor at any time and there was no evidence of conjunctival irritation.
S
1
I2 TIBE
(NOMS)
Fig. 2 (Lazar, Lieberman, Furman and Leopold). Changes in aqueous humor concentration of Hetrazan base with time after single topical instillation of 2 drops of 5% Hetrazan dihydrogen citrate.
INTRAVENOUS ADMINISTRATION
One hour after a single intravenous dose of 5 |ig HetDC/kg bodyweight or 10 (ig HetDC/kg the drug could not be detected in the aqueous or vitreous humors. After a single
218
AMERICAN JOURNAL OF OPHTHALMOLOGY
t serum
o aqueous
a vitreous
A
B
5-
^
A U G U S T , 1968
5432I
2Ii—i
0 15 30
t »t
r 60
A »
i—i—i 0 15 30 TINE (minutes)
r 60
t t t
Fig. 4 (Lazar, Lieberman, Furman and Leopold). Serum, aqueous humor and vitreous humor concentrations of Hetrazen base following the repeated intravenous administration of 10 ug Hetrazan dihydrogen citrateAg body weight (Graph A ) and S ug Hetrazan dihydrogen citrate (Graph B ) . The sign, f, indicates time of injections.
20 v-g/kg dose of HetDC, serum concentrations fell rapidly. Detectable quantities were found one hour after injection in both the aqueous and vitreous humors and fell over the next hour to low concentrations (fig. 3 ) . Repeated intravenous injections of large doses of Hetrazan (fig. 4 ) do not give as high aqueous humor concentration at one hour as found one hour after the topical administration of 5% HetDC. However, as after the single 20 |*g/kg intravenous dose, the drug was found within the vitreous humor. DISCUSSION
The disease is transmitted by various species of the black fly, Simulium, infected with onchocerca larvae. The fly deposits the larvae in the human subcutaneous tissues where they grow into adult worms, frequently forming skin nodules. The female worms deposit the microfilariae which migrate throughout the subcutaneous tissues of the body, but are not found in the bloodstream. The microfilariae penetrate the conjunctiva and apparently enter the eye in the area of the corneoscleral limbus. In other parts of the globe they have been found in the superficial sclera, but the deep portions of the sclera appear inpenetrable. They spread into the subepithelial cornea, the superficial stroma, the iris, ciliary body, choroid and lens. Optic atrophy is frequent but its pathogenesis is incompletely understood. It is considered most often to be of the consecutive type. Microfilariae can be seen in the aqueous humor, even in eyes without clinical 6
6
Onchocerciasis is a disease which affects large segments of the population living in endemic areas. The true extent of the disease is unknown but the magnitude of the problem can be appreciated by the estimates of 10 million" and 38 million affected Africans. 10
11
VOL. 56, NO. 2
219
OCULAR PENETRATION OF HETRAZAN TABLE 1
PERCENT DISTRIBUTION OF OCULAR LESIONS I N PATIENTS, 40 YEARS OF AGE AND OLDER, WITH PROVEN ONCHOCERCIASIS*
West Africa Plains No. of subjects Diminished visual acuityt Punctate keratitis . Type I Type II Type III Type IV Sclerosing keratitis Chronic iritis Iris atrophy Chorioretinal lesions § Stage I Stage II Stage III Optic atrophy 4
Forest
349
23.6% 7.0% 14.6% 14.6% 1.0% 16.6% 11.6% 33.2% 7.1% 8.2% 6.7% 18.0%
15.7% 5.2% 8.5%
— — 5.3% 2.1%
4.2% 7.7% 5.5% 2.2% 3.3%
Guatemala 125 12.2% 30.1% 8.2%
—
1.4% 41.8% 14.4% 28.7% 6.6% 8.2% 5.7% 1.8%
* Modified from Monjusiau, A.G.M, Lagraulet, J. and Gockel, C. W.: Ref. Ne. 12. Proven by positive biopsies, subcutaneous nodules and/or anterior chamber microfilariae, t Visual acuity equal to or worse than 20/200 in one or both eyes. t Type I: 0.5-1 mmfluffysubepithelial opacities. Type II: 1 mm subepithelial white dots with surrounding halos. Type III: Irregular punctate opacities. Type IV: Small discoid lesions, i Stage I: Irregular pigmentation. Stage II: Pigment migration. Stage III: Depigmentation and "sclerotic" choroidal vessels.
lesions." It has been hypothesized that the inflammatory reaction is caused by dying or dead microfilariae. * Table 1 shows the distribution of ocular lesions in individuals 40 years of age or older with proven onchocerciasis. Since the incidence of ocular onchocerciasis is known to increase with age, the figures given in Table 1 are high. Blindness in endemic areas may reach as high as 15%* to 30%. Keratitis and iritis are responsible for the major loss of vision.* Onchocerciasis is often termed river blindness because it is prevalent in the vicinity of rapidly flowing rivers, the breeding area of Simulium flies. Attempts at decreasing the population density of the vector Simulium have met with limited success. Two different methods have been used to destroy the adult worms. Individuals have been denodulated surgically.*' However, not all worms are found within nodules and as a massive therapeutic approach, this method is too timeconsuming. Suramin, a urea derivative, is toxic to the adult worm. • Unfortunately, solutions of the drug are extremely unstable, 1
5
5
5
3
5
must be administered intravenously, and can be nephrotoxic. Renal function should be followed over a five- to eight-week period while the drug is being used. The above properties of Suramin are not favorable for massive chemotherapy. Hetrazan has been therapeutically the most successful drug because of its low toxicity. As previously mentioned, the reactions during Hetrazan administration produce a great deal of discomfort. These side-effects are thought to be due to tissue reaction to dead microfilariae, ' * and are so frequent that their occurrence after the administration of Hetrazan has been used as evidence of onchocerciasis (Mazzotti test).* These reactions can be diminished with concomitant use of systemic corticosteroids or antihistamines. The oral dose of Hetrazan used in the treatment of onchocerciasis is 2 to 3 mg/kg of body weight three times daily. The use of similar quantities in normal volunteers over afive-dayperiod has been found to result in maximum serum concentrations of 3 to 5 jxg 5
5 1
8
220
AMERICAN JOURNAL OF OPHTHALMOLOGY
Hetrazan base/ml after the second day of treatment. At this blood level adequate amounts of Hetrazan would appear clinically to reach the eye, as evidenced by transient worsening of the conjunctivities and iritis and by disappearance of microfilariae from the eyes of infected subjects. In this study, serum levels of Hetrazan comparable to those found in human subjects, could not sustain with a single intravenous dose, probably because of rapid renal excretion. However, similar serum levels were sustained for brief periods by repeated intravenous injections, resulting in higher aqueous humor concentrations than after single intravenous doses. Aqueous humor concentrations in excess of those achieved by the intravenous route were found after topical or subconjunctival administration and would appear adequate for antifilarial activity. 8
8
An ophthalmic preparation of Hetrazan may prove valuable in treating patients refusing systemic therapy, and also as prophylaxis when administered intermittently. By keeping the microfilarial count low within the conjunctiva and subconjunctival tissues of the lid, it may be possible to decrease the number of microfilariae entering the eye. W e suggest that if field trials are initiated, very low concentrations of the topical drug be used at first. Topical corticosteroids could be administered to control excessive reaction to dying microfilariae. No conjunctival irritation was noted during any of the present experiments and there was no apparent discomfort to the animals. However, further toxicity studies will have to be pursued before human evaluation is attempted. 8
SUMMARY
Hetrazan, a drug used orally in the treatment of onchocerciasis, has been demon-
A U G U S T , 1968
strated in high aqueous humor concentrations in rabbit eyes after topical instillation. Possible benefits of an ophthalmic preparation of this drug in treating and preventing onchocerciasis are discussed. Fifth Avenue and 100th Street (10029) ACKNOWLEDGMENT
W e want to thank Drs. S. A. Szumski, R. G. Kelly and R. Hewitt of Lederle Laboratories Division, American Cyanimid Company for their technical advice and cooperation during this experiment. REFERENCES 1. Hewitt, R. I , Kushner, S , Stewart, H. W , White, E., Wallace, W . S. and SubbaRow, Y . : Experimental chemotherapy of Filariasis. III. Effect of 1 -diethyl-carbamy 1-4-methylpiperazine hydrochloride against naturally acquired filarial infections in cotton rats and dogs. J. Lab. Clin. Med. 32:1314, 1947. 2. Expert Committee on Onchocerciasis, First Report, W H O Techn. Rep. Ser, No. 87,1954. 3. Nnochiri, E. : Observations on the treatment of onchoceriasis in an endemic area of Western Nigeria. Tr. Roy. Soc. Trop. Med. Hyg. 58:413, 1964. 4. Lazar, M . : Personal observation made at Governmental Eye Clinic, Monrovia, Liberia, 1964-1966. 5. Onchoceriasis: Epidemiology and control, W H O Chron. 20:378, 1966. 6. Rodger, F. C. : The pathogenesis and pathology of ocular onchocerciasis. Am. J. Ophth. 49:327, 1960. 7. Neumann, E. and Gunders, A. E. : Ocular lesions of onchocerciasis in Liberia. Am. J. Ophth. 56:573, 1963. 8. Lubran, M . : Determination of Hetrazan in biological fluids. Brit. J. Pharmacol. 5:210, 1950. 9. Stoll, N. R . : This wormy world. J. Parasit. 33 :1, 1947. 10. Rodger, F. C. : The pathogenesis and pathology of ocular onchoceriasis. Am. J. Ophth. 49:104, 1960. 11. McLean, C. M . : Ocular onchoceriasis in Northern Ghana: A treatment survey. Brit. J. Ophth. 43:477, 1959. 12. Monjusiau, A . G. M , Lagraulet, J , d'Haussy, R. and Göckel, C. W . : Aspects of ophtalmologiques de l'onchocercose au Guatemala et en Afrique occidentale, Bull. W H O 32:339,1965. 13. Rodger, F. C. : A review of recent advances in scientific knowledge of the symptomatology, pathology and pathogenesis of onchocercal infections. Bull. W H O 27 :429, 1962.
RETICULUM
CELL SARCOMA
Mikroglia. Arch. Psychiat. Nervenkr. 206 r662, 1965. 15. Miller, A . A . and Ramsden, F. : Primary reticulosis of the central nervous system. Acta Neurochir. 11:439,1963. 16. Yuile, C. L. : Case of primary reticulum cell sarcoma of the brain. Arch. Path. 26:1036, 1938. 17. Del Rio-Hortega, P. : The Microscopic Anatomy of Tumors of the Central and Peripheral Nervous System. Springfield, Thomas, 1962, p. 27. 18. Kinney, T. D. and Adams, R D. : Reticulum cell sarcoma of the brain. Arch. Neurol. Psychiat. 50:552, 1943. 19. Abbot, K. H . and Kernohan, J. W . : Primary sarcomas of the brain. Arch. Neurol. Psychiat 50:43, 1943.
215
20. Dougherty, T. F. : Studies on the cytogenesis of microglia and their relation to cells of the reticulo-endothelial system. Am. J. Anat 74:61, 1944. 21. Russell, D. S. and Rubinstein, L. J. : Pathology of Tumors of the Nervous System. Baltimore, Williams and Wilkins, 1963, p. 66. 22. Albites, V . E. : Primary reticulum-cell sarcoma of the brain. J. Neurosurg. 20:777, 1963. 23. Onofrio, B. M , Kernohan, J. W . and Uihlein, A : Primary meningeal Sarcomatosis. Cancer 15:1197,1962. 24. Hogan, M . J , Spencer, W . H . and Hoyt, W . F. : Primary reticuloendothelial sarcomas of the orbital and cranial meninges : Ophthalmologic aspects. Am. J. Ophth. 61:1146, 1966.
O C U L A R P E N E T R A T I O N O F H E T R A Z A N IN R A B B I T S MOSHE LAZAR, M . D , * THEODORE W . LIEBERMAN, M . D . , MOSES F U R M A N , M . S . AND IRVING H .
LEOPOLD, M . D .
New York Hetrazan ( l-diethylcarbamyl-4-methylpip- t i o n ' ' ' may make initial therapy intolerable erazine) (fig. 1) is an antifilarial agent intro- and subsequent treatment undesirable to the duced by Hewitt and co-workers in 1947. patient.* The drug is used in the treatment of the Since the greatest morbidity of the disease round-worm infection, onchocerciasis, a prev- is due to ocular anterior segment infestation alent cause of ocular morbidity in Africa, with microfilariae, * and since the major Mexico, Guatemala and Venezuela. As an oral route of the microfilariae into the eye appreparation, Hetrazan has proven to be highly pears to be through the conjunctiva,*' it was effective in transiently reducing the number considered that an ophthalmic preparation of of microfilariae found in skin biopsies or seen Hetrazan might be a useful adjunct to the in the aqueous humor of individuals harbor- present therapeutic armamentarium against ing the worm, Onchocerca volvulus. ' The onchocerciasis. This study, a first step in the drug is customarily administered daily over a development of such a preparation, was unseveral-week period. After a course of treat- dertaken to determine the penetration of Hement the microfilariae are practically nonex- trazan into the eye after its ocular adminisistent in skin-snip biopsies, but after several tration. months begin to increase in number due to MATERIALS AND METHODS the ineffectiveness of Hetrazan in eradicatHetrazan dihydrogen citrate (HetDC) ing the adult worms. Consequently, repeated powder}" was used in all experiments. Solutreatments over many years are required to tions were prepared on a weight-to-volume control the disease. Transient pruritis, basis of HetDC to distilled water. The pH edema, dermatitis, arthralgias, conjunctiviwas adjusted to 7.0 with concentrated soties and fever during Hetrazan administradium hydroxide. One hundred and twenty albino and pigFrom the Department of Ophthalmology, Mount mented rabbits weighting from 3 to 4 kg Sinai School of Medicine, the City University of 2
4
1
5
6
7
z z
New York. This investigation was supported by U S P H S Grant N B 06579 from the National Institute of Neurological Diseases and Blindness. * O n leave from Tel-Hashomer Hospital, Israel.
t Contributed by Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York.
216
AMERICAN JOURNAL OF OPHTHALMOLOGY
C H — 2
Fig. 1 (Lazar, Lieberman, Furman and Leopold). Molecular structure of Hetrazan base, 1-diethylcarbamyl-4-methylpiperazine.
A U G U S T , 1968
0 II
- C - N
CH -N 3
C H — 2
were used to study the aqueous humor, vitreous humor and serum concentrations of Hetrazan after topical, subconjunctival and intravenous administration of the drug. Each rabbit was used for a single aqueous and vitreous Hetrazan concentration determination. Topical instillations were made to both eyes of alert animals without anesthesia. The lids were held to prevent blinking for 30 seconds after administration. Subconjunctival injections were given to both eyes of animals anesthetized with intravenous pentobarbital. Intravenous injections of Hetrazan were given in the marginal ear vein after pentobarbital anesthesia. Anterior chamber taps were performed on anesthetized animals. The eyes were washed thoroughly with distilled water before the anterior chambers were entered. Aqueous humor was aspirated with a 26-gauge needle attached to a tuberculin syringe. The aqueous humor from both eyes of a single animal was pooled for Hetrazan determinations. Vitreous humor was aspirated with a 20-gauge needle attached to a 6.0 cc syringe immediately after the aqueous humor tap. Blood samples were obtained by heart puncture. All samples were analyzed for drug concentration within two hours after collection. Hetrazan in the aqueous, vitreous and serum was determined by a modification of the method of Lubran. In the technique used, Hetrazan forms a colored salt with the dye bromthymol blue. The colored salt is extracted into benzene in which bromthymol blue itself is insoluble. The concentration of
/ \
2 5 n
C2 H
5
Hetrazan in the benzene layer is determined spectrophotometrically against a standard curve. The exact technique is as follows : T o 0.5 ml of aqueous or vitreous humor, 0.5 ml of 4.0 N sodium hydroxide and 2.0 ml of benzene* were added and vigorously shaken for 15 seconds with a vortex mixer. The benzene layer was separated by centrifugation. T o an aliquot of the benzene layer 1.0 ml of a 0.05% bromthymol blue solutionf was added. The mixture was shaken for 15 seconds with a vortex mixer and then centrif uged 10 minutes to separate the benzene layer. The optical density of the benzene layer was measured at 400 mu in a Beckman DB spectrophotometer. A standard curve was prepared using the above procedure and known amounts of Hetrazan. Through preliminary determinations of the range of Hetrazan concentrations to be expected during the experiment, the unknown samples were adjusted to read on the straight-line portion of the standard curve. For Hetrazan determinations in the serum 1.0 ml of serum and double the amounts of the reagents given above were used. When blank samples of aqueous humor, vitreous humor and serum were run through the above procedure and compared to distilled water, they gave optical density readings corresponding to concentrations of less than 0.5 u-g of Hetrazan base per ml.
8
•Certified benzene, 99 Mol % pure, thiophenefree (Fisher Scientific Company). t Bromthymol blue (Fisher Scientific Company) was made up in 0.1 M phosphate buffer (pH 7.0) and washed twice with analytical grade benzene.
VOL.
OCULAR PENETRATION O F HETRAZAN
66. N O . 2
217
RESULTS
Each value given in the results is the average of five or more separate determinations.
10-
O--0
serum aqueous humor
A--0
vitreous humor
TOPICAL APPLICATION
After a single topical administration of two drops (approximately 0.12 ml) of 5% HetDC (2.5% Hetrazan base) the ocular penetration of Hetrazan was rapid (9.5 |ig of Hetrazan base/ml aqueous humor at onehalf hour). Within four hours the aqueous humor concentration had fallen to less than 0.5 jig base/ml (fig. 2 ) . No Hetrazan could be detected in the vitreous humor. With a single instillation of two drops of a 1 % solution of HetDC (0.5% base), 2 jig base/ml aqueous humor were found at one hour. This compared to 5 (tg base/ml aqueous humor found at one hour after the instillation of two drops of the 5% HetDC solution. Animals were treated with repeated topical doses of Hetrazan to determine if there was any accumulation of the drug in the aqueous humor. The animals received two drops of 5% HetDC every three hours four times daily. The anterior chambers were tapped in one group of animals four hours after the second dose on the fourth day of treatment and in another group of animals four hours after the first dose on the fifth day of treat-
p
1
1
0
1/2
I
2
t
TINE (hours)
Fig. 3 (Lazar, Lieberman, Furman and Leopold). Changes in serum, aqueous humor and vitreous humor concentrations of Hetrazan base after a single intravenous injection of 20 jig Hetrazan dihydrogen citrateAg body weight The sign f indicates time of injection.
ment. A s in the single dose experiment, no Hetrazan within experimental limits (less than 0.5 |ig Hetrazan base/ml aqueous) could be detected four hours after topical dose. There was no evidence of ocular irritation in any animal receiving topical solutions. SUBCONJUNCTIVAL INJECTIONS
One hour after the subconjunctival administration of 0.1 ml of 5% HetDC, 11 (ig Hetrazan base/ml aqueous humor was detected. After two hours the concentration dropped to 3.5 u,g Hetrazan base/ml aqueous humor. At four hours, less than 1 u,g Hetrazan base/ml aqueous humor was found. No Hetrazan was detected in the vitreous humor at any time and there was no evidence of conjunctival irritation.
S
1
I2 TIBE
(NOMS)
Fig. 2 (Lazar, Lieberman, Furman and Leopold). Changes in aqueous humor concentration of Hetrazan base with time after single topical instillation of 2 drops of 5% Hetrazan dihydrogen citrate.
INTRAVENOUS ADMINISTRATION
One hour after a single intravenous dose of 5 |ig HetDC/kg bodyweight or 10 (ig HetDC/kg the drug could not be detected in the aqueous or vitreous humors. After a single
218
AMERICAN JOURNAL OF OPHTHALMOLOGY
t serum
o aqueous
a vitreous
A
B
5-
^
A U G U S T , 1968
5432I
2Ii—i
0 15 30
t »t
r 60
A »
i—i—i 0 15 30 TINE (minutes)
r 60
t t t
Fig. 4 (Lazar, Lieberman, Furman and Leopold). Serum, aqueous humor and vitreous humor concentrations of Hetrazen base following the repeated intravenous administration of 10 ug Hetrazan dihydrogen citrateAg body weight (Graph A ) and S ug Hetrazan dihydrogen citrate (Graph B ) . The sign, f, indicates time of injections.
20 v-g/kg dose of HetDC, serum concentrations fell rapidly. Detectable quantities were found one hour after injection in both the aqueous and vitreous humors and fell over the next hour to low concentrations (fig. 3 ) . Repeated intravenous injections of large doses of Hetrazan (fig. 4 ) do not give as high aqueous humor concentration at one hour as found one hour after the topical administration of 5% HetDC. However, as after the single 20 |*g/kg intravenous dose, the drug was found within the vitreous humor. DISCUSSION
The disease is transmitted by various species of the black fly, Simulium, infected with onchocerca larvae. The fly deposits the larvae in the human subcutaneous tissues where they grow into adult worms, frequently forming skin nodules. The female worms deposit the microfilariae which migrate throughout the subcutaneous tissues of the body, but are not found in the bloodstream. The microfilariae penetrate the conjunctiva and apparently enter the eye in the area of the corneoscleral limbus. In other parts of the globe they have been found in the superficial sclera, but the deep portions of the sclera appear inpenetrable. They spread into the subepithelial cornea, the superficial stroma, the iris, ciliary body, choroid and lens. Optic atrophy is frequent but its pathogenesis is incompletely understood. It is considered most often to be of the consecutive type. Microfilariae can be seen in the aqueous humor, even in eyes without clinical 6
6
Onchocerciasis is a disease which affects large segments of the population living in endemic areas. The true extent of the disease is unknown but the magnitude of the problem can be appreciated by the estimates of 10 million" and 38 million affected Africans. 10
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OCULAR PENETRATION OF HETRAZAN TABLE 1
PERCENT DISTRIBUTION OF OCULAR LESIONS I N PATIENTS, 40 YEARS OF AGE AND OLDER, WITH PROVEN ONCHOCERCIASIS*
West Africa Plains No. of subjects Diminished visual acuityt Punctate keratitis . Type I Type II Type III Type IV Sclerosing keratitis Chronic iritis Iris atrophy Chorioretinal lesions § Stage I Stage II Stage III Optic atrophy 4
Forest
349
23.6% 7.0% 14.6% 14.6% 1.0% 16.6% 11.6% 33.2% 7.1% 8.2% 6.7% 18.0%
15.7% 5.2% 8.5%
— — 5.3% 2.1%
4.2% 7.7% 5.5% 2.2% 3.3%
Guatemala 125 12.2% 30.1% 8.2%
—
1.4% 41.8% 14.4% 28.7% 6.6% 8.2% 5.7% 1.8%
* Modified from Monjusiau, A.G.M, Lagraulet, J. and Gockel, C. W.: Ref. Ne. 12. Proven by positive biopsies, subcutaneous nodules and/or anterior chamber microfilariae, t Visual acuity equal to or worse than 20/200 in one or both eyes. t Type I: 0.5-1 mmfluffysubepithelial opacities. Type II: 1 mm subepithelial white dots with surrounding halos. Type III: Irregular punctate opacities. Type IV: Small discoid lesions, i Stage I: Irregular pigmentation. Stage II: Pigment migration. Stage III: Depigmentation and "sclerotic" choroidal vessels.
lesions." It has been hypothesized that the inflammatory reaction is caused by dying or dead microfilariae. * Table 1 shows the distribution of ocular lesions in individuals 40 years of age or older with proven onchocerciasis. Since the incidence of ocular onchocerciasis is known to increase with age, the figures given in Table 1 are high. Blindness in endemic areas may reach as high as 15%* to 30%. Keratitis and iritis are responsible for the major loss of vision.* Onchocerciasis is often termed river blindness because it is prevalent in the vicinity of rapidly flowing rivers, the breeding area of Simulium flies. Attempts at decreasing the population density of the vector Simulium have met with limited success. Two different methods have been used to destroy the adult worms. Individuals have been denodulated surgically.*' However, not all worms are found within nodules and as a massive therapeutic approach, this method is too timeconsuming. Suramin, a urea derivative, is toxic to the adult worm. • Unfortunately, solutions of the drug are extremely unstable, 1
5
5
5
3
5
must be administered intravenously, and can be nephrotoxic. Renal function should be followed over a five- to eight-week period while the drug is being used. The above properties of Suramin are not favorable for massive chemotherapy. Hetrazan has been therapeutically the most successful drug because of its low toxicity. As previously mentioned, the reactions during Hetrazan administration produce a great deal of discomfort. These side-effects are thought to be due to tissue reaction to dead microfilariae, ' * and are so frequent that their occurrence after the administration of Hetrazan has been used as evidence of onchocerciasis (Mazzotti test).* These reactions can be diminished with concomitant use of systemic corticosteroids or antihistamines. The oral dose of Hetrazan used in the treatment of onchocerciasis is 2 to 3 mg/kg of body weight three times daily. The use of similar quantities in normal volunteers over afive-dayperiod has been found to result in maximum serum concentrations of 3 to 5 jxg 5
5 1
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AMERICAN JOURNAL OF OPHTHALMOLOGY
Hetrazan base/ml after the second day of treatment. At this blood level adequate amounts of Hetrazan would appear clinically to reach the eye, as evidenced by transient worsening of the conjunctivities and iritis and by disappearance of microfilariae from the eyes of infected subjects. In this study, serum levels of Hetrazan comparable to those found in human subjects, could not sustain with a single intravenous dose, probably because of rapid renal excretion. However, similar serum levels were sustained for brief periods by repeated intravenous injections, resulting in higher aqueous humor concentrations than after single intravenous doses. Aqueous humor concentrations in excess of those achieved by the intravenous route were found after topical or subconjunctival administration and would appear adequate for antifilarial activity. 8
8
An ophthalmic preparation of Hetrazan may prove valuable in treating patients refusing systemic therapy, and also as prophylaxis when administered intermittently. By keeping the microfilarial count low within the conjunctiva and subconjunctival tissues of the lid, it may be possible to decrease the number of microfilariae entering the eye. W e suggest that if field trials are initiated, very low concentrations of the topical drug be used at first. Topical corticosteroids could be administered to control excessive reaction to dying microfilariae. No conjunctival irritation was noted during any of the present experiments and there was no apparent discomfort to the animals. However, further toxicity studies will have to be pursued before human evaluation is attempted. 8
SUMMARY
Hetrazan, a drug used orally in the treatment of onchocerciasis, has been demon-
A U G U S T , 1968
strated in high aqueous humor concentrations in rabbit eyes after topical instillation. Possible benefits of an ophthalmic preparation of this drug in treating and preventing onchocerciasis are discussed. Fifth Avenue and 100th Street (10029) ACKNOWLEDGMENT
W e want to thank Drs. S. A. Szumski, R. G. Kelly and R. Hewitt of Lederle Laboratories Division, American Cyanimid Company for their technical advice and cooperation during this experiment. REFERENCES 1. Hewitt, R. I , Kushner, S , Stewart, H. W , White, E., Wallace, W . S. and SubbaRow, Y . : Experimental chemotherapy of Filariasis. III. Effect of 1 -diethyl-carbamy 1-4-methylpiperazine hydrochloride against naturally acquired filarial infections in cotton rats and dogs. J. Lab. Clin. Med. 32:1314, 1947. 2. Expert Committee on Onchocerciasis, First Report, W H O Techn. Rep. Ser, No. 87,1954. 3. Nnochiri, E. : Observations on the treatment of onchoceriasis in an endemic area of Western Nigeria. Tr. Roy. Soc. Trop. Med. Hyg. 58:413, 1964. 4. Lazar, M . : Personal observation made at Governmental Eye Clinic, Monrovia, Liberia, 1964-1966. 5. Onchoceriasis: Epidemiology and control, W H O Chron. 20:378, 1966. 6. Rodger, F. C. : The pathogenesis and pathology of ocular onchocerciasis. Am. J. Ophth. 49:327, 1960. 7. Neumann, E. and Gunders, A. E. : Ocular lesions of onchocerciasis in Liberia. Am. J. Ophth. 56:573, 1963. 8. Lubran, M . : Determination of Hetrazan in biological fluids. Brit. J. Pharmacol. 5:210, 1950. 9. Stoll, N. R . : This wormy world. J. Parasit. 33 :1, 1947. 10. Rodger, F. C. : The pathogenesis and pathology of ocular onchoceriasis. Am. J. Ophth. 49:104, 1960. 11. McLean, C. M . : Ocular onchoceriasis in Northern Ghana: A treatment survey. Brit. J. Ophth. 43:477, 1959. 12. Monjusiau, A . G. M , Lagraulet, J , d'Haussy, R. and Göckel, C. W . : Aspects of ophtalmologiques de l'onchocercose au Guatemala et en Afrique occidentale, Bull. W H O 32:339,1965. 13. Rodger, F. C. : A review of recent advances in scientific knowledge of the symptomatology, pathology and pathogenesis of onchocercal infections. Bull. W H O 27 :429, 1962.