Pharmacokinetics is the study of in vitro absorption, distribution, biotransformation and excretion in the tissue or organism on which a drug acts. In order to determine the non-toxic dose level of a drug prior to any clinical intervention by intravitreal injection, a histopathological analysis, including clinical observation, electroretinography, electron and light microscopy and evaluations on elimination life of the drug and clearance from the vitreous must be implemented.
Administration of methotrexate by means of oral, intravenous, intramuscular and intrathecal routes is already included in the routine regimen, while recent studies reported intravitreal use.[2–5] Topical administration on eye will also take its place among other treatment methods in the near future. As any possible toxicity, associated with this local treatment method, due to a pre-existing ocular pathology or a retinal impairment from prior therapy has not been determined, we aimed to investigate the distribution of the drug, duration of efficacy and elimination of half-life following the intravitreal injection of methotrexate in an experimental rabbit model.
Materials and Methods
The pharmacokinetics of the serial intravitreal injections of methotrexate on the retina was studied on a total of 42 eyes from 21 adult New Zealand white rabbits; male and female, weighing between 1500 and 2500 g, upon approval of the Ethical Board, and the research followed the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in ophthalmic and vision research.
The highest frequently used intravitreal dose of methotrexate in the clinical studies on human was 400 μg. It is already known that the antineoplastic effect of methotrexate is dose-dependent. Therefore, our experimental study was based on the administration of high dose in rabbits‘ eyes, targeting to reduce the number and frequency of serial injections of the drug throughout the treatment. The present study included analysis of the pharmacokinetics of the intravitreal injection of 800 μg methotrexate.
Each examination and surgical procedure in all rabbits was carried out under general anesthesia with an intramuscular injection of ketamine hydrochloride 35 mg/kg and Xylazine hydrochloridine 5 mg/kg. Fifteen minutes after instillation of one drop of phenylephrine HCL 2.5% (Mydfrin, Alcon Lab, Fort Worth, TX, USA) and tropicamide 1% (Tropamid Forte, Bilim Lab, Mefar Ilac San.A.S.,Istanbul, Turkey) into both eyes of each rabbit, anterior segment, vitreous and retina were examined by an indirect ophthalmoscope (Heine Omega 180, Heine Optotechnik, Herrsching, Germany) using a blepharostat on the eyelids.
A 50 mg/2 ml methotrexate without preservative (Methotrexate “Ebewe”, Liba Lab. A.S) was diluted by 4.25-ml distilled water to obtain methotrexate 8 mg/ml. A volume of 0.1 ml of the preparation (methotrexate 800 μg, 1.76 μmol) was injected into the vitreous of both eyes of the rabbits by an insulin injector with a 26-G needle under general anesthesia. Intravitreal injection was performed 3-mm posterior to the limbus from the upper temporal quadrant. Measurement of the ocular pressure and retinal examinations of the rabbits were repeated following the injection.
The retinal examination was repeated at seven different time points (30 minutes, 4.5, 6, 9, 24.5, 53.5 and 72.5 hours) following the injection of the drug with the same routine as mentioned earlier, and the intraocular pressures were measured. The 200 μl content of the vitreous was aspirated by a 26-G insulin needle at 3-mm posterior to the surgical limbus in sets of six eyes of three rabbits at different time intervals mentioned here. The rabbits were sacrificed after completion of the procedures (aspiration of 200 μl of vitreous).
The vitreous specimens aspirated were transferred into ependorf tubes, and then maintained at -70°C until analysis. The methotrexate levels in the vitreous humor were quantified using the fluorescence polarization immunoassay method (TDx/FLx, Abbott Laboratories, Abbott Park, IL, USA).
Euthanasia was performed via intravenous administration of a high-dose anaesthetic agent to the rabbits after surgical procedures. For quantification of the drug level, the baseline, i.e., time point 0.01, was taken as 30 minutes, which was the initial measurement expected to have a homogenous distribution of the agent following the intravitreal injection. The following measurements were carried out at hours 4, 5.5, 8.5, 24, 53 and 72, respectively. All statistical calculations were performed using the SPSS 10.1.
During the examination before the intravitreal injection of methotrexate, it was observed that the structures of cornea, anterior segment and lens were normal for both eyes of the rabbits and the retina was efficiently luminous. Intraocular pressures ranged from 8 to 16 mmHg with a mean value of 11.8 ±1.8 mmHg. However, the intraocular pressures measured following the injection of methotrexate ranged from 17 to 36 mmHg with a mean value of 23.5 ±4.7 mmHg.
The pharmacokinetics of methotrexate 800 μg administered was evaluated. When the calculations were based on the molecular weight of methotrexate, which was 454.5 g, the administered dose was found to be 1.76 mol. As the unit of quantification is mmol/L (molarity) for the drug, we considered our initial measurements as primary time point (hour 0.01). Measurements at 30 minutes were between 1105.58 and 1598.28 μM with a mean value of 1321.9033 μM (±185.5879). When the baseline was based on the measurements at 30 minutes, the virtual distribution volume was calculated by the following formula;
Vd= the quantity of drug administered / the concentration detected.
The mean distribution volume in the rabbit's eye was 1.33 cc following the intravitreal injection of methotrexate. With baseline as the measurements of 30 minutes, methotrexate concentrations in the vitreous were summarized in Table 1, and Fig. 1.
A mathematical relation was found between the drug level and the time of using an exponential association [Fig. 2].
Drug level= 1426.73 e-0.1182(time)
Calculations based on the mathematical relation demonstrated that the elimination half-life of the intravitreal administration of methoraxate was 5.8629≈ 5.9 hours.
Permeability of drugs administered via systemic routes into the eye is very restricted because of blood-eye barriers. Although posterior uveal capillaries and pigmented ciliary body capillaries are fenestrated type allowing drugs to achieve relatively high concentrations in this localization, tight junctions in the vascular endothelium of iris and non-pigmented ciliary body epithelium inhibit drugs to achieve higher concentrations in vitreous and aqueous humors. Therefore, local administration of the drugs has been preferred and developed for ophthalmological chemotherapy and regimens. It is also likely that local treatment is more efficient for isolated ocular involvement of the systemic diseases.
De Smet et al., studied the pharmacokinetics of intravitreal injections of methotrexate and thiotepa in a blind patient with recurrent ocular lymphoma, who previously received radiotherapy and high-dose chemotherapy and refused enucleation. They found that the cytotoxic effect of methotrexate in the vitreous humor lasted 5 days following the intravitreal injection, and ocular lymphoma was in remission. In another study of De Smet et al., in case of intravenous administration of methotrexate for 24 hours, the methotrexate levels reached to 55 μM at 7 hours while methotrexate concentrations were below the cytotoxic level in the serum and cerebrospinal fluid at 74 hours; however, the methotrexate level in the aqueous humor was 1.2 μM, i.e., above the cytotoxic concentration. High penetration of methotrexate into the eye and its slower clearance from the aqueous humor, compared to blood and cerebrospinal fluid, unlike many other chemotherapeutics, have been attributed to a possible disruption of the blood-aqueous barrier due to inflammation. Furthermore, it has been reported that the patient partially responded to the treatment of ocular pathology. It has also been indicated in the literature that the blood-brain barrier can be temporarily disrupted by means of hypertonic infusion of mannitol, increasing the cranial expansion of the agent in the chemotherapy protocols.
It has been reported that the methotrexate level in the blood was 10 times of the vitreous level and the methotrexate level in the aqueous humor was 10 times of the vitreous level following an intravenous infusion of high dose (15.6 g) methotrexate for 4 hours, and that the efficacy levels were not achieved in vitreous by intravenous administration of high-dose methotrexate. It has also been added that the cranial and anterior segment involvement of the patient responded well to the treatment, but the vitreous involvement persisted.
There are some studies in the literature indicating that intravitreal injections of methotrexate 400 μg twice a week, followed by injections of once a week, once every 2 weeks and once a month until the number of cells get reduced in the vitreous humor produces successful results in the ocular lymphoma.[13–15] Helbig et al. reduced the frequency of serial injections of intravitreal methotrexate (0.4 mg) and dexametazone (0.4 mg) to once a week for 4 weeks, followed by injections of once a week until the malignant cells were eliminated from the eye, and they achieved remission at 2 years. However, it is not possible to determine if any retinal impairment developed had been developed or not because of underlying pathologies.
In a study evaluating the in vitro activity of methotrexate under a developmental therapeutic program of the National Cancer Institute, it has been reported that methorexate concentrations between 1 and 0.1 μM inhibited the growth of tumor cells by a mean of 50%. In our study, we found that methotrexate levels were persistent above 1 μM for 61.5 hours and subsequently they were reduced to less than 0.1 μM at 81 hours in the vitreous humor following an intravitreal injection of methotrexate 800 μg in the experimental rabbit model.
In a study on the pharmacokinetics of the intravitreal injection of methotrexate, Velez et al. reported that the distribution volume of methotrexate was 2.16 ml. However, we found that the distribution volume of methotrexate was 1.33 ml in the rabbit eye, which is consistent with the literature reporting a range from 1.2 to 1.5 ml for the distribution of volume in the rabbit eye. In the study of Velez et al., the methotrexate level in the vitreous was measured at 1.5 hours following the injection, and those measurements were taken as the baseline for evaluations. Our initial measurements were performed in a shorter period of time, at 30 minutes, as the calculation errors resulting from distribution and absorption could be as low as possible at that time. We attribute this inconsistency regarding the distribution volume of methotrexate in the rabbit eye to the difference at time points for measurements.
The calculations we made based on the mathematical relation which were completely in compliance with the drug levels, demonstrated that the elimination half-life of methotrexate in the rabbit vitreous was approximately 5.9 hours whereas the elimination half-life of methotrexate in the rabbit vitreous was reported as 7.6 hours in the study carried out by Velez et al. In this publication, the values were not reported by hours and the results obtained by the predicted curve in the graphics are not adequately compatible.
Such measurements define the drug level in vitreous, and as already known, healthy vitreous do not include any cellular elements. However, in ocular lymphoma high levels of methotrexate in the vitreous will only act on the target cells which are pathological. Determination of drug levels in tissues is also required in order to determine the distribution of methotrexate from the vitreous to the tissues. Therefore, we preferred to use histopathological analysis as an objective evidence of toxicity at the second phase of our study based on the idea that the toxic values reported in the blood were not correlated with the vitreous levels.
Local administration of methotrexate in isolated ocular pathologies could be indicated only after its pharmacokinetics, toxicity or safety in such interventions has been established. Further studies are needed for determination of optimal dosage of intravitreal methotrexate with minimal adverse effects.
The major limitation of the study is the lack of electrophysiological study. Further study is needed to investigate the effect of methotrexate on retinal physiology.
As a conclusion, the present study showed that vitreous levels of methotrexate after 800 μg intravitreal injections can be calculated by a mathematical equation, at each hour.
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Conflict of Interest: None declared.