Pharmacokinetics And Distributions Of Bevacizumab By Intravitreal Injection Of Bevacizumab-PLGA Microspheres In Rabbits

Objective:Investigating the pharmacokinetics and distributions of bevacizumab by intravitreal injection of prepared bevacizumab-PLGA microspheres in order to appraise the safety and prospect of this kind of bevacizumab sustained release dosage form,and to provide evidences for clinical application of it.Methods:Fifteen healthy New Zealand albino-rabbits were used in this study. The eyes of each rabbit received an intravitreal injection. The left eyes were injected by prepared bevacizumab-PLGA microspheres (containing about 1.25mg bevacizumab). The right eyes were injected by bevacizumab solution(containing about 1.25mg bevacizumab).The anterior segment and ocular fundus were examined by slit-lamp microscope and direct ophthalmoscope before and after injection.Rabbits were randomly selected at Days 3,7,14,28,and 42 after intravitreal injection,three animals each day, the aqueous humor and vitreous of both eyes was aspirated about 0.05ml respectively, which were frozen at -80°C immediately.Then both eyes were immediately enucleated at each time and fixed in 10% formalin.For quantitating of free bevacizumab concentrations,ELISA kit was used.The parameters of pharmacokinetics of bevacizumab in aqueous humor and vitreous of both eyes were calculated through 3p97 software.The data were analyzed by t-test.Paraffin sections of both eyes were used to observe the distribution of bevacizumab in the tissues.Results:1.The results of ocular examination:All the experimental animals were healthy during the whole proceduals.There are no obvious complications in the eyes after intravitreal injection,such as inflammation,corneal opacity,cataract,vitreous opacity, retinal hemorrhage et al.2. Pharmacokinetic parameters:A vitreous concentration of bevacizumab reached a peak concentration of 249±0.13 and 156±0.20μg/ml in left (received bevacizumab- PLGA microspheres) and right eyes (received solubal bevacizumab) after 3 days of injection,respectively.Vitreous concentration of bevacizumab declined during time. Concentrations of 45±0.14 and 20±0.23μg/ml of bevacizumab were maintained in the vitreous of left and right eyes after 28 days.The final concentrations after 42 days of injection were 14±0.22 and 3.6±0.19μg/ml in the eyes that received bevacizumab- PLGA microspheres and solubal bevacizumab, respectively. Area under the drug concentration time curve for bevacizumab-PLGA microspheres was 2-fold higher when compared with solubal bevacizumab.The aqueous humor concentrations of bevacizumab after 3 days also reached a peak concentrition of 19±0.22 and 16±0.17μg/ml in the left and right eyes,respectively.The concentritions went down to 8±0.17 and 2.3±0.04μg/ml 14 days after injection,and the final concentrions fell to 1.2±0.03 and 0.3±0.04μg/ml 42 days after injection. The half-life of intravitreal injection of bevacizumab-PLGA microspheres is 9.6 days in vitreous and 10.2 days in aqueous humor,and the half-life of intravitreal injection of solubal bevacizumab is 3.91 days in vitreous and 4.1 days in aqueous humor.There were statistical significant in the comparison results of the bevacizumab in the vitreous and in the aqueous humor between the left and right eyes (P<0.05).The AUC0-t of the sustained release dosage form was 1-fold higher than the solubal form.The relative bioavailability was raised significantly.3. Immunofluorescence staining:The figues showed that bevacizumab was distributed in the retina,choroid,iris,ciliary body and anterior chamber angle in both eyes,especially in the vascular tissues after intravitreal injection.Strongest staining (+++) was seen at Day 3 and Day 7 in both eyes.The intensity of staining in the left eyes was becoming weakened at Day 14 and Day 28,which was recorded as (++).Weak staining (+) could also be detected at Day 42. In the right eyes,bright staining (++) was seen at Day 14 which was weaker than the left eyes.Only very weak staining (+) was detected at Day28,and no staining can be seen at Day 42.Conclusion: There were no obvious toxic and side effects in the eyes after intravitreal injection of bevacizumab-PLGA microspheres (containing about 1.25mg bevacizumab),which may certify the security for intravitrieal administration of this new kind of sustained release dosage form.It may provide a reliable basis for the development of this new dosage form. The penetration and distribution of free bevacizumab released from the microspheres were similar to the traditional dosage form.But the intensity of staining was stronger and longer than the latter over time.These results demonstrated that the free bevacizumab that slow released could combined with the VEGF to prolong the duration of effects.From the data of pharmacokinetic parameters and time-concentration curves we can see the effect of slow release of the bevacizumab-PLGA microspheres.On the whole,the bevacizumab- PLGA microspheres may become a new dosage form for the therapy of neurovascular diseases,in order to reduce the frequency of intravitreal injection and degrade the probability of operative risks.