Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro.

PURPOSE: The effects of gravity on bevacizumab or the recommended head position after intraocular bevacizumab injection have not been reported. To evaluate the effect of gravity on bevacizumab in vitro, we added bevacizumab to the upper part of a test tube filled with balanced salt solution (BSS) and examined its distribution over time.
MATERIALS AND METHODS: Sixty-four test tubes were divided equally into two groups; group 1 (32, collected from upper part of the tube) and group 2 (32, collected from lower part of the tube). Each test tube was filled with 5 mL BSS before bevacizumab (1.25 mg/0.05 mL) was added, and then stored at 36°C. Bevacizumab concentration in 8 test tubes from each group was measured at 12, 24, 48, and 168 h using an enzyme-linked immunosorbent analysis (ELISA) kit. Mann-Whitney and Jonckheere-Terpstra tests were used for statistical analysis.
RESULTS: Bevacizumab concentration was significantly higher in Group 2 than in Group 1 at 12, 24, 48, and 168 h (12, 24, 48, and 168 h; P < 0.01 each; Mann-Whitney test). The mean change in bevacizumab concentration over time tended to increase in Group 1 (P < 0.01; Jonckheere-Terpstra test), but tended to decrease in Group 2 (P < 0.01; Jonckheere-Terpstra test).
CONCLUSIONS: The significant differences in concentration between the upper and lower parts even after a considerable amount of storage time showed that bevacizumab did not dissolve immediately and diffused evenly throughout the solution. It appeared that more bevacizumab settled in the lower part of the tube than in the upper part because of gravitational force. However, the concentration difference between the upper and lower parts decreased as bevacizumab gradually diffused over time, indicating that the difference in concentration due to gravity was more significant at the beginning of bevacizumab injection.

Introduction

Proliferative diabetic retinopathy (PDR) can cause vitreous hemorrhage, retinal detachment, and neovascular glaucoma, and can even lead to blindness [1]. Vitrectomy is a surgical treatment for restoring vision in PDR patients [2]. At the end of vitrectomy in PDR patients, bevacizumab (Avastin®; Genentech, San Francisco, CA, USA) is injected intraocularly as a relatively safe method to effectively reduce recurrent intraocular hemorrhage [3]. Moreover, intraocular bevacizumab injection is known to be a safe and effective method to treat vitreous hemorrhage occurring after vitrectomy for PDR patients [4]. For this reason, bevacizumab injection is often administered after vitrectomy in patients with PDR. We also typically administer an intraocular bevacizumab injection after vitrectomy in patients with PDR, and then observe using a surgical microscope whether the injected drug collects at the bottom of the eye. So, we questioned whether bevacizumab dissolves and disperses rapidly and evenly throughout the vitreous chamber when injected into balanced salt solution (BSS)-filled eyes after vitrectomy.

To date, there have been numerous studies on the half-life, clearance rate, and overall pharmacokinetics of intraocular bevacizumab injections after vitrectomy, but there have not been studies on the intraocular distribution by weight [5-7]. Hence, using an indirect method initially, we examined the distribution of bevacizumab over time following its addition into the upper part of a test tube filled with BSS. We aimed to provide insights into the intraocular distribution of bevacizumab by weight following post-vitrectomy injection, and to present our opinions about postoperative head position.

Materials and methods

Sixty-four test tubes were divided into two groups of 32 test tubes each. Each borosilicate glass test tube (diameter: 10mm, length: 100mm) was filled with 5 mL of BSS (BSS Plus, Alcon laboratories, Fort Worth, TX). A 1-cc syringe with a 30-gauge needle was used to inject 1.25 mg/0.05 mL bevacizumab (Avastin; Genentech, San Francisco, CA, USA). By using the 1-mL syringe positioned vertically with the needle downwards from 0.5 cm above the surface of the solution in each test tube. Bevacizumab was added at a rate of two drops per second while carefully making sure that the needle did not touch the tube’s internal wall. Following the drug injection, the tubes were immediately covered and stored undisturbed in CO2 INCUBATOR (MCO 175, Sanyo, Japan) at a temperature of 36.0°C and CO2 of 0.0%. The concentration was measured in eight tubes from each group after 12, 24, 48, and 168 h of storage. The solution was collected only once from each tube: the upper part was collected from one tube and the lower part from another tube. For the upper layer, the solution was collected 0.5 cm below the surface, and for the lower layer, the solution was collected 0.5 cm above the floor. From the upper (Group 1) and lower (Group 2) parts of the tube, 0.2 mL of solution was collected by using a micropipette to avoid dispersion of the drug. Microcapillary tips (Denville scientific, Metuchen, NJ) were used to collect the lower layer of the solution, while carefully minimizing disturbances of the upper layer or its mixture with the lower layer. Bevacizumab's concentration was analyzed using an enzyme-linked immunosorbent analysis (ELISA) kit (Protein Detector ELISA Kit; KPL, Inc., Gaithersburg, MD, USA), which was used immediately after calibration of the concentration according to the manufacturer’s calibration guidelines.

Statistical analysis was performed using IBM SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant.

Results

Bevacizumab concentrations in the samples collected from the 64 test tubes [Group 1 (32, from the upper part) and Group 2 (32, from the lower part)] were analyzed. No samples were lost or contaminated. Table 1 and Fig 1 show concentration of bevacizumab over time in the upper and lower parts of the test tube following bevacizumab injection. The details of individual concentration measurements for each tube at each time point are shown in S1 Table. We compared the concentrations in Groups 1 and 2 at 12, 24, 48, and 168 h. A significant difference was observed between the two groups at all points, with a higher bevacizumab concentration in Group 2 than in Group 1 (12 h, P < 0.01; 24 h, P < 0.01; 48 h, P < 0.01 and 168 h P < 0.01 by Mann–Whitney test). The mean changes in bevacizumab concentration in Group 1 and Group 2 were calculated. The bevacizumab concentration in Group 1 showed a significant increasing trend over time (P < 0.01 by Jonckheere–Terpstra test), while Group 2 showed a significant decreasing trend (P < 0.01 by Jonckheere–Terpstra test). The difference in bevacizumab concentration between the two groups showed a gradually decreasing pattern over time, but this decrease was not statistically significant (P = 0.275 by Jonckheere–Terpstra test).

Table 1

Mean bevacizumab concentrations in samples collected from the upper (Group 1) and lower (Group 2) parts of the test tube.

Group	Concentration (μg/mL)
	12 h	24 h	48 h	7 days
Group 1	67.28 ± 19.61(33.5–82.4)	86.69 ± 19.85(50.4–110.5)	73.04 ± 17.64(43.4–98.5)	103.93 ± 14.61(74. 2–120.4)
Group 2	443.33 ± 31.39(403.6–426.70)	452.44 ± 25.31(412.4–488.8)	442.06 ± 24.14(405.6–466.7)	393.90 ± 28.04(348.3–421.5)
*Difference	376.05	365.75	369.02	289.97

Values are mean ± standard deviatio (range), unless otherwise indicated

*Difference = mean value of Group 2 –mean value of Group 1

Fig 1

Box plot of bevacizumab concentrations in Group 1 (upper part of the tube) and Group 2 (lower part of the tube).

*: P < 0.001. P values were calculated using the Mann–Whitney test.

Discussion

This study showed that the bevacizumab concentration in the lower part of the tube was significantly higher than that in the upper part of the tube from 12 to 168 h post injection, even when the drug was injected from above the surface.

The significant differences in concentration between the upper and lower parts even after a considerable amount of time had passed showed that bevacizumab does not dissolve immediately and diffuse evenly throughout the solution. It appears that more bevacizumab settles in the lower part of the tube relative to the upper part because of gravitational force. Furthermore, the mean concentration of bevacizumab tended to significantly increase in Group 1 over time, but tended to significantly decrease in Group 2, indicating that the concentration difference between the upper and lower parts decreased as bevacizumab gradually diffused over time. Thus, this indicates that the difference in concentration due to the effect of gravity is more significant particularly at the beginning of bevacizumab injection.

Several factors affect the change in bevacizumab concentration when it is injected directly into the vitreous chamber, including convection currents, diffusion, temperature, and volume. Jooybar et al. reported that during intravitreal injection, the position, needle size, and injection speed could cause differences in the distribution of the injected drug within the vitreous chamber [8]. In the present study, the samples were stored close to body temperature. The test tubes were filled with 5 mL of BSS, which is equivalent to the vitreous volume. Bevacizumab at a concentration of 0.05 mL, which is consistent with the amount used for intraocular injection, was injected using a needle of constant size and at a constant injection speed. Under these conditions, we observed a significant difference between the upper and lower parts of the mixture over a long period, which suggests that similar results may occur in clinical practice.

Although no research on the effect of gravity on bevacizumab has been conducted to date, previous studies have reported the effects of head position and gravity on the intraocular injection of other drugs. Lim et al. administered intravitreal injections of gentamicin, which is heavier than BSS, in rabbits that underwent vitrectomy. The eyeballs were collected after keeping the rabbits in a fixed position for 30 min, and then evaluated. The study reported significant injuries to the retinal tissue located inferiorly and indicated that gravity affects intravitreal injection of gentamicin. According to these results, they recommended that patients should be placed in an appropriate position during intravitreal injection to minimize foveal damage [9]. Jaissle et al. reported that gravity could lead to the accumulation of crystals in the inferior part of the vitreous humor following intraocular triamcinolone injection. The study showed that deposition could occur at the posterior pole of the retina depending on the patient’s head position, and this form of deposition could be more likely after vitrectomy [10]. Of course, while it requires the results of additional animal experiments or in vivo clinical trials, bevacizumab showed a higher concentration at the inferior side due to gravity as demonstrated in our study. We believe that these factors can be considered similar to the above drug during intraocular injection of bevacizumab after vitrectomy. For example, if the patient is asked to maintain a supine position after injection, a higher concentration of the drug could be delivered to the fovea. In case of a problem in the anterior eye, such as neovascular glaucoma, the patient could be made to keep prone position after injection.

The limitation of this study is that the experimental conditions were not identical to real-world clinical situations. The surface area/length/shape of the tubes used in this study are different from that of an actual vitreous chamber. Moreover, patients are not in a completely fixed, stationary position in real-world conditions, and their posture or movement could cause the solution to mix. In addition, the surrounding tissues could affect the absorption of the drug, resulting in a relatively smaller gravity-induced concentration gradient. Furthermore, errors in the measurement of bevacizumab concentration due to inevitable mixing of samples while collecting a relatively small amount of sample (0.2 mL) from each layer should be taken into consideration. As the sample was collected by passing through the upper layer of the solution, we cannot completely rule out a possibility that this method may have affected the lower layer’s bevacizumab concentration even if microcapillary tips had been used carefully. However, we believe it is very unlikely that this sampling method, would have affected our results that the lower layer’s bevacizumab concertation was statistically significantly higher because the upper layer was found to have a lower bevacizumab concentration. Nevertheless, given that the patient must rest in a fixed position after vitrectomy, we believe that bevacizumab will be present at a higher concentration in the inferior part of the vitreous chamber at least in the early stage.

Further studies on the effect of gravity on bevacizumab, including model eye, animal experiments, and clinical trials, are required in the future. The results of the present in vitro distribution study can lay the foundation for future studies. In addition, further research should be conducted to verify whether this gravity-dependent distribution is evident for intraocular bevacizumab injection in patients who have not undergone vitrectomy. This would help determine the optimal postoperative head position for the vast number of patients who undergo intraocular bevacizumab injection worldwide.

The details of individual concentration measurements for each tube at each time point.

(XLSX)

Click here for additional data file.

22 Jul 2019

PONE-D-19-17798

Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro

PLOS ONE

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Reviewer #1: The findings of the authors are interesting. However, based on the data presented, there seems to be no way to apply these purely in vitro findings to clinical practice at this time. Therefore, the concluding sentence of results is an overstatement and not supported by the results presented, “These factors should be taken into account during intraocular bevacizumab injection after vitrectomy.” Recommend this be eliminated or modified substantially to directly and accurately reflect the results presented.

Similarly for the comments made in lines 137-138: there is no data in the current manuscript to support the claim that “similar results would be observed in clinical practice.” This should be removed or substantively modified. Also for lines 153-154, there is no data to indicate that the current results “need to be taken into account during intraocular injection.” Again, over reach by the authors beyond a reflection of their actual, presented data. It would be acceptable to mention these issues as possibilities requiring more study, particularly in vivo studies. But, to conclude that human treatment should be directly modified based on this work alone should be avoided.

The authors state that a “micropipette” was used to collect the samples from the tubes at the end of the prespecified waiting period. How was this performed for the “lower layer” without disturbing the “upper layer” in the process. Or, was all of the upper layer removed and then the lower layer was accessed? Please clarify exactly how the samples were harvested prior to ELISA analysis. This seems to be partially addressed in lines 163-166, but clarity in the Methods would be valuable to readers.

Reviewer #2: The study aims to evaluate the effect of gravity on bevacizumab in vitro.The authors added bevacizumab to the upper part of a test tube filled with balanced salt solution (BSS) and examined its distribution over time. They found significant differences in concentration between the upper and lower parts of the test tubes, even after a considerable amount of time had passed which shows that bevacizumab did not dissolve immediately and diffuse evenly throughout the solution. They reasoned that more bevacizumab settles in the lower part of the tube relative to the upper part because of gravitational force.

The biggest problem of this study is they used test tubes to conduct the study which is so different from the actual environment in the eye. This will make the data difficult to be interpreted in the "in vivo" condition.

Other comments:

1. Please describe the material of test tubes, which will be a possible factor to affect the distribution of medications

2. Please describe the meaning of all the abbreviations

3. In discussion, authors stated that bevacizumab was injected at a constant injection speed, however, in material and methods parts, the way of injection was not described, which would also cause the difference in distribution. Please also state further how to keep a constant injection speed.

4. The method of temperature control should be mentioned

5. In the first paragraph of discussion, authors stated that the concentration was higher in the upper part after 6 to 168 hours after injection, however, the first measurement time stated in your methods was 12 hours after the injection.

Minor points:

6. Line 56: "bevacizumab injection is often prescribed after vitrectomy", may change to "Bevacizumab injection is often given, or performed, after vitrectomy

7. Line 60: “whether bevacizumab dissolves and disperses rapidly and evenly throughout the vitreous chamber when injected into balanced salt solution (BSS) filling the vitreous chamber after vitrectomy”, may change to “when injected into balanced salt solution (BSS) filled eyes”

8. Line 65: “Through this, we aimed…”, you can delete the word “Through this” and start the paragraph with “We aimed…”

9. Line 90: “Bevacizumab concentrations...” should be written as “Bevacizumab’s concentrations...”

Reviewer #3: I believe this research may have other applications besides DR. For instance, Rop and Srn.

Can the authors please comment on thoughts on the same concept with an intact vitreous?

It would be interesting to do an animal model for the distribution of antivegf in eyes with intact vitreous.

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Reviewer #2: No

Reviewer #3: Yes: Clio Armitage Harper III

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19 Sep 2019

Response to reviewers

Reviewer #1: The findings of the authors are interesting. However, based on the data presented, there seems to be no way to apply these purely in vitro findings to clinical practice at this time. Therefore, the concluding sentence of results is an overstatement and not supported by the results presented, "These factors should be taken into account during intraocular bevacizumab injection after vitrectomy." Recommend this be eliminated or modified substantially to directly and accurately reflect the results presented.--> We agree with your comment and therefore, we have deleted the relevant sentence from the Abstract.

Similarly for the comments made in lines 137-138: there is no data in the current manuscript to support the claim that "similar results would be observed in clinical practice." This should be removed or substantively modified. --> We have revised the relevant sentence. Also for lines 153-154, there is no data to indicate that the current results "need to be taken into account during intraocular injection." --> We have made the necessary revision. Again, over reach by the authors beyond a reflection of their actual, presented data. It would be acceptable to mention these issues as possibilities requiring more study, particularly in vivo studies. But, to conclude that human treatment should be directly modified based on this work alone should be avoided.

The authors state that a "micropipette" was used to collect the samples from the tubes at the end of the pre-specified waiting period. How was this performed for the "lower layer" without disturbing the "upper layer" in the process. Or, was all of the upper layer removed and then the lower layer was accessed? Please clarify exactly how the samples were harvested prior to ELISA analysis. This seems to be partially addressed in lines 163-166, but clarity in the Methods would be valuable to readers.--> Thank you for your comment. To collect the lower layer, microcapillary tips (Denville Scientific, Metuchen, NJ) were inserted slowly and carefully to ensure that the upper layer was not mixed with the lower layer. We have described this in the Methods.

As we collected the sample from the lower layer by passing through the upper layer of the solution, we cannot completely rule out the possibility that this method may have affected the lower layer’s bevacizumab concentration. However, we believe that it is highly unlikely that this sampling method would have affected our results, as the lower layer’s bevacizumab concertation was statistically significantly higher. We have described this in the Discussion.

Reviewer #2: The study aims to evaluate the effect of gravity on bevacizumab in vitro. The authors added bevacizumab to the upper part of a test tube filled with balanced salt solution (BSS) and examined its distribution over time. They found significant differences in concentration between the upper and lower parts of the test tubes, even after a considerable amount of time had passed which shows that bevacizumab did not dissolve immediately and diffuse evenly throughout the solution. They reasoned that more bevacizumab settles in the lower part of the tube relative to the upper part because of gravitational force.

The biggest problem of this study is they used test tubes to conduct the study which is so different from the actual environment in the eye. This will make the data difficult to be interpreted in the "in vivo" condition.--> We have toned down the Conclusions to better reflect the results. We have also stated our limitations and mentioned that animal experiments or in vivo clinical trials are necessary.

Other comments:

1. Please describe the material of test tubes, which will be a possible factor to affect the distribution of medications --> We have added the necessary information in the second line of the Methods.

2. Please describe the meaning of all the abbreviations

3. In discussion, authors stated that bevacizumab was injected at a constant injection speed, however, in material and methods parts, the way of injection was not described, which would also cause the difference in distribution. Please also state further how to keep a constant injection speed.--> Thank you for your detailed review. We have provided the necessary information in the Methods.

4. The method of temperature control should be mentioned-> We have provided the necessary information in the Methods.

5. In the first paragraph of discussion, authors stated that the concentration was higher in the upper part after 6 to 168 hours after injection, however, the first measurement time stated in your methods was 12 hours after the injection. --> Originally, we collected data from 6 h, but we just indicated data from 12 h because it did not affect our results. Thank you for pointing out our mistake; we have made the necessary change.

Minor points:

6. Line 56: "bevacizumab injection is often prescribed after vitrectomy", may change to "Bevacizumab injection is often given, or performed, after vitrectomy -> We have made the necessary change.

7. Line 60: "whether bevacizumab dissolves and disperses rapidly and evenly throughout the vitreous chamber when injected into balanced salt solution (BSS) filling the vitreous chamber after vitrectomy", may change to "when injected into balanced salt solution (BSS) filled eyes" -> We have made the necessary change.

8. Line 65: "Through this, we aimed…", you can delete the word "Through this" and start the paragraph with "We aimed…" -> We have made the necessary change.

9. Line 90: "Bevacizumab concentrations..." should be written as "Bevacizumab's concentrations..." -> We have revised the relevant sentence.

Reviewer #3: I believe this research may have other applications besides DR. For instance, Rop and Srn.

Can the authors please comment on thoughts on the same concept with an intact vitreous?

It would be interesting to do an animal model for the distribution of antivegf in eyes with intact vitreous.

-> Thank you for your detailed review.

We are planning to conduct additional animal experiments or in vivo clinical trials.

This study's experimental conditions are not identical to real-world clinical situations, but the results of the present in vitro distribution study lay a foundation for future studies.

Submitted filename: Response letter.docx

Click here for additional data file.

23 Sep 2019

Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro

PONE-D-19-17798R1

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27 Sep 2019

PONE-D-19-17798R1

Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro

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