# Abstract

 

## Background The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs). 

## Methods **Ex-vivo treatment of AVM tissues:** Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured. **Pilot trial:** AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured. 

## Results **Ex-vivo treatment of AVM tissues:** Doxycycline at 10 and 100 μg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo (total: control vs 10 vs 100 μg/ml = 100 ± 6 vs 60 ± 16 vs 61 ± 9%; active: 100 ± 8 vs 48 ± 16 vs 59 ± 10%). **Pilot trial:** 10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group (total: 2.18 ± 1.94 vs 3.26 ± 3.58, P = .50; active: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25). 

## Conclusions A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues. # Ex-vivo treatment of cultured AVM tissues We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study. # AVM-I AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro. There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure [1](#)). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues. <figure> <p><img src="" /></p> <figcaption><strong>Tissue viability assessed by LDH (lactate dehydrogenase) release.</strong> There was a gradual increase in LDH release (mean ± SD) over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability. However, there was no difference among different treatment groups.</figcaption> </figure> Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure [2](#)). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. <figure> <p><img src="" /></p> <figcaption><strong>H&E staining of ex-vivo cultured AVM tissues.</strong> On day 0 (before the treatment), although cutting and mincing of the AVM tissues appeared to cause minor tissue injury and distortion, a majority of blood vessels was intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore.</figcaption> </figure> Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P < .05) (Figure [ 4A](#)). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P < .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P < .05). (Figure [4B](#)) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure [3](#). <figure> <p><img src="" /></p> <figcaption><strong>Representative zymogram showing MMP-9 and MMP-2 standard, control AVM tissues, AVM tissues treated with 10 μg/ml doxycycline, and AVM tissues treated with 100 μg/ml doxycycline.</strong> In AVM tissues, there were proteolytic bands corresponding to pro-MMP-9 (≈97 kDa) and active-MMP-9 (≈88 kDa).</figcaption> </figure> <figure> <p><img src="" /></p> <figcaption><strong>Active and total MMP-9 levels after 48 hours of ex-vivo doxycycline treatment.</strong> <strong>AVM-I:</strong> Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 There was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. <strong>AVM-II:</strong> Doxycycline 10 μg/ml significantly decreased active and total MMP-9. There was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. (mean ± SD)</figcaption> </figure> # AVM-II AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery. Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1--10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4. There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P < .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P < .05) (Figure [ 4C](#) &[4D](#)). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. # Pilot clinical trial 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design. 10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table [1](#). Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted. ::::table-wrap

::: caption
Characteristics of AVM patients enrolled in a pilot clinical study to
test effects of oral doxycycline treatment on MMP-9 in AVM lesions.
:::

Group Age Gender S-M score\* AVM size\*\* Draining veins History of hemorrhage Number of embolization treatments

______________________________________________________________________

Dox 50 M 4 38 Superficial & Deep No 1
Dox 48 F 4 30 Deep Yes 0

Dox 52 M 4 42 Superficial & Deep Yes 2

Dox 43 M 3 29 Superficial & Deep No 1

Dox 23 M 4 34 Superficial & Deep No 2
Dox 28 M 4 40 Superficial Yes 2
Dox 56 M 2 10 Superficial No\
Dox 41 F 3 30 Superficial No 1
Dox 62 M 2 41 Superficial No 1
Dox 42 M 2 2 Superficial Yes 0

Placebo 53 M 2 28 Superficial & Deep No 1
Placebo 17 M 1 18 Superficial No 1
Placebo 48 F 2 13 Deep No 0
Placebo 22 M 3 19 Deep Yes 0

- Spetzler-Martin score (15), \*\*AVM maximum diameter (10). Dox =
  doxycycline:::: There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure [5](#)). <figure> <p><img src="" /></p> <figcaption><strong>Effects of oral doxycycline treatment on MMP-9 in AVM tissues.</strong> There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group. (mean ± SD)</figcaption> </figure>


Extracted trial result:
Outcome: matrix metalloproteinase-9 (MMP-9) (continuous)
Intervention: doxycycline (100 mg)
Comparator: placebo
Intervention group size: 10
Intervention mean: 2.18
Intervention standard deviation: 1.94
Comparator group size: 4
Comparator mean: 3.26
Comparator standard deviation: 3.58
Notes: found in abstract