text:
"A Piece Of Your Mind" Review- A Drama That Could've Been So Much Better, Here Is What Ruined Its Chances At Success
Published April 30, 2020 2:53 am By Jass K.Categorized as Kdrama Reviews
"A Piece Of Your Mind" aired its last episode last night and since I've seen it and completed the series successfully, I thought it would be a good idea to talk about it and why I personally couldn't fully support it despite the actors' stellar performances.
Now I know some of you are already triggered but there are reasons for what I said above, so before you judge, read this article. I know I am probably of the few who liked this drama but not enough to call it a 'great drama.' Those who stopped watching did so because of valid reasons and those who persisted and loved it also have valid reasons.
Note: this will be a spoiler review of the series.
Of course, we're talking about the script here; I have no issues with the acting or the directing or anything else pertaining to production for that matter.
Before we begin, I would like to point this out:
Dismissing Koreans decision to stop watching this drama is honestly very childish, it's very understandable in this case, in this case particularly.
Sometimes its competition, or terrible acting and shitty marketing but this time, there were clearly reasons valid enough for people to stop tuning in. Just because you decided to continue and felt rewarded in having done so, doesn't invalidate the decision of those who decided to drop the drama.
"A Piece Of Your Mind" was reduced from 16 to 12 episodes due to poor reception. A very terrible thing to happen to any production but I understand why it happened, today, I will discuss my personal issues with the series and explain why I believe it could've been one of the best but ended being average.
The problematic creepy Ha Won
I've stated this before, but Ha Won's way of thinking is deeply flawed and concerning. My issue with "A Piece Of Your Mind" wasn't that he behaved this way, but the way the show portrayed it felt problematic.
It's okay if the show recognizes he has deep psychological issues but they briefly address it then dismiss it and move on. A lot of things he says and does in the first half of this drama were very creepy.
Ha Won lives his life longing for a person he's never been romantically involved with; he misses her for 10 years after she clearly rejects him and tells him she's getting married to another man. If their relationship was platonic, I think I it would've made more sense, they spent a long time together, they're like family.
Ha Won is stuck in the past refusing to move on from her, he's unable to love another woman and continues to dream of one day meeting her, he stalks her on social media and eventually enlists the help of Seo Woo to make this happen.
The show portrays Ha Won like the hopeless romantic but if you're not fully aware and paying attention you'll almost fall for his toxic and creepy way of thinking. At some point in the plot, we found out he told her to come find him if she's miserable, like dude, are you serious? How full are you of yourself? She doesn't own you shit.
Nothing explains why he behaves that way and continues to do so despite being a levelheaded human being in all other aspects of life. "A Piece Of Your Mind" is about why its characters behave a certain way, we find out the way they think throughout the drama, but Ha Won, the one who needed to be explored the most, isn't even explored.
His qualities contradict each other fundamentally. Ha Won is a 'nice' character but if a man like him showed up to me in real life, I'd put out a restraining order.
I also didn't like the fact that he recorded his first love voice without her knowledge or consent and continued to use her voice as an experiment and a project to work on among his peers.
If this happened to my family, I'd be livid. Saying he wants to know and wants to speak to the device to understand Jisoo and live through her perspective feeling everything she felt is very weird and creepy. Aside from being an unattainable goal since she's already dead, it's very selfish and problematic.
And even after her death, Ha Won is still unable to move on until something switches in his brain or to put it precisely the writer decided so after finding out people weren't going to stick around to see this, he falls for Seo Woo against all odds and starts a romantic relationship with her.
"A Piece Of Your Mind" is about moving on from tragedy, it's about choosing to live and finding solace in the people you love. It's also about communicating your feelings to others. however, the entire premise of "A Piece Of Your Mind" relies on a man who couldn't move on from someone, the fact that shortly after she died he begins to have feelings for another woman is odd to me, like how could he just change like that? The fact that she's dead should make him feel even more miserable and lead to more problematic behavior but his love for Seo Woo suddenly heals him…. get outta here!
The 'Healing' Device
"A Piece Of Your Mind" has a plot point that was very confusing. I learned that Koreans don't like confusing plots, "The King: Eternal Monarch," too, is facing backlash for being too confusing, it even saw a dip in ratings because of this, there is no way "A Piece Of Your Mind" was going to survive that critique point.
"A Piece Of Your Mind" doesn't explicitly explain but shows you, which I actually like but the thing is, it's showing something that is very difficult to comprehend unless explained.
Eventually, we learn that Ha Won is working on devices that could help humans move on with their lives and heal; the devices are AI machines of people's thoughts. The device eventually picks up on the owner's habits, speech and personality.
There is also something called 'a point of reaction,' something that needs to be said in order to fully activate the device.
The bigger issue than Ha Won's behavior is this plot point which is by far one of the most absurd I've seen for a plot that's about life and reality.
If this was a fantasy drama, my tone would be very different but "A Piece Of Your Mind" is about humans, life and death. It's grounded in reality, so to introduce such a foreign concept that isn't even clearly explained until episode 6 to 7 is a grave mistake from a writer who's been working since 2001, yes, 2001.
I don't understand the screenwriter decisions here, in my opinion; her decisions for the plot ruined what could've easily been an amazing project.
She took advantage of the fact that Jung Hae In has a huge following dedicated enough to be patient with the slow pacing and illogical theme, but eventually, it backfired because why would busy people waste their times when there are better dramas on?
The 'healing device' is an important point in "A Piece Of Your Mind" and is not something to be glossed over or poorly explained as well. It needed to have been fully established and explained as logically as possible so viewers could easily follow along and enjoy the ride instead of wondering what it all meant.
Jisoo's Device Without Logic
This ties to the 'healing device' point and is something I haven't seen discussed enough at all. It should be the center of a lot of conversations about the plot but despite its importance for the advancement of it, it doesn't make an ounce of sense.
Jisoo dies and Ha Won programs a device with her voice, somehow the device replicates her feelings, thoughts and a bunch of situations there is no way on earth a device could've obtained.
And yes, I know the device conversed with Ha Won and Seo Woo and that's how it got a lot of information but it's still one sided and in no way a clear explanation of how it knows so much about things no one told it about…
This is me assuming the 'healing device' is an actual thing that existed in real life. I still can't comprehend how the screenwriter thought of this. Jisoo died, she only recorded a couple of times, and no matter how sophisticated a device is, it won't be able to generate memories on its own. However, it somehow converses with characters as if it knows Jisoo's logic when no one knows, she's dead.
It also knows about situations that Jisoo never mentioned such as the ones about her husband, or how she felt after ending meetings with Ha Won and Seo Woo. HOW?
I just can't comprehend this part and every single time it's brought up, it stands like the elephant in the room and I unable to take this drama as seriously as I should. A lot of issues happen because of Jisoo's device and it creates scenarios and drives the plot forward so the least it could do is make sense.
"A Piece Of Your Mind" is good, it's not revolutionary or out of this world, it's just fine.
It needs patience and if you wait until the second half, you'll get what you were looking for.
It's a good healing drama for those who enjoy such content; my favorite parts were the ones that didn't focus on the device but rather on people's emotions and insecurities.
My favorite character is Seo Woo. I just loved loved Chae Soo Bin's performance here and felt so bad for her, her feelings felt genuine and real. She's easily my favorite part about this drama. She and Jung hae in had good chemistry.
Every single actor performance in this drama is epic; I think they truly made this drama watchable. I don't know if I would have stayed if the actors were different. You can feel their passion and that's something to appreciate.
I also liked the flow of conversations a lot; it felt that these characters were human with flaws, every conversation felt unique despite some of them being so ordinary.
I also liked how much it focused on things all of us deal with, what's considered trivial things are front and center. To someone who's struggling with the loss of their loved ones, this drama will strike a chord.
The aesthetics are pleasing, the setting and attention to details is also amazing. The OST perfectly matches the aesthetics and feel of the drama.
Overall, "A Piece Of Your Mind" could've been better, it could've easily become one of the best of 2020 but the screenwriter took things for granted.
A little banter about Jung Hae In's choices
If you don't follow Jung Hae In, you probably won't understand this section. But for those who do, you'll easily understand what I mean.
Jung Hae In gives a Jung Hae In-ess performance here; there is nothing new or exciting about it. I've seen all of his projects since he became famous and this drama is very similar to the majority of characters he played in his recent works, which disappoints me because I expected a different approach or at least a different acting method overall.
This doesn't mean he was terrible, he did pretty well but if you watched any of his recent projects, there is nothing exciting anymore about watching him basically play the same character.
Jung Hae In is a good actor but I am starting to wonder whether he's too scared to get out of his comfort zone or confortable doing only these types of roles. It's up to him to pick his projects but if he continues to play the same roles, people wouldn't have a reason to be excited about what he puts out next.
He needs a shocking role to shake his image; an OCN drama would be nice. I want him to play a despicable character that is legitimately bad, I am so curious to see if he can pull it off. I don't want him to play a stereotypical bad guy, but a 4D psycho.
What pains me the most about "A Piece Of Your Mind" is the potential it had, but at the same time, as I previously explained, I don't blame those who decided to drop the drama. There are serious flaws with the script that can be easily noticed with the naked eye, but there are also lots of great things about this drama.
What did you guys personally think about "A Piece Of Your Mind"? do you agree or disagree with me?
Tagged A Piece of Your Mind
L nandi says:
May 1, 2020 11:26 am at 11:26 am
Yes I almost agree with whatever you said. Ha won was infact a little bit creepy. And his character development doesn't make any sense. A person who was not over his first love suddenly changes after her death does not not add up. The device makes less sense. I guess there is an explanation which we never get to see. Seo Woo was indeed charming. Ha Won is also a great character and except for the creepy part I completely understand what Ji soo was to him. And also I think he asked her to call him when she was miserable because Ji Soo loved him as her friend and saw him as family. So I don't think there's wrong in that part. Ha won in the second part of the drama was great too. The way he falls in love and takes care of Seo Woo. It's just not the same person. The first part Ha Won can never become the second part Ha won in that short span of time. But both were beautiful in their own way. Allover I lived watching it. But it could have been greater than this.
qistina najiha says:
May 1, 2020 7:54 pm at 7:54 pm
after reading the whole article i know that i wasn't alone. this article gives a very satisfying feeling and i can't disagree on any of your points. i also look forward for any new improvisation on jung hae in next role
MARTINE SYNAKOWSKI says:
I agree about all you say….
I feel same exactly, i was so happy to find my 2 prefered actors. I disovered Chae Soo Bin, in "I'm not a robot", and since i have seen all her dramas… She is without any doubt one of the best Korean Dramas actress…. She is not same to others, she has this "little +" missing on others…
I don't lie if i say that Jung Hae In, is my very very prefered actors , and i'm thinking exactly as you, i wait him for a "bad" but not too "bad" boy … lol. I was near happy when i saw him to fight with Jisso's husband, i feel i has a big potentiel in this kind of acting
I'm waiting now, for both of them future projects….
So sorry for my english, i'm french but hope every one will understand. Thank to you Jasmine media .
Devon says:
March 18, 2021 9:22 am at 9:22 am
I agree with your points on the plot. I disagree with your comments on Jung Hae In's performance. He's a very meticulous performer and television requires a lot of subtlety. You need to look at his body language and postures between each of his projects to see the differences in his characters. He takes on a different physicality with each and has different postures and movements for each character. If you can't spot them, then you're not looking close enough. As a trained performer with a lot of experience, it's very easy for me to see these things. He's a great actor who changes a lot between his various roles but it's more subtle and naturalistic. If you want to see him in something different, then watch him in "The Age of Blood." You won't even know it's him unless you look closely. In "Age of Blood" his performance has characteristics of some of the stock, larger than life, characters you see in action films, including the cocky bravado and snappy come-backs. He's also just plain hot in that film. He's handsome as it is, but in "Age of Blood" he's dangerously handsome. You should check it ou.
Amy Amano says:
May 11, 2022 12:03 am at 12:03 am
You have started with incorrect facts of this story. It's fine if you didn't like it. But you are just showing that you weren't paying attention to what was actually going on. Ji Soo and Ha Won were never lovers. They were best friends. They were not in contact for a few years, then met up so she could tell him she was getting married. Then they didn't meet for 9 more years. He didn't stalk her social media or know who she married. That's just to start.


text:
There are three basic types of signature in DROID:

  * file extensions (least reliable, just matches the file extension of a file)
  * binary signatures (very reliable, looks for complex patterns inside a file)
  * container signatures (extremely reliable - looks for internal files and patterns inside them)

Binary signatures are compiled by the PRONOM registry into binary signature XML for publication to DROID.  Container signatures cannot currently be compiled by PRONOM into signature XML files.  This can make developing container signatures harder than binary signatures, as the signature file XML is hand written (see `sigtool` description in [README](README.md#sigtool) for details of a tool which can produce this XML automatically).

## Syntax
The signature syntax compiled into XML by PRONOM is the original syntax defined for DROID, and should be backwards compatible with all versions of DROID.  This syntax is used in binary signature files.

Container signatures support a wider syntax than binary signatures, for example allowing whitespace and ASCII strings. For the most part, this extended syntax just makes the signatures more human readable.  In a few cases some new capabilities are supported that PRONOM can't currently compile for binary signatures.

All of the syntax can be used in either binary or container signatures in DROID itself, but PRONOM won't be able to compile container syntax into XML for binary signatures if you want to submit them to TNA.

Container specific syntax is marked in the descriptions below.

### Value matching
The following syntax can be used to match bytes.

#### Bytes:  00 FF
Bytes to match are written as two digit, case insensitive hex values.
```
ff Fe A1 00
```

#### Strings:  'Latin1'
**Container signature syntax**
Strings of ISO-8859-1 characters (Latin1) can be written enclosed in single quotation marks.
```
'version:4'
```

A single character enclosed in single quotes can also be used as in place of a byte value:
```

30 '0'   # These both encode the character zero, one is a hex byte, the other is the character directly.
```
_Note:_ due to a bug in the byteseek library, only ASCII characters are currently supported by DROID.  Characters over 127 may cause incorrect compilation, as it attempts to render the characters in the system default character set, which is probably not ISO-8859-1.

#### Byte ranges: \[n:m]
To match a byte within a range of values, we can use a byte range. This is written as two byte values separated with a colon, all enclosed in square brackets.  Bytes are specified as 2 digit hex values.
```
[30:39]
```

**Container signature syntax**
Using a hyphen is also permitted in range separators for container signatures, and you can specify the range using single character string literals:
```
[30-39]
['0'-'9']
```

#### Inverted ranges: \[!n:m]
To match a byte which is outside a range of values, we can invert a byte range, by placing an exclamation mark immediately after the first square bracket.
```
[!61:7a]
```
**Container signature syntax**
Using a hyphen is also permitted in inverted range separators for container signatures, and you can specify the range using single character string literals:
```
[!61-7a]
[!'a'-'z']
```

#### Bitmask: \[&FF]
To match a particular set of bits in a byte, we can use a bitmask, which is a hex value.  It will match all bytes which have the same bits set to one in the bitmask.  For example, to match all the bytes which have the first bit set to one, we could write:
```
[&01]
```
And to match all the bytes without the first bit set, we can invert it like this:
```
[!&01]
```
This will match all the bytes which have the 8th or 4th bit set (10001000):
```
[&88]
```
_Note:_ the bitmask is not a standard part of the original PRONOM syntax; it originates in the byteseek matching library used by DROID.  However, it is now being used in both binary and container signatures, although older versions of DROID won't be able to parse it in binary signatures.

### Alternatives: (00|F0|3C)
If there is a set of different values that can match, they can be specified inside round brackets, with the alternatives separated by the | character.
```
(00|F0|3C)
```
Alternatives can be longer than just a single byte.  In this example, three byte sequences could be matched.  Alternative sequences don't have to be the same length as each other:
```
(00 01 | B0 B1 B2 | C0 C1 C2 C3)
```
**Container signature syntax**
Older binary signatures should only use hex bytes inside alternatives.  Container syntax supports any byte matching values inside an alternative, except another nested set of alternatives.
```
('start' | 'end' | 01 FF 32 'EOF' [30:39])
```

#### Multi-byte sets: (00|C2|DE) or \[00 C2 DE]
The standard PRONOM binary syntax only supports multi-byte sets by specifying them as a set of alternative bytes.  For example:
```
(00|C2|DE)
```

**Container signature syntax**
Multi byte sets let you specify any set of bytes within square brackets.  For example:
```
[00 C2 DE]
```
They can be inverted just like ranges (which the alternatives syntax doesn't support):
```
[!00 C2 DE]
```
It can also include ranges or any other value matching syntax within the set:
```
[00 C2 DE 'A'-'Z' &01]
```
Strings can be used to specify particular byte values.  Note that the string itself doesn't match - the set matches any of the characters in the string:
```
['A'-'Z' 'aeiou']
```

### Wildcards
The following syntax can be used to specify sequences of unknown bytes, including variable or unlimited ranges of unknown bytes which cause DROID to search for the next part of the expression.

#### Any byte ??
To match any byte, we can use two question marks:
```
01 02 ?? 04
```

#### Unlimited gaps *
To specify that there's a unlimited gap between two parts of an expression (subject to how far DROID is configured to actually scan or the end of the data, whichever comes first), we can write an asterisk.

The following expression will first match _30 31 23 33 4E_, and it will then search for _43 2A B1 D4 CC EF_ until the end of the data:
```
30 31 23 33 4E * 43 2A B1 D4 CC EF
```

#### Fixed gaps {n}
To specify that there's a gap of one or more bytes we don't care about, we can write the size of the gap as a decimal number, surrounded by curly brackets:
```
01 02 03 {128} FF FE
```

Note that writing _{1}_ is equivalent to writing _??_.

#### Variable gaps {n-m}
To specify that there's a range of possible bytes we don't care about, we can write the range as two numbers separted by a hyphen, surrounded by curly brackets:
```
01 02 03 {128-256} FF FE
```

#### Min to many gaps {n-*}
To specify there must be a minimum gap, but after that it could be unlimited, we can write:
```
01 02 03 {128-*} FF FE
```
This is equivalent to writing a fixed gap followed by an unlimited gap:
```
01 02 03 {128} * FF FE
```

#### Whitespace
**Container signature syntax**
All whitespace (space, tab, newline, carriage return) between elements is ignored when parsing.  You could write a single expression as:

```
00 01
'version:' ??
FE FF
```

redpajama_set_name: RedPajamaGithub


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Research ArticleArticle
HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans
Kajsa P. Kanebratt and Tommy B. Andersson
Drug Metabolism and Disposition January 2008, 36 (1) 137-145; DOI: https://doi.org/10.1124/dmd.107.017418
Kajsa P. Kanebratt
Tommy B. Andersson
HepaRG is a highly differentiated cell line that displays several hepatocyte-like functions, including drug-metabolizing enzymes. In this study, the HepaRG cells were characterized and evaluated as an in vitro model to predict cytochrome P450 (P450) enzyme induction of drugs in humans. Exposure of HepaRG cells to prototypical inducers resulted in induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA, as well as phenacetin O-dealkylase, bupropion hydroxylase, diclofenac 4′-hydroxylase, and midazolam 1′-hydroxylase activities. The observed induction is consistent with the previously reported expression of the nuclear receptors pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor, which are necessary for a P450 induction response. To avoid problems with toxicity and solubility, the induction potency of test compounds was evaluated by calculating the concentrations leading to a 2-fold increase of baseline mRNA or enzyme activity levels (F2 values) instead of EC50 values from full dose-response curves. For CYP3A4 mRNA, the obtained F2 values were related to the in vivo exposure [area under the plasma concentration versus time curve (AUC)] of the inducer (AUC/F2). This score was then correlated with the decrease in AUC for a CYP3A probe drug, administered before and after treatment with the inducing agent. By using this method an excellent correlation (R2 = 0.863) was obtained, which implies that the degree of CYP3A induction in vivo can be predicted from CYP3A4 mRNA induction in HepaRG cells. The present study shows that HepaRG cells are a valuable model to be used for prediction of induction of drug-metabolizing P450 enzymes in vivo in humans.
The cytochrome P450 (P450) enzymes have been shown to be responsible for the metabolism of the majority of therapeutics and are therefore important for first-pass elimination (bioavailability), clearance, and drug-drug interactions. Drug-drug interactions may be the result of both inhibition and induction of P450 enzymes. P450 inhibition studies using human liver microsomes or recombinant enzymes and model substrates for specific P450s are well established in vitro methods for quantitative predictions of inhibition potency in vivo (Ito et al., 1998; Bjornsson et al., 2003). On the contrary, quantitative predictions of induction in vivo from in vitro results are less well established. Because induction is subject to important species differences, prediction of possible induction in humans cannot rely on animal studies. Human hepatocytes have been suggested by several reports as a preferred model for induction studies (Li et al., 1997; Madan et al., 2003). Nevertheless, primary human hepatocytes are flawed with several drawbacks, such as experimentally demanding methods, phenotypic changes during culture, scarce availability, interindividual variation, and the need to evaluate multiple donors, which makes the model arduous. The demanding technique is probably the reason why no quantitative predictions of P450 induction in vivo using human hepatocytes have been published.
Induction of P450s generally occurs at the transcriptional level and is mediated by receptors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), and the aryl hydrocarbon receptor (AhR). PXR is a transcription factor that is widely accepted as the major determinant of CYP3A4 gene regulation by xenobiotics (Lehmann et al., 1998; Gibson et al., 2002) and has also been established to be important for CYP2C9 induction (Chen et al., 2004), whereas CAR has been proposed to regulate the expression of CYP2B6 (Sueyoshi et al., 1999; Wang et al., 2004). However, during recent years there has been increasing evidence of an extensive cross-talk between nuclear receptors, indicating that CAR and PXR affect the regulation of all three P450 enzymes (CYP2B6, CYP2C9, and CYP3A4) (Lin, 2006). The CYP1A enzymes are regulated by AhR, and prototypical AhR ligands are planar, hydrophobic, and halogenated hydrocarbons such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (Denison and Nagy, 2003).
Reporter gene assays for PXR and AhR have been developed and used as convenient high-throughput tools for the determination of a compound's ability to induce CYP3A and CYP1A. Results from PXR reporter gene assays were recently successfully used to classify clinically used drugs as CYP3A inducers or noninducers (Persson et al., 2006; Sinz et al., 2006). However, because induction of P450 enzymes by drug molecules does not only depend on a single transcription factor but is mediated by a combination of several transcription factors in the cell, a human hepatocyte-like cell line would be a superior in vitro model for evaluation of P450 induction. A promising cell line is the HepaRG cells, which display several hepatocyte-like functions including drug-metabolizing enzymes, nuclear receptors, and hepatic drug transporters and can be differentiated into a hepatocyte-like morphology (Aninat et al., 2006; Le Vee et al., 2006). The HepaRG cells have also been shown to respond to prototypical P450 inducers such as 3-methylcholantrene, rifampicin, and isoniazid (Aninat et al., 2006).
The aim of the present study was to further characterize the P450 induction response in HepaRG cells and to develop a method that can be used to predict the extent of P450 induction in vivo.
Chemicals. Carbamazepine, dexamethasone, diclofenac, dimethyl sulfoxide (DMSO), nifedipine, phenobarbital, rifampicin, sulfinpyrazone, and Williams' E medium without phenol red were purchased from Sigma Chemical Co. (St. Louis, MO). Paracetamol, phenacetin, and primaquine were purchased from Aldrich Chemical Co. (St. Louis, MO). Hyperforin was purchased from Apin Chemicals Ltd. (Oxon, UK). Williams' medium E with phenol red and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA). Omeprazole and troglitazone were provided by AstraZeneca R&D Mölndal (Mölndal, Sweden). 4′-Hydroxydiclofenac was obtained from BD Gentest (Woburn, MA), and 1′-hydroxymidazolam was purchased from Ultrafine (Manchester, UK). SuperScript III First-Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) and TRIzol reagent were acquired from Invitrogen (Carlsbad, CA). Midazolam was purchased from Larodan Fine Chemicals AB (Malmö, Sweden). Bupropion was purchased from Kemprotec Ltd. (Middlesbrough, UK), and hydroxybupropion was provided by Toronto Research Chemicals Inc. (North York, ON, Canada). LDH opt, reference LD401, was purchased from Randox Laboratories Ltd. (Crumlin, UK), and Triton X was purchased from Acros Organics (Geel, Belgium). The primers and probe used in this study were provided by Applied Biosystems (Cheshire, UK). Taqman Assay on Demand and Taqman Universal Master Mix were purchased from Applied Biosystems (Foster City, CA). All the other chemicals were of analytical grade and highest quality available.
Cell Culturing and Induction. The differentiated HepaRG cells (passages 14–17) were purchased from Biopredic International (Rennes, France). Twenty-four-well plates, seeded with 0.05 million cells, were used for all the experiments. The cells were initially grown in Williams' medium E with Glutamax I, supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml bovine insulin, and 50 μM hydrocortisone hemisuccinate. At confluence, 2% DMSO was added to the medium to differentiate the cells into a hepatocyte-like morphology. The cells were cultured in differentiation medium for 3 weeks before shipment to AstraZeneca. At arrival, the medium was renewed, and the cells were given 24 h to recover before the medium was changed to basal HepaRG medium (Williams' medium E with Glutamax I, supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 4 μg/ml bovine insulin, and 50 μM hydrocortisone hemisuccinate). The cells were cultured in the basal medium for 5 days before the induction started, and the medium was renewed every 24 to 48 h.
The cells were induced with rifampicin (0.004–4.0 μM), omeprazole (0.04–40 μM), troglitazone (0.024–25 μM), primaquine (0.04–40 μM), phenytoin (0.04–40 μM), phenobarbital (0.20–200 μM), carbamazepine (0.24–250 μM), dexamethasone (0.24–250 μM), nifedipine (0.24–250 μM), sulfinpyrazone (0.24–250 μM), and hyperforin (0.24–250 μM). Test compounds were dissolved in DMSO and added to the plates in basal HepaRG medium with a final DMSO concentration of 0.1% in all the incubations. Control incubations contained basal HepaRG medium and 0.1% DMSO. For mRNA measurements, the cells were harvested in TRIzol reagent (0.5 ml/well) after 24-h exposure to the study compounds, and experiments were repeated at three different occasions on separate batches of differentiated HepaRG cells. P450 activities were measured directly in the plates after 48-h exposure to the study compounds, and experiments were performed in duplicate and repeated at two different occasions on separate batches of differentiated HepaRG cells.
Viability. The viability of the cells in the dose-response experiments was tested by measuring the lactate dehydrogenase (LDH) activity before and after 24-h exposure to the study compounds. LDH was measured by using a commercial reagent kit (LDH opt) and was performed with a Cobas Bio centrifugal analyzer (Hoffman-La Roche, Basel, Switzerland).
RNA Extraction. Total RNA from HepaRG cells was prepared using TRIzol reagent according to manufacturers' instructions. Quantity and purity of the RNA were determined spectrophotometrically using a GenQuant pro RNA/DNA calculator (Biochrom, Cambridge, UK). Electrophoretic separation of 0.5 μg of total RNA on a 1% agarose gel run in Tris borate-EDTA buffer (0.09 M Tris-borate, 2 mM EDTA, pH 7.8) at 80 mV for 1 h allowed integrity assessment of the isolated RNA. Two sharp ribosomal RNA bands and absence of RNA debris were set as quality criteria to proceed to cDNA synthesis.
cDNA was prepared from 1 μg of total RNA using the SuperScript III First-Strand Synthesis System for reverse transcription-PCR with random hexamer primers according to the manufacturer's protocol.
Real-Time PCR. Real-time PCR for human P450 mRNA levels was performed using a 7500 Sequence Detector (Applied Biosystems, Foster City, CA) and manufacturer-designed Assay on Demand for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and glyceraldehyde-3-phosphate dehydrogenase. For human acidic ribosomal phosphoprotein (huPO), gene-specific double fluorescent labeled probes were used as reported previously (Persson et al., 2006). The reaction mixture (25 μl/well) contained 30 ng of cDNA, 1×Taqman Universal Master Mix, optimized concentrations of primers and probes (huPO), or 1.25 μl of Assay on Demand and RNase free water. The thermal cycle conditions were identical for all the genes analyzed and had initial steps of 50°C for 2 min and a 10-min step at 95°C, followed by 40 PCR cycles of 95°C for 15 s and 60°C for 1 min. Each sample was analyzed in triplicate, and data were analyzed using the 7500 Sequence detector software version 1.3.1 (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase and huPO were used as endogenous controls, and the amount of mRNA was determined relative to that from control samples.
Activity Measurements. For cells used for activity measurements, induction medium was renewed after 24 h. After 48 h, induction medium was removed, and the cells were washed twice with Williams' medium E without phenol red; thereafter, 500 μl of activity medium was added to each well. The activity medium consisted of Williams' medium E without phenol red, pH 7.4, and bupropion (100 μM) or a mixture of phenacetin (26 μM), diclofenac (9 μM), and midazolam (3 μM). The compounds were dissolved in methanol and added to a Falcon tube. The methanol was evaporated under nitrogen gas, and the compounds were dissolved in Williams' medium E without phenol red so that the activity medium did not include any organic solvent. After 15, 30, 45, and 60 min (for bupropion) or 0.5, 1, 2, and 3 h (for the mixture), 100 μl of sample was taken out. Fifteen microliters of cold acetonitrile with 0.8% formic acid was added, and the samples were analyzed for the CYP2B6 metabolite hydroxybupropion or the CYP1A2 metabolite paracetamol, the CYP2C9 metabolite 4′-hydroxydiclofenac, and the CYP3A4 metabolite 1′-hydroxymidazolam.
After the last sample was taken, the final medium was removed, and the cells were lysed with 200 μl of 1% Triton X. The wells were scraped, and the lysate was used for protein determination by a modified Lowry procedure (Markwell et al., 1978).
The samples were analyzed at separate occasions by means of liquid chromatography (LC)/mass spectrometry. The LC system consisted of an HP 1100 series LC pump and column oven (Agilent Technologies, Santa Clara, CA) combined with an HTS PAL injector (CTC Analytics, Zwingen, Switzerland). For hydroxybupropion, 4′-hydroxydiclofenac and 1′-hydroxymidazolam LC separations were performed on a reversed-phase HyPurity C18 column (2.1 × 50 mm, 5 μm, ThermoQuest, Runcorn, UK) with a HyPurity C18 precolumn at 40°C and with a flow rate at 750 μl/min. The mobile phase consisted of 0.1% (v/v) formic acid (A) and 0.1% (v/v) formic acid in acetonitrile (B). The organic modifier content B was increased linearly from 5 to 80% B over 3 min and then back to 5% B in 0.2 min.
For paracetamol, chromatography was performed on a Zorbax Eclipse XDB-C8 column (4.6 × 150 mm, 5 μm; Agilent Technologies) with a HyPurity C18 precolumn using the same system and mobile phase. The organic modifier content B was increased linearly from 2 to 30% B over 5 min, then from 30 to 80% over 2 min, and then back to 2% B in 0.1 min. The retention times of hydroxybupropion, 4′-hydroxydiclofenac, 1′-hydroxymidazolam, and paracetamol were 2.0, 2.9, 2.4, and 6.4 min, respectively. Detection was performed with a triple quadrupole mass spectrometer, API4000, equipped with electrospray interface (Applied Biosystems/MDS Sciex, Concord, ON, Canada). The mass spectrometry parameters were optimized using each analyte. The compound dependent parameters were as follows: the collision energy was set at 20, -15, 39, and 21 V for hydroxybupropion, 4′-hydroxydiclofenac, 1′-hydroxymidazolam, and paracetamol, respectively. Collision-activated dissociation gas was at 5, 5, 7, and 5, respectively. The multiple reaction monitoring transitions chosen were 256.1 > 237.8 for hydroxybupropion, 309.9 > 265.9 for 4′-hydroxydiclofenac, 342.0 > 202.7 for 1′-hydroxymidazolam, and 152.3 > 110.0 for paracetamol. A dwell time of 200 ms was used. Instrument control, data acquisition, and data evaluation were performed using Applied Biosystems/MDS Sciex Analyst 1.4 software.
Metabolite formation was calculated in the linear range of the time curve, and for hydroxybupropion the formation rate at 30 min was used. For paracetamol, 4′-hydroxydiclofenac, and 1′-hydroxymidazolam, the formation rate at 2, 1, and 1 h was used, respectively.
Curve-Fitting and Calculation of Induction Response. All the curve-fittings were carried out with XLfit 4.1.1 (ID Business Solutions, Emeryville, CA). Dose-response data were fitted to a Hill equation for one-site dose-response as follows:
where Emin is the background, Emax the maximum effect, EC50 is the concentration giving 50% of Emax, k is the slope of the curve, and C is the drug concentration. A fit to the data points is created even if a plateau in the effect is not reached, but Emax values are only reported if a plateau is obtained. From this fit the F2 values also were calculated. The F2 value is the concentration leading to a 2-fold increase of the baseline levels. This approach was used by Weiss and Haefeli (2006) and found useful for evaluation of inhibition of P-glycoprotein.
The F2 values were related to the in vivo exposure of the test drug, represented by the area under the plasma concentration versus time curve (AUC). This score, AUC/F2, was then ranked from lowest to highest value.
In vivo induction is measured by administration of a drug metabolized by the enzyme of interest, called a probe drug. Examples of probe drugs are caffeine for CYP1A2, tolbutamide for CYP2B6, and midazolam for CYP3A4. The probe drug is administered before and after administration of the potential inducer, and the induction is measured as the decrease in AUC of the probe drug. The correlation between in vitro and in vivo induction in the present study was assessed by plotting AUC/F2 versus the percentage decrease in AUC of a coadministered CYP3A probe drug. The data were fitted to an equation as follows:
Viability. The viability of the cells was analyzed by measuring LDH activity in the medium before and after 24-h exposure. No effect on the cells, as evidenced by no increased LDH activity in the medium, was detected after incubations with carbamazepine, dexamethasone, omeprazole, phenobarbital, primaquine, rifampicin, and troglitazone. Hyperforin, nifedipine, phenytoin, and sulfinpyrazone increased LDH activity at all the tested concentrations, although not in a dose-related fashion, and dose-response curves were still achieved. Hyperforin, nifedipine, and troglitazone displayed the highest induction at 62.5, 0.4, and 6.25 μM, respectively, whereas the induction response was weaker at the highest concentration (250, 2, and 25 μM, respectively). The weaker induction response by troglitazone at the highest concentration was not reflected by LDH leakage, suggesting that the induction response by troglitazone in this study was not affected by acute cell toxicity.
Induction of P450 mRNA. In initial experiments, HepaRG cells were treated with omeprazole, 2,3,7,8-tetrachloro-dibenzo-p-dioxin, phenobarbital, and rifampicin. Results indicated that the induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA peaked after 24 h. No induction was seen for CYP2D6 mRNA, and the mRNA for CYP2D6 was not analyzed in subsequent experiments. Therefore, the HepaRG cells were exposed to the study compounds rifampicin, omeprazole, troglitazone, primaquine, phenytoin, and phenobarbital for 24 h for mRNA experiments. Representative dose-response curves for CYP1A1 and CYP1A2 mRNA induction by omeprazole and for CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA induction by rifampicin are shown in Fig. 1. In Tables 1 and 2, the F2 values and the -fold induction at the highest response measured for CYP1A1, CYP1A2, and CYP2B6 mRNA by the test compounds are presented. When a full dose-response curve was obtained and no sign of toxicity or solubility problems was observed, the EC50 value also was calculated. CYP1A1 and CYP1A2 mRNA was induced by omeprazole and primaquine, whereas no effect was seen by the other tested compounds (Table 1). CYP2B6 mRNA was strongly induced by rifampicin, omeprazole, troglitazone, phenytoin, and phenobarbital, whereas primaquine did not affect CYP2B6 transcription (Table 2). Induction of CYP2C8, CYP2C9, and CYP2C19 mRNA was seen after exposure to rifampicin (Table 3), whereas omeprazole, troglitazone, phenytoin, and primaquine did not significantly affect the CYP2C mRNA levels. Phenobarbital gave a weak induction of CYP2C mRNA, but the average -fold induction at the highest concentration was low (2.6-, 1.7-, and 2.5-fold for CYP2C8, CYP2C9, and CYP2C19, respectively), and no average F2 values could be calculated. A larger set of compounds was tested for CYP3A4 mRNA induction. Rifampicin, omeprazole, troglitazone, phenytoin, phenobarbital, carbamazepine, dexamethasone, hyperforin, nifedipine, and sulfinpyrazone all induced CYP3A4 mRNA, whereas primaquine did not affect CYP3A4 mRNA levels (Table 4).
F2 values and -fold induction at highest concentration tested (given in parenthesis) for induction of CYP1A1 and CYP1A2 mRNA in HepaRG cells treated with rifampicin, omeprazole, troglitazone, phenytoin, primaquine, or phenobarbital F2 is the concentration resulting in a 2-fold increase of baseline mRNA levels. EC50 values are presented when achieved. Values are mean ± S.D. of three separate batches of differentiated HepaRG cells.
F2 values and -fold induction at highest concentration tested (given in parenthesis) for induction of CYP2B6 mRNA in HepaRG cells treated with rifampicin, omeprazole, troglitazone, phenytoin, primaquine, or phenobarbital F2 is the concentration resulting in a 2-fold increase of baseline mRNA levels. EC50 values are presented when achieved. Values are mean ± S.D. of three separate batches of differentiated HepaRG cells.
F2 values and -fold induction at highest concentration tested (given in parenthesis) for induction of CYP2C8, CYP2C9, and CYP2C19 mRNA in HepaRG cells treated with rifampicin F2 is the concentration resulting in a 2-fold increase of baseline mRNA levels. Values are mean ± S.D. of three separate batches of differentiated HepaRG cells.
F2 values, -fold induction at highest concentration tested (given in parenthesis), and AUC/F2 for induction of CYP3A4 mRNA in HepaRG cells treated with rifampicin, omeprazole, troglitazone, phenytoin, primaquine, phenobarbital, carbamazepine, dexamethasone, hyperforin, nifedipine, or sulfinpyrazone F2 is the concentration resulting in a 2-fold increase of baseline mRNA levels. EC50 values are presented when achieved. Values are mean ± S.D. of three separate batches of differentiated HepaRG cells. Published values of in vivo exposure (AUC) for the same drugs are listed.
Induction of P450-Dependent Activities. The induction of P450-specific enzyme activities was more pronounced after 48-h exposure than after 24-h exposure to the test compounds; therefore, 48-h incubation was used for enzyme activity experiments. Induction of phenacetin O-dealkylase activity by omeprazole and bupropion hydroxylase, diclofenac 4′-hydroxylase, and midazolam 1′-hydroxylase activities by rifampicin gave clear dose-response curves over the concentration ranges tested (Fig. 2). In Table 5, the F2 values, the -fold induction at the highest response measured, and, when obtained, EC50 values for phenacetin O-dealkylase, bupropion hydroxylase, and midazolam 1′-hydroxylase activities after treatment with rifampicin, omeprazole, troglitazone, phenytoin, primaquine, and phenobarbital are presented.
F2 values and -fold induction at highest concentration tested (given in parenthesis) for induction of phenacetin O-dealkylase, bupropion hydroxylase, and midazolam 1′-hydroxylase activities in HepaRG cells treated with rifampicin, omeprazole, troglitazone, phenytoin, primaquine, or phenobarbital F2 is the concentration resulting in a 2-fold increase of baseline activities levels. EC50 values are presented when achieved. Values are mean ± S.D. of duplicates in two separate batches of differentiated HepaRG cells.
Induction of the CYP1A2-dependent phenacetin O-dealkylase activity was only detected by omeprazole. Bupropion hydroxylase activity, which is catalyzed by CYP2B6, was induced by rifampicin, phenytoin, and phenobarbital. Omeprazole and troglitazone did not result in measurable effects on bupropion hydroxylase activity, although they induced CYP2B6 mRNA. CYP2C9-catalyzed diclofenac 4′-hydroxylase activity was induced by rifampicin, and the average -fold induction at the highest concentration was 1.7-fold. CYP3A4-dependent midazolam 1′-hydroxylase activity was strongly induced by rifampicin and phenobarbital and weakly by troglitazone.
In Vitro-in Vivo Correlation. To investigate the relationship between P450 induction in HepaRG cells and induction in vivo, the same approach as for the previously reported PXR assay was applied (Persson et al., 2006), in which the in vivo exposure of the test compound was related to the EC50 from the PXR reporter gene assay (AUC/EC50). This method correctly identified compounds known to induce CYP3A in vivo. We recalculated the previously published PXR results to F2 values and found the same ranking and correct classification of the tested compounds when using F2 values instead of the EC50 values, which indicates that the F2 values could be a useful indicator for assessing induction potential.
Representative results of mRNA induction in HepaRG cells. Induction of CYP1A1 and CYP1A2 by omeprazole (A), CYP2C8, CYP2C9, and CYP2C19 by rifampicin (B), and CYP2B6 and CYP3A4 by rifampicin (C).
In the present study, the in vivo AUC for the investigated drugs was related to F2 values (AUC/F2) obtained for the induction of CYP3A4 mRNA in the HepaRG cells (AUC, F2, and AUC/F2 values are listed in Table 4). By using the results from HepaRG cells, the same ranking of compounds was obtained as with the AUC/F2 values from the PXR reporter gene assay (Persson et al., 2006). In Fig. 3, the AUC/EC50 and the AUC/F2 from activation of PXR in the reporter gene assay and the AUC/F2 from CYP3A4 mRNA induction in HepaRG cells are shown normalized to rifampicin, which was set to 100%. The same ranking of compounds was obtained for all three ratios.
Induction of CYP3A4 mRNA in HepaRG cells by drugs was investigated as a quantitative measure of induction potency of the same drugs in vivo. Ten compounds (listed in Table 4) were selected on the basis that acceptable information on their induction potential in vivo is published. The in vivo induction is represented by the decrease in AUC for a CYP3A probe drug, administered before and after treatment with the inducing agent. The in vivo probes used for the correlation were midazolam for rifampicin (Backman et al., 1996a, 1998), phenytoin (Backman et al., 1996b), carbamazepine (Backman et al., 1996b), and hyperforin (St. John's wort) (Wang et al., 2001); verapamil for phenobarbital (Rutledge et al., 1988) and sulfinpyrazone (Wing et al., 1985); terfenadine for troglitazone (Loi et al., 1998); and triazolam for dexamethasone (Villikka et al., 1998). These probe drugs are moderate or high clearance drugs (Greenblatt et al., 1998; Wong et al., 1998; Lin, 2006), and the clearance and bioavailability are dependent on CYP3A4 metabolism. Omeprazole and nifedipine have not been found to induce CYP3A in vivo (Soons et al., 1992; Bowles et al., 1993). The correlation of AUC/F2 values from CYP3A4 mRNA induction and the in vivo induction (Fig. 4) show an excellent correlation (R2 = 0.863). When excluding hyperforin, a constituent of St. John's wort, the correlation factor increased to 0.943 (Fig. 4).
Using unbound AUC values gave a slightly lower in vitro-in vivo correlation (R2 = 0.859, data not shown). When the maximum plasma concentration in vivo (Cmax and Cmax,u) was used instead of AUC and AUCu, the R2 values were 0.741 and 0.812, respectively.
The corresponding evaluation of the predictive value for the induction of the other investigated P450s in the HepaRG cells is hampered by the lack of in vivo data.
HepaRG is a new human hepatoma cell line developed from a liver carcinoma. The aim of the present study was to further evaluate the induction properties of the HepaRG cells and to develop a method to predict the magnitude of induction in vivo. In the present study, we show that the drug-metabolizing enzymes CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 are inducible in the HepaRG cells. The compounds tested are known to mediate the induction response via the AhR, CAR, and/or PXR. Thus, the results are consistent with the reported expression of relevant transcription factors in the HepaRG cells (Aninat et al., 2006).
The results are from three separate batches of differentiated HepaRG cells. The small variations in -fold induction and F2 values obtained after exposure to test compounds indicate that the HepaRG cells will give reproducible and consistent results.
CYP1A1 and CYP1A2 mRNA were highly induced in HepaRG cells by the tested compounds previously known to induce CYP1A. The EC50 value for induction of CYP1A1 mRNA by omeprazole (5.9 μM) was slightly lower than the EC50 value (18 μM) for the activation of AhR by omeprazole previously reported in a reporter gene assay (Persson et al., 2006). The F2 values for the induction of phenacetin O-dealkylase activity was in the same order of magnitude as the F2 values for induction of CYP1A1 and CYP1A2 mRNA. Although the -fold induction of CYP1A1 and CYP1A2 mRNA was extensive after exposure to 40 μM omeprazole, the corresponding -fold induction of phenacetin O-dealkylase activity at 48-h exposure in the present study was only 3.5-fold. Omeprazole has in many in vitro studies been used as a positive control for activation of AhR and induction of CYP1A. However, omeprazole, or any other drug on the market that induces CYP1A or activate AhR in vitro, has not been found to induce CYP1A in vivo at relevant concentrations. Thus, any translation of the in vitro results for CYP1A induction to the in vivo situation is uncertain.
CYP2B6 mRNA and bupropion hydroxylase activity was highly inducible in HepaRG cells by rifampicin, phenobarbital, and phenytoin. Rifampicin and phenytoin are considered to be selective activators of PXR and CAR, respectively, whereas phenobarbital activates both receptors (Moore et al., 2000; Wang et al., 2004). The present results, together with previously reported mRNA expression of PXR and CAR (Aninat et al., 2006), indicate that HepaRG cells express both a functional PXR and a functional CAR.
In in vivo studies using P450-selective probes, the induction of CYP2C9 results in lower induction than induction of CYP3A when treating subjects with the same activators, such as rifampicin. This implies that CYP2C9 will be induced to a lesser degree than CYP3A by the same compounds. In accordance with the in vivo findings, the F2 values were higher and the -fold induction lower for CYP2C9 mRNA induction in HepaRG cells by rifampicin compared with CYP3A4 mRNA induction. The same phenomenon has also been shown in induction studies with primary human hepatocytes (Gerbal-Chaloin et al., 2001).
Induction of CYP2C8 and CYP2C19 mRNA in HepaRG cells by rifampicin is in the same range as induction of CYP2C9 mRNA, which corresponds with results from human hepatocytes (Gerbal-Chaloin et al., 2001). Both CYP2C8 and CYP2C19 are also induced in vivo by rifampicin (Zhou et al., 1990; Niemi et al., 2004; Park et al., 2004).
CYP3A4 was highly induced by all the compounds known to activate PXR and/or CAR. The EC50 for rifampicin induction of CYP3A4 mRNA (0.42 μM) and midazolam 1′-hydroxylase activity (0.12 μM) in HepaRG cells is consistent with the EC50 value obtained in the PXR reporter gene assay (0.20 μM) by the same drug (Persson et al., 2006), which suggests that PXR is the major receptor regulating the rifampicin induction of this gene product.
One important application of in vitro P450 induction studies in drug discovery is to predict the risk for induction of drug metabolism in vivo. In a previous study it was shown that the EC50 values from a PXR reporter gene assay could be used to distinguish between CYP3A inducers and noninducers when related to the in vivo exposure of the test compound (Persson et al., 2006). In the present study, F2 values from CYP3A4 mRNA induction related to in vivo AUC of the test compounds were found to give the same correct ranking of inducers and noninducers. The results suggest that instead of EC50 values from full dose-response curves, F2 values could be used to evaluate the induction response in the cell system. This is an advantage because an Emax many times cannot be achieved or may be confounded as a result of toxicity or solubility problems. For example, troglitazone did not affect viability but showed a decrease in CYP3A4 induction response at the highest concentration, which may affect the Emax value and as a consequence the EC50.
The AUC/F2 values for CYP3A4 mRNA induction were further evaluated as possible predictors of the magnitude of induction in vivo. In the present study we used results from published in vivo induction studies utilizing different probe drugs for measuring CYP3A4-dependent clearance. The probe drugs used in the current in vivo studies are all intermediate or high clearance drugs, metabolized and cleared mainly by CYP3A4. Therefore, they should be affected to a similar extent by CYP3A4 induction (Lin, 2006). Such an assumption is supported by the fact that the effect of rifampicin on AUC for the high/medium clearance drugs midazolam, verapamil, and triazolam after p.o. dosing is comparable (Backman et al., 1996a, 1998; Fromm et al., 1996; Villikka et al., 1997). An excellent correlation was found between the decrease of in vivo AUC for CYP3A probe drugs and AUC/F2 for the tested drugs. Thus, the results indicate that the AUC/F2 values for CYP3A4 mRNA response in HepaRG cells could be used as a reliable in vitro measure for evaluating CYP3A induction potency of compounds in vivo.
In the in vitro-in vivo correlation analysis, a component of St. John's wort, hyperforin, was included. The manufacture of herbal remedies is not as rigorously controlled as production of drugs, which results in varying concentration of the components in different extracts. This means that in vivo data for hyperforin could be confounded by varying concentrations of hyperforin in the extracts. If hyperforin is excluded from the correlation, a higher correlation factor is obtained, which could indicate that this may be the case. Besides hyperforin, the compound deviating the most from the correlation is dexamethasone, a more potent inducer in vivo than what was predicted from AUC/F2 values. The reason for this is obscure and proposes further investigations of this compound.
The Fa2N-4 cells were investigated by Ripp et al. (2006) as a model for predicting CYP3A induction in vivo. The in vitro Emax and EC50 values for a number of potential CYP3A drug inducers were related to in vivo Cmax for the same drugs. The induction score obtained showed an excellent correlation with the degree of induction in vivo. In the present study on HepaRG cells, using Cmax instead of AUC values for calculation of induction scores gave a poorer correlation with the degree of induction in vivo for the tested drugs.
According to the Food and Drug Administration guideline (http://www.fda.gov/cder/guidance/6695dft.pdf), a test compound should reach an induction level equal to or greater than 40% of a positive control to be considered as relevant for the in vivo situation. In the present study all the compounds known to induce CYP3A4 in vivo reached at least 40% of rifampicin induction, except the well known CYP3A inducer phenytoin. Phenytoin has previously been shown to be a weak inducer in PXR reporter gene assays and human hepatocytes, exhibiting an induction response close to or less than 40% of rifampicin response (Luo et al., 2002; Persson et al., 2006). Therefore, substances like phenytoin could be overlooked if using a conservative cut-off value for in vivo relevance. In the present study, 40 μM phenytoin gave an induction response that was only 14% of maximum rifampicin induction. However, using the AUC/F2 value for the CYP3A4 mRNA induction response in HepaRG cells correctly predicted the degree of induction in vivo by phenytoin. Furthermore, strong in vitro CYP3A4 inducers like omeprazole and nifedipine (50 and 92% of maximum rifampicin induction, respectively) were correctly classified as noninducers when using AUC/F2 values. These results emphasize the importance to relate the in vitro results to in vivo exposure for both seemingly "strong" and "weak" inducers in the in vitro system.
In conclusion, we have shown that HepaRG cells respond to PXR, CAR, and AhR activators, resulting in induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4. Further, it was shown that the F2 values for CYP3A4 mRNA induction in HepaRG cells could be used not only to classify compounds as inducers or noninducers of CYP3A but also to predict the extent of induction in vivo in humans. Thus, the HepaRG cell line could be used as an important in vitro model for investigations of enzyme induction in drug discovery.
Representative results of induction of enzyme activities in HepaRG cells. Induction of phenacetin O-dealkylase (CYP1A2) activity by omeprazole (A), bupropion hydroxylase (CYP2B6) activity by rifampicin (B), diclofenac 4′-hydroxylase (CYP2C9) activity by rifampicin (C), and midazolam 1′-hydroxylase (CYP3A4) activity by rifampicin (D).
Ranking of in vitro results. The AUC/EC50 from PXR assay (white bars), AUC/F2 from PXR assay (gray bars) [data from (Persson et al., 2006)], and AUC/F2 CYP3A4 mRNA in HepaRG cells (black bars). The study compounds were normalized to the rifampicin response, which were set to 100%.
Correlation of AUC/F2 of CYP3A4 mRNA in HepaRG cells with percentage decrease of in vivo AUC for CYP3A probe drugs (whole line), R2 = 0.863. The equation used is described under Materials and Methods. Correlation when hyperforin is excluded (dashed line), R2 = 0.943. Compound abbreviations: CBZ, carbamazepine; DEX, dexamethasone; HYP, hyperforin; NIF, nifedipine; OME, omeprazole; PB, phenobarbital; PHY, phenytoin; RIF, rifampicin; SULF, sulfinpyrazone; TRO, troglitazone.
We thank Gisela Häggblad, Anne-Cristine Carlsson, and Dr. Lennart Nilsson for measurement of LDH activity.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.017418.
ABBREVIATIONS: P450, cytochrome P450; PXR, pregnane X receptor; CAR, constitutive androstane receptor; AhR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; LDH, lactate dehydrogenase; huPO, human acidic ribosomal phosphoprotein; LC, liquid chromatography; AUC, area under the plasma concentration versus time curve.
The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition January 1, 2008, 36 (1) 137-145; DOI: https://doi.org/10.1124/dmd.107.017418
Identification of payload-containing catabolites of ADCs
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Prince Albert is once again backing the campaign to Orange Wolverhampton and end violence against women and girls.
The iconic statue in Queen Square is proudly sporting an orange sash in support of the annual 16 day campaign which began on Sunday (25 November).
And for the first time, he has been joined by Lady Wulfruna - with the statue of the founder of Wolverhampton which stands outside St Peter's Collegiate Church also wearing a sash in support of the campaign.
Businesses, community groups and individuals are organising a wide range of activities to raise awareness of gender based violence, primarily committed against women and girls, including domestic abuse, female genital mutilation, forced marriage, so-called 'honour based' violence and sexual violence - and also the help and support that is available to victims in Wolverhampton.
Kathy Cole-Evans, Strategy Coordinator and General Manager of Wolverhampton Domestic Violence Forum, said: "It's great that we are able to use both of these iconic statues to promote this very important campaign to the people of Wolverhampton.
"There were over 6,500 incidents of domestic violence recorded by police in Wolverhampton in 2017 to 2018 and the vast majority of victims were women, so this is a major issue and one that we all need to be aware of.
"But the Orange Wolverhampton campaign is not just about highlighting the problem, but also ensuring that victims do not suffer in silence and are aware of and feel able to get the help and support they need."
Meanwhile, people can visit the Orange Wolverhampton pop-up shop in the Wulfrun Centre on Thursday (29 November) from 10am to 4pm. Professionals from different organisations will be meeting shoppers at the shop, at the Cleveland Street entrance, to talk about ending violence against women and girls, and will be providing free leaflets, information, and advice along with orange ribbons for people to wear to show their support.
Orange Wolverhampton is the city's contribution to the United Nations' annual Orange The World campaign, which takes place every year between 25 November, International Day for the Elimination of Violence Against Women and Girls, and 10 December, UN Human Rights Day.
Councillor Hazel Malcolm, the City of Wolverhampton Council Cabinet Member for Public Health and Wellbeing, said: "We are delighted to once again be supporting the United Nations' Orange the World campaign and turning Wolverhampton orange is an important part of our work to raise awareness of the issues and to make sure people know how to get the help and support they need."
Follow the Orange Wolverhampton campaign on Twitter and share your support and photos using the hashtag #OrangeWolves and by following Type=links;Linkid=9893;Title=WVsafeguarding;Target=_blank;, or via the Wolverhampton Safeguarding Boards' Type=links;Linkid=7295;Title=Facebook;Target=_blank; page.


text:
A Estação Ferroviária de Braga é uma interface do Ramal de Braga, que serve a localidade de Braga, em Portugal. A construção de uma estação em Braga esteve planeada desde a década de 1860, primeiro como parte da Linha do Minho e posteriormente como terminal de um ramal próprio. Entrou ao serviço em 21 de Maio de 1875. A estação foi alvo de obras de remodelação na década de 1930, e foi profundamente modificada na década de 2000, no âmbito do projecto de modernização e electrificação do Ramal de Braga, que permitiram a introdução dos comboios Alfa Pendular até Braga.

Caracterização

Localização e acessos
A estação localiza-se junto à localidade de Braga, com acesso pelo Largo da Estação.

Serviços
A estação de Braga acolhe serviços de passageiros das tipologias Alfa Pendular, Intercidades e Urbano

Classificação, vias e plataformas
Segundo o Directório da Rede Ferroviária Portuguesa 2005, lançado pela Rede Ferroviária Nacional em 13 de Outubro de 2004, a estação ferroviária de Braga tinha a classificação B. Segundo o Diretório da Rede 2016, de 12 de Dezembro de 2014, a estação contava com seis vias de circulação, com comprimentos entre os 400 e 230 m, enquanto que as plataformas tinham 232 a 220 m de extensão e 80 cm de altura.

A estação de Braga é um dos pontos extremos do Eixo Ferroviário Norte-Sul, um importante corredor composto por várias linhas férreas entre Braga, Porto, Lisboa e Faro.

História

Pré-história

No local onde se ergue a Estação de Braga existiu um balneário pré-romano, redescoberto já no séc. XXI aquando da construção na nova estação.

Século XIX

Antecedentes e primeiros projectos
Até meados do Século XIX, a região interior do Minho tinha graves problemas de comunicações, devido a uma rede viária pouco desenvolvida, com poucas estradas e em más condições. As principais estradas ligavam aos portos fluvais, sendo a navegação fluvial o meio de transporte mais utilizado na região. No entanto, os rios tinham problemas na navegação, como aconteceu com o Cávado, que conheceu vários projectos para aumentar a sua navegabilidade, de forma a se tornar numa importante via comercial até Braga, mas sempre sem sucesso. Assim, grande parte do comércio entre o Porto e Braga era feito através da costa e depois pelo Rio Lima, sendo o resto do percurso feito por estrada. Esta situação começou a mudar na década de 1850, quando o governo iniciou um ambicioso programa de desenvolvimento das vias de comunicação, ao abrigo do qual foram melhoradas as estradas, incluindo uma do Porto a Braga, permitindo a criação de um serviço de diligências, e instalada uma rede telegráfica ligando Braga ao Porto e outras localidades.

Ainda durante a década de 1840 tinham surgido várias propostas para a construção de caminhos de ferro na região do Minho, incluindo uma de Hardy Hislop para ligar o Porto a Valença.

Planeamento e inauguração
No entanto, o grande impulso para a introdução do comboio em Portugal foi dado na década seguinte, com os programas de obras públicas do governo, e em 1857 o conde de Réus apresentou uma proposta para uma linha de Porto a Vigo. Em 1864, o governo fez uma proposta de lei à Câmara dos Deputados para construir esta linha, e em 2 de Julho de 1867 foi publicada uma lei que autorizou o governo a construir e explorar uma linha do Porto a Braga e à fronteira com a Galiza, tendo a construção desta linha sido ordenada por um decreto de 14 de Junho de 1872. Foram feitos vários projectos para a linha, com percursos diferentes, que inicialmente seguiam mais pelo litoral mas depois se tornaram mais interiorizados, de forma a servir melhor Braga, embora no final a cidade tenha ficado prejudicada em relação a Viana do Castelo, que tinha estação directa na linha, enquanto que Braga necessitava de um ramal. O percurso original, elaborado por um engenheiro português às ordens do Conde de Réus, seguia pela costa a Norte da Vila do Conde, enquanto que um segundo estudo, feito por ordem do governo, já fazia a linha entrar seguir a costa apenas junto a Esposende, com um ramal para Braga, que seguia o vale do Rio Este. Depois foi feito um terceiro projecto, que servia melhor Barcelos e Vila Nova de Famalicão, e iniciava o Ramal de Braga em Nine, enquanto que um quarto projecto se debruçou principalmente no lanço entre Barcelos e Viana do Castelo.

O Ramal de Braga foi inaugurado em 20 de Maio de 1875, e aberto à exploração no dia seguinte, pela divisão do Minho e Douro dos caminhos de ferro estatais. Após a inauguração, foi construída uma estrada entre a estação e o centro de Braga, que originalmente denominava-se de Rua do Corvo e foi posteriormente renomeada para Rua Andrade Corvo.

Primeiros anos
A construção do caminho de ferro levou à extinção dos serviços de mala-posta que existiam entre o Porto, Guimarães e Braga.

Em 1888, existia uma carreira de diligências ligando a estação de Braga às Termas do Gerês, num percurso de quatro horas.

Em Janeiro de 1897, Joaquim Augusto Mouzinho de Albuquerque viajou de comboio por vários pontos do país depois de ter regressado de África, tendo passado por Braga, onde foi festivamente recebido na gare por uma grande multidão.

Século XX

Décadas de 1900 e 1910
Durante o Outono de 1903, João Franco utilizou o comboio em várias viagens de propaganda ao longo do país, incluindo uma até Braga.

Em 1913, a estação de Braga estava ligada por carreiras de diligências até Palmeira, Lago, Ponte do Bico, Rendufe, Amares, Dornelas, Bouro, Gerês, Ruivães, Vieira do Minho, Rossas, Monsul, Póvoa de Lanhoso, Cabeceiras de Basto e Alvão, e Vila Verde, Portela do Abade, Pico de Regalados, Ponte de Lima, Ponte da Barca, Arcos de Valdevez e Monção, e Taipas, Caldelas e Guimarães.

Em 1914, uma viagem do Porto a Braga de comboio demorava cerca de 2 horas e 20 minutos.

Em 28 de Maio de 1916, as populações das freguesias de Braga dirigiram-se à cidade para protestar contra a carestia de alimentos provocada pela Primeira Guerra Mundial, tendo os militares e a Guarda Nacional Republicana sido destacados para guardar a estação de Braga, onde tinha sido armazenada uma grande quantidade de milho.

Décadas de 1920 e 1930
Em 1921, a Câmara Municipal de Braga foi autorizada a construir uma linha de eléctricos desde a estação até à Ponte do Prado.

Em 1926, a Direcção dos Caminhos de Ferro do Minho e Douro estava a explorar uma linha de autocarros entre a estação de Braga e o Santuário do Bom Jesus do Monte. Em 1927, a Companhia dos Caminhos de Ferro Portugueses passou a explorar as linhas que anteriormente pertenciam à rede dos Caminhos de Ferro do Estado.

Em 1933, a estação sofreu obras de recuperação e melhoramentos, e no ano seguinte realizaram-se obras parciais de reparação, e foi instalada a iluminação eléctrica na segunda plataforma. Em 1935, existia um serviço de camionagem entre esta estação e a localidade de Gerês.

Década de 1940
Em 5 de Fevereiro de 1949, entraram ao serviço novas carruagens Schindler nos comboios entre Porto e Braga.

Décadas de 1950 e 1960
Em meados do século, o corredor entre a cidade e a estação não tinha capacidade para o intenso tráfego automóvel, pelo que a Câmara Municipal de Braga iniciou um programa faseado para o seu desenvolvimento, que incluiu o alargamento e o arranjo paisagístico das bermas. Em 1955, já tinha sido concluída a fase da transformação do Campo das Hortas num jardim, estando ainda em obras a ligação ao Largo da Estação.

Durante o II Plano de Fomento, entre 1959 e 1964, a Companhia dos Caminhos de Ferro Portugueses planou a electrificação de vários troços da rede ferroviária nacional, incluindo de Ermesinde a Braga.

Em 6 de Maio de 1967, foi apresentado um plano de investimentos para a modernização das vias férreas na zona do Porto, que incluía a electrificação da via férrea entre Ermesinde e Braga.

Décadas de 1980 e 1990
Em 1982, o papa João Paulo II viajou de comboio entre Santa Apolónia e Braga, durante uma visita a Portugal. Na década de 1980, foi instalado um núcleo museológico na estação de Braga, que durante algum tempo albergou a a locomotiva mais antiga em Portugal.

Em 29 de dezembro de 1992, um comboio com cimento não travou na estação, avançando cerca de 50 metros, atravessando a rua. Esmagou um carro e provocou uma vítima mortal.

Nos finais da década de 1980, a operadora Caminhos de Ferro Portugueses iniciou um grande programa de modernização das linhas férreas em território nacional, que se debruçou especialmente sobre os serviços suburbanos das cidades do Porto e Lisboa, incluindo o Ramal de Braga. Com efeito, o corredor entre Porto e Braga, apesar de ser um importante eixo suburbano, apresentava graves deficiências, que reduziam o seu papel como estrutura de articulação entre zonas urbanas. Com efeito, a ideia de electrificar o ramal não era novidade, tendo sido proposta desde os anos 30, quando foi sugerida a electrificação das linhas suburbanas do Porto como forma de desenvolver o tráfego dos comboios.

A modernização do ramal estava inserida no Itinerário do Porto a Braga e Vale do Ave, que abrangia a duplicação, electrificação e renovação da via férrea entre Ermesinde e Braga, com rectificação do traçado, no sentido de reduzir o tempo de viagem dos comboios entre Porto e Braga: o tempo de percurso dos serviços Intercidades deveria ser reduzido de 1 hora e 5 minutos para 36 minutos, enquanto que os suburbanos expressos rápidos deveriam levar 48 minutos em vez de 1 hora e 25 minutos. Além disso, seria possível criar novos comboios entre São Bento e Braga, como um serviço semi-rápido cadenciado parando em Campanhã, Ermesinde, São Romão, Trofa, Famalicão e Nine, e todas as estações e apeadeiros no ramal de Braga, um serviço suburbano expresso em horário adicional, fora das horas de ponta, servindo Campanhã, Ermesinde, Trofa e Famalicão, ou serviços suburbanos regulares de maior frequência e com maiores comboios, durante as horas de ponta. Com estas medidas, estava previsto para o horizonte de 2012 um aumento de 200% na procura dos passageiros no lanço entre Nine e Braga.

Além da sua importância como corredor suburbano, o Ramal de Braga também fazia parte de um grande eixo transversal, que ligava Braga a Faro, que era de grande relevância no âmbito da política nacional de transportes, uma vez que abrangia cerca de 85 % da população portuguesa. Com a instalação do caminho de ferro pela Ponte 25 de Abril, seria possível ir de comboio directamente de Braga a Faro por Lisboa.

Em 1997, o projecto de remodelação passou a ser executado pela Rede Ferroviária Nacional, entidade criada para assegurar a gestão das infraestruturas ferroviárias em Portugal.

Em 3 de Novembro de 2002, foi encerrada a circulação dos comboios no ramal de Braga, para se iniciarem as obras de modernização, que se previa, nessa altura, estarem concluídas em Fevereiro de 2004. Foi organizado um serviço de autocarros para substituir o percurso dos comboios urbanos e Intercidades, que no entanto iria demorar o dobro do tempo e teria menos paragens. Previa-se nessa altura que o projecto de modernização entre Nine e Braga iria custar cerca de 100 milhões de Euros, mas iria melhorar consideravelmente a qualidade dos serviços, através da introdução de novos comboios, especialmente o Alfa Pendular, e seriam remodeladas as estações e apeadeiros. A estação de Braga seria uma das que teriam maiores intervenções, pois iria contar com um novo edifício de passageiros, e plataformas de 220 m de comprimento, dois terços dos quais com cobertura.

Século XXI
A estação de Braga foi remodelada em 2003, no âmbito do projecto "Estações com Vida" da Rede Ferroviária Nacional, que tinha como objectivo a transformação das estações em espaços de dinamização social, cultural e económica das localidades onde se inserem. No caso da estação de Braga, foi construído um novo edifício com espaços comerciais e de serviços, como restaurantes e lojas, e a instalação de jardins e de parques de estacionamento. O edifício integra ainda, em espaço musealizado, um balneário pré-romano, identificado nas escavações arqueológicas prévias às obras. A nova estação foi inaugurada a 21 de Abril de 2004, no mesmo dia em que o Ramal de Braga foi reaberto após obras de modernização, realizadas pela Rede Ferroviária Nacional. Após a modernização, Braga passou a ser servida pelos comboios Alfa Pendular.

Com o processo de modernização, o antigo edifício deixou de ser utilizado como interface ferroviária, tendo sido cedido pela Rede Ferroviária Nacional à Câmara Municipal de Braga, e remodelado para alojar várias instituições de cariz cultural e turístico, como a Companhia de Teatro de Braga, o Orfeão de Braga e o Turismo do Porto e Norte de Portugal. Este projecto custou cerca de 300 mil Euros, tendo o edifício sido inaugurado, após as obras de requalificação, em 5 de Julho de 2013. Já em 2010, parte do antigo edifício da estação tinha sido ocupado por uma escola de formação na área das artes visuais. Mais recentemente, em julho de 2017, um candidato independente à freguesia local, Seco Magalhães, lamentava o «abandono e degradação progressiva» do edifício.

Ligações projectadas a outras linhas
Desde os finais do Século XIX que se planeou a construção de mais vias férreas com início em Braga, tendo os projectos mais importantes sido os das linhas do Alto Minho, até Monção, e de Braga a Guimarães. Em 10 de Agosto de 1897, o Ministro da Fazenda, Mariano de Carvalho, apresentou um projecto de lei acerca do arrendamento dos caminhos de ferro do estado, onde o governo era autorizado a abrir concurso para a construção de várias linhas, incluindo uma de Braga a Ponte da Barca, Arcos de Valdevez e à fronteira com Espanha, e outra de Braga a Chaves, seguindo pelo vale do Rio Cávado e por Ruivães.

Em 16 de Novembro de 1898, a Gazeta dos Caminhos de Ferro noticiou que a comissão que tinha sido encarregada de estudar os projectos na Rede Complementar ao Norte do Mondego já tinha apresentado as suas propostas para novas linhas, incluindo uma de Braga a Cavez por Guimarães. Em 16 de Janeiro de 1899, a Gazeta relatar que tinha sido aberto um inquérito administrativo, para informar o público sobre os projectos ferroviários dos Planos das Redes Complementares ao Norte do Mondego e Sul do Tejo, incluindo as linhas de Braga a Ponte da Barca, em via larga, que seria a continuação do Ramal de Braga, e de Braga a Guimarães, em via estreita. Em finais de 1899, o engenheiro Cachapuz, em representação de um sindicato de capitalistas italianos, pediu ao conselho de administração dos Caminhos de Ferro do Estado a concessão de vários caminhos de ferro no Norte do país, incluindo as linhas de Braga a Monção, e de Braga a Guimarães. O Plano da Rede Complementar ao norte do Mondego, decretado em 15 de Fevereiro de 1900, alterou o projecto de Braga a Ponte da Barca, que passou a ser de via estreita, com futuro prolongamento até Monção.

Em 27 de Abril de 1903, o Ministro das Obras Públicas, Conde de Paçô-Vieira, determinou que Braga deveria ser o princípio das Linhas do Alto Minho, e de Braga a Guimarães, ambas em via métrica. Em Fevereiro de 1910, já tinha sido aprovado o projecto definitivo para o troço entre Braga e Arcos de Valdevez da Linha do Alto Minho.

A concessão passou destas duas linhas, em conjunto com a Linha do Vale do Lima, passou por várias empresas, tendo sido finalmente passada para a Companhia dos Caminhos de Ferro do Norte de Portugal, que não as pôde construir devido a problemas financeiros, causados pela Grande Depressão e pelo desenvolvimento do transporte rodoviário.

Entretanto, em 1908 estava a ser planeado o Caminho de Ferro de Braga a Vidago.

O Decreto n.º 18:190, de 28 de Março de 1930, introduziu o Plano Geral da Rede Ferroviária, que tinha como objectivo reorganizar a rede portuguesa de caminhos de ferro, e listar os vários projectos para novas linhas; entre os troços programados, estava a Transversal do Minho, de Entre-os-Rios a Arcos de Valdevez com passagem por Braga, e a Linha do Cávado, de Braga a Esposende.

Ver também
Comboios de Portugal
Infraestruturas de Portugal
Transporte ferroviário em Portugal
História do transporte ferroviário em Portugal

Bibliografia

Leitura recomendada

Ligações externas

Braga
Braga
Fundações em Portugal em 1875
Estações ferroviárias inauguradas em 1875

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