This is a very good point! My mother had Hormone Positive Breast Cancer so, I can never take hormones of any kind, raises my risk too high so, birth control pills are OUT.
I think that when the stakes are high (such as the possibility of a stroke!), it can be easy to abandon critical thought. That there would be ANY chance of something terrible happening is frightening. And yet, taking a step back from it and looking at the numbers makes the most sense.
You know what they say…There are lies, damn lies, and statistics!
True information…keep watch to see if b/c pills trigger more attacks. They're in some people's "stackable triggers" list.
I *love* having an IUD. I have the Mirena, and I don't menstruate on it. It's great – and it's meant no hormonal migraines for me.
Hormonal trigger is my biggest, too. I've been on the pill for 20 years. To some degree it's been worth it to prevent "accidental" babies. But now I'm 40, had a recent annual exam that produced a high blood pressure reading (higher than readings I get at home regularly) and an "abnormal" PAP…..so it's time for an alternative course of action. I have a cyst on an ovary and have opted to have my tubes tied while the Doc is in there taking care of the cyst. Killing two birds with one scalpel and reducing my threshold for migraines. I'll still get them but I refuse to let them run my life.
Thanks for keeping your site going, I've enjoyed reading every single "page" of it and it's SO good to know I'm not weird. Ha ha! Good luck to you all!
Things like "eight times more likely" can sound just plain scary. I'm glad you're having a chance to re-evaluate. It's always good to have options. I had fewer Migraines when on BC pills, when pregnant, when nursing, and now that I'm on bio-identical HRT, my Migraines are less severe. For me, my cycles were always irregular so anything that modified or enhanced my hormones has been helpful. Of course, some women don't react that way at all. I guess you don't know until you try. Good luck!


text:
Canada's Delayed Decision Has Effectively Blocked Huawei From 5G in the Country
by Christopher Baugh – Wed, August 26, 2020
Canada has effectively blocked Huawei from the country's next-generation 5G wireless networks, even if it hasn't specifically said so.
Canada is the last remaining member of the Five Eyes intelligence sharing network that hasn't officially blocked Huawei from 5G networks. While it hasn't decided on the matter yet, Canada's long delay in making such a decision essentially forces Canadian telecoms to exclude the Chinese company, reads a new report from Reuters.
The strategy allows Canada to stay on the right side of both China and the United States as they fight over Huawei. Canada and its Five Eyes allies — the United Kingdom, New Zealand and Australia — are under pressure from fellow member the United States to squeeze out Huawei on security grounds.
"The absence of a solution will eventually settle all problems," said a source directly familiar with the approach taken by the Liberal government of Canadian Prime Minister Justin Trudeau.
Earlier this month, the Trump administration announced further US restrictions on Huawei in order curb its access to commercially available chips. As such, Canadian telecoms now feel without choice but to crack dowan on Huawei in 5G networks, say the report's sources.
"They've done the political calculus and said 'The best thing for us is to do nothing and if we do nothing we don't upset the Chinese, we don't upset the Americans,'" said a source familiar with what government officials are saying.
Huawei said in a statement that it believed Ottawa "when it says it is taking the time to make a considered decision."
Uber to Expand Ridesharing to All of Quebec This Fall
Wed, August 26, 2020

redpajama_set_name: RedPajamaCommonCrawl


Hex #39c0a1 or rgb(57,192,161) in RGB space is the tint of greenish cian. It has hue angle of 166.22 degrees, value = 75 and saturation = 70. #39c0a1 can be obtained by mixing 4 colors: 2 drops of yellow, 1 drop of blue, 5 drops of cyan, 1 drop of black. Nearest safe hex - #33cc99. Below you can see the block with #39c0a1 color and its structure: in procentage ratio and in drops of pigments. Click "TRY" button to move #39c0a1 to the mixer and play with it.
The funnel shows the process of mixing multiple colors step by step. Here you can see the mix of 22.2% of yellow, 11.1% of blue, 55.6% of cyan, 11.1% of black colors.
We find few colors similar to #39c0a1. For more information about color - click on the circle.
Below you can see the model of the mix of #39c0a1 with pure water. The more water added to the mixture, the mixture will be less saturated.


text:
Baker Mayfield Leads Comeback as Browns Beat Josh Allen, Bills 19-16
Tyler Conway@jtylerconwayTwitter LogoFeatured ColumnistNovember 10, 2019
It wasn't pretty, but the Cleveland Browns have won for the first time in more than a month.
Nick Chubb and Kareem Hunt provided a strong one-two punch at running back, and Baker Mayfield took care of the ball as the Browns earned a 19-16 win over the Buffalo Bills on Sunday.
Stephen Hauschka missed a potential game-tying 53-yard field goal—his second miss of the game—with 17 seconds remaining to clinch the game.
Cleveland held Buffalo to 344 yards of total offense, while Josh Allen continued to struggle as a passer. The loss drops Buffalo to 6-3 on the season, jeopardizing the Bills' hold on one of two wild-card spots.
The Browns entered losers of four straight games since their 2-2 start and appeared to be reeling. Mayfield, one of the league's biggest disappointments, went through several facial-hair iterations last week alone in hopes of fixing his woes.
Hunt, who returned from an eight-game suspension, totaled 74 yards and gave the Browns dynamism as a pass-catcher out of the backfield.
Notable Stats
QB Josh Allen: 22-of-41 passing, 266 YDS (6 CAR, 28 YDS, 2 TD)
RB Devin Singletary: 8 CAR, 42 YDS (3 REC, 8 YDS)
WR John Brown: 5 REC, 77 YDS
WR Cole Beasley: 4 REC, 74 YDS
QB Baker Mayfield: 26-of-38 passing, 238 YDS, 2 TD
RB Nick Chubb: 20 CAR, 116 YDS (2 REC, 5 YDS)
RB Kareem Hunt: 4 CAR, 30 YDS (7 REC, 44 YDS)
WR Odell Beckham Jr: 5 REC, 57 YDS
WR Jarvis Landry: 9 REC, 97 YDS, 1 TD
Passing Game Continues to Hold Bills Back
Buffalo Bills @BuffaloBills
QB Josh Allen: "This loss hurts. Our defense played well today, but I didn't make enough plays for our team." #BUFvsCLE https://t.co/5ooSbntu8N
The ceiling for this team is limited as long as Josh Allen is the starting quarterback. While he's effective with his feet, his passing should probably make Bills fans wistful for the halcyon days of Tyrod Taylor under center.
Allen consistently misses open targets, tries to use his arm to fit the ball into impossible windows and nearly cost the Bills the game with a fumble deep inside the red zone in the fourth quarter. Only Jon Feliciano's quick hands saved Allen from a potentially game-losing turnover.
John Brown and Cole Beasley are a solid (albeit unspectacular) one-two punch at the receiver spot, and Devin Singletary has helped give life to the running game.
Stephen Hauschka is the root cause of the Bills' loss Sunday; they win if he connects on his two field goal attempts, which came from 34 and 53 yards out. But the Bills haven't been able to finish drives all season because they can't count on Allen to make passes into small windows.
The Browns Are Still Too Talented to Be This Bad
SportsCenter @SportsCenter
Baker Mayfield for the lead and the Browns hang on for the win! 🔥 (via @NFL) https://t.co/OMyZf4JwMZ
Hunt sure didn't look like he's lost a step in his return to football, giving the Browns perhaps the league's best running back duo. Odell Beckham didn't suddenly lose his magic on the flight from New York to Cleveland, and Jarvis Landry is among the best underneath targets in football.
Mayfield had one of his best games of the season, completing 26 of his 38 passes for 238 yards and two touchdowns, but it still amounted to just 19 points. Compiling 368 yards of total offense against this Bills defense is impressive; they came into the league third in yards allowed (298.3 per game). Chubb's 116-yard performance continued his season-long trend of churning out big games despite the circumstances.
There are so many pieces here, all healthy and ready to be competitive. Mayfield's decision making has been bad all season, but the lack of victories and discipline comes down to coaching. The Browns ran six plays inside the Bills 2-yard line in the first half and found a way to turn the ball over on downs—a seeming schematic impossibility.
A win is a win, but it's hard to not watch this team and wonder why it's not better.
The Browns host the Steelers on Thursday night. The Bills travel to Miami to play the Dolphins next Sunday.


text:
Q: PHP: unusual table from array Suppose that you have an array like this one:
Array([0] => Array([first_name] => Stack [last_name] => Overflow)
      [1] => Array([first_name] => Andrew [last_name] => Oliveira)
     );

It is easy to populate a table on the following way:
 first name
   last name
<? foreach($info as $key => $data) { ?>

   <?= $data['first_name'] ?>
   <?= $data['last_name'] ?>

 <? } ?>

The above produces a table like this:
first name || last name
========================
Stack-------|| Overflow
========================
Andrew----|| Oliveira
========================

And so on... But how would you do to do a table like this, given the same array:
person 0 || person 1
========================
Stack-----|| Andrew
========================
Overflow|| Oliveira
========================

A: Something like this?
<?php
// create line with "Person X"
echo '
';
$s = sizeof ($info);
for ($i=0;$i<$s;++$i) {
  echo 'Person '.$i.'	';
}
echo '

';

// create line with first name
foreach ($info as $data) {
  echo ''.$data['first_name'].'	';
}
echo '

';

// create line with last name
foreach ($info as $data) {
  echo ''.$data['last_name'].'	';
}
echo '
';
?>

A: Yes, very easy in fact, but use this method:
<?php $COUNT_INFO=count($info); ?>
first name
    last name

        <? for($i=0; $i<$COUNT_INFO; $i++) { ?>
        <?= $info[$i]['first_name'] ?>
        <? } ?>

        <? for($i=0; $i<$COUNT_INFO; $i++) { ?>
        <?= $info[$i]['last_name'] ?>
        <? } ?>

A: Make two arrays, one for first names and another for last names. After that, use foreach to loop through them and concatenate the first and the second line of your table.
foreach($array as $i=>$data){
    $headers .= "Person $i	";
    $first_names .= "".$data["first_name"]."	";
    $last_names .= "".$data["first_name"]."	";
}

echo "

$headers

$first_names

$last_names

";

There you go! Just one loop! :)

A: The answer of @Vitor42 is ok, but if you want something more generic and you are sure that the data is always complete try with:

<?php
$info = array(
    array('first_name'=>'Stack','last_name'=>'Overflow'),
    array('first_name'=>'Andrew','last_name'=>'Oliveira')
);
$sHtmlHead = '
';
$arrHtmlBodies = array();
$nSize = sizeOf($info);

for($i=0;$i<$nSize;$i++){
    $sHtmlHead .= 'Person '.$i.'	';
    $arrTemp = $info[$i];
    $j = 0;
    foreach($arrTemp as $data){
        if(!isset($arrHtmlBodies[$j])) $arrHtmlBodies[$j] = '
';
        $arrHtmlBodies[$j] .= ''.$data.'	';
        if($i==($nSize-1)) $arrHtmlBodies[$j] .= '
';
        $j++;
    }
}
echo $sHtmlHead; ?>

<?php
echo implode('',$arrHtmlBodies); ?>

If you are not sure that the data is complete try with:

<?php
$info = array(
    array('first_name'=>'Stack','last_name'=>'Overflow'),
    array('first_name'=>'Andrew','last_name'=>'Oliveira'),
    array('first_name'=>'Eli'),
    array('last_name'=>'Astocaza')
);
$sHtmlHead = '
';
$arrHtmlBodies = array();
$nSize = sizeOf($info);
$arrKeys = array_keys($info['0']);
$nSizeKeys = sizeOf($arrKeys);
for($i=0;$i<$nSize;$i++){
    $sHtmlHead .= 'Person '.$i.'	';
    $arrTemp = $info[$i];
    $j = 0;
    for($j=0;$j<$nSizeKeys;$j++){
        if(!isset($arrHtmlBodies[$j])) $arrHtmlBodies[$j] = '
';
        $arrHtmlBodies[$j] .= ''.$info[$i][$arrKeys[$j]].'	';
        if($i==($nSize-1)) $arrHtmlBodies[$j] .= '
';
    }
}
echo $sHtmlHead; ?>

<?php
echo implode('',$arrHtmlBodies); ?>

redpajama_set_name: RedPajamaStackExchange


We have more than 10 years of retail pharmacy experience and we strongly believe that true pharmacy care starts with the exceptional service. That means we work closely with patients, doctors and insurance carriers to provide accurate health care.
Today at First Choice Pharmacy, we realize that insurance and the paper work – all these are overwhelming when it comes to filling your prescriptions. We will make every effort to make sure your visit to our pharmacy is enjoyable.
First Choice Pharmacy specializes in Senior Care Medicine and Compounding. To know more about our services, please visit the Services page.


This classic dessert gets a delicious vegan makeover!
Preheat oven to 350°F. Lightly brush a 13" x 18" with coconut oil and line with parchment paper. In a stand mixer, whip the 'aquafaba' with the cream of tartar until stiff peaks begin to form and gradually beat in the sugar. Sift the flour and corn starch together and fold into the 'aquafaba' mixture and mix gently until smooth, but careful not to deflate the batter. Stir in 1 teaspoon vanilla extract.
Spread the batter into the prepared baking pan and bake for 20-25 minutes, until cake is set and slightly browned. Cool cake 10 minutes before removing from pan, and then invert onto a parchment lined cooling rack and cool completely. Peel off the parchment paper on the top and allow cake surface to dry out while it cools.
Prepare the 'soak' by mixing the espresso, maple syrup, rum and coffee liquor together and set aside. Blend the tofu, honey and remaining 2 teaspoons vanilla extract in a blender or food processor until smooth. Season with a pinch of salt and grated nutmeg. Store in the refrigerator until ready to assemble the dessert.
Cut the cake with a circle cutter or glass that you are serving it in. Cut 24 cake circles, using scraps if necessary, depending on the size of your serving glass. In 8 ounce ramekin or serving glass, place a cake layer, brush with 1 of the espresso soak. Spread with 1½ tablespoons whipped topping and top with another cake round. Brush that cake round with 1 tablespoon soak, and spread with 1½ tablespoons tofu filing and top with cake. Brush that cake round with 1 tablespoons soak and then top with another 1½ tablespoon whipped topping. Dust the top with cocoa. Repeat these steps to make 8 individual desserts. Top each with a chocolate covered espresso bean, if desired.


The NSW Taxi Council announced today that the NSW Taxi Industry will be joining a class action case against Uber, along with Western Australia and Queensland. The Class action is being led by high profile lawyers, Maurice Blackburn. The class action in NSW follows a claim in Victoria that has already commenced.
CEO of the NSW Taxi Council, Martin Rogers said, "Thousands of our members – hard-working Australians, have been impacted by Uber's actions. Taxi licence owners, operators and drivers have suffered enormously, both emotionally and financially, causing a significant impact on their livelihood. The NSW Taxi Council welcomes the opportunity for our members to recover some of their vast losses".
The NSW Taxi Council has been actively working with Lawyers from Maurice Blackburn over the last six months, assisting with their investigation into the class action. Taxi Owners, Operators and Drivers have all met with representatives from Maurice Blackburn as part of the investigation process.
Mr Rogers said, "A class action is a positive step for the NSW Taxi Industry, as Uber needs to be held accountable for the time it operated in NSW outside of any regulatory framework".
Taxi licence owners, operators and drivers are now able to visit the Maurice Blackburn website to register their interest.
For any media enquiries, please contact Nick Abrahim, Deputy Chief Executive Officer at the NSW Taxi Council.


text:
Global (AU)
UNHCR Representation in Canberra
Human lives, Human rights
World leaders voice strong support for new refugee deal at UN General Assembly
Countries hosting large numbers of refugees join UNHCR and World Bank in support of ambitious model to respond to displacement crises.
By Marta Martinez | 24 September 2018 | Español | Français | عربي
South Sudanese refugee Peter Batali runs a community initiative that helps young Ugandans access online learning platforms. Uganda allows refugees to work, start businesses and own property. © UNHCR/Michele Sibiloni
NEW YORK – UNHCR, the UN Refugee Agency, hosted a gathering with political leaders from four continents, including Bangladesh, Costa Rica, Turkey and the World Bank today at the UN General Assembly High Level Week in support of the international agreement known as the global compact on refugees, a robust and systematic model to improve the lives of refugees and their host communities.
The global compact is expected to be endorsed by members of the UN General Assembly in December 2018, after two years of intense consultations UNHCR has led with UN member states, international organizations, experts, civil society, and refugees.
The UN High Commissioner for Refugees Filippo Grandi praised the role of host countries as first responders to refugee emergencies, like Bangladesh, Uganda, Niger or Mali, among others.
"These countries have kept their borders open at a time when in the world this is not something that we can take for granted anymore," Grandi said. "We are facing an increasing tendency to reject, to close, to push back."
"Host countries have kept their borders open at a time when in the world this is not something that we can take for granted anymore."
2017 saw record figures of forced displacement — 68.5 million people worldwide were forced to leave their homes, of whom 25.4 million became refugees in other countries.
Almost nine in 10 of the world's refugees are hosted in developing countries, where they often live in remote areas with significant development challenges. The impact on local services, infrastructure and resources "is colossal," Grandi said.
UN High Commissioner for Refugees Filippo Grandi greets Bangladesh Prime Minister Sheikh Hasina at a High Level Meeting on the Global Compact for Refugees in New York. © UNHCR/Lorey Campese
The global compact envisions specific measures benefiting both refugees and hosts, such as strengthening the hosting country's health and educational infrastructure, as well as potentially revitalizing economies.
The prime minister of Bangladesh Sheikh Hasina shared the experience of how the country has been helping Rohingya refugees fleeing persecution in the neighbouring country of Myanmar.
Bangladesh has received over 727,000 Rohingya refugees in one year and it has allocated 600 acres of land to provide shelter, as well as covering their basic needs. "The resources invested in the Rohingya refugees have taken a toll on our environment and our economy," Hasina said.
"Inaction is not an option."
The Bangladeshi leader stressed the need for the international community to "share the burden" of the global refugee crisis. "World leaders must come forward with political will and commitment," she added.
"No government has the right to ignore this problem," said the Turkish minister of Foreign Affairs Mevlüt Çavuşoğlu.
Çavuşoğlu pointed at the crisis in the Mediterranean sea, where over 1,600 people have already died in 2018 trying to reach Europe. He advocated for "sustainable solutions to the migration crisis" like the global compact on refugees. "The current situation is unsustainable. Closing borders is not the right answer," he said.
"Refugees and migrants contribute to economic development and enrich cultural diversity," said the vice-president of Costa Rica Epsy Campbell Barr. The Central American country has been experiencing a rising flow of refugees from Nicaragua in recent months.
Barr reminded the representatives in the room of their shared responsibility: "A small country like ours is committed to the systematic efforts of receiving dozens of refugees daily. We anxiously look forward to the adoption of these global agreements."
"We need to remember that this is about people, about refugees who dream of better lives for themselves and their families."
The global compact also envisions measures such as resettlement and other pathways to third countries to help ease pressures on the countries hosting the largest numbers. The High Commissioner expressed concern about the shrinking figures of resettlement admissions. "We must reverse that course," Grandi said. "It is another expression of solidarity."
The involvement of development actors is key in the success of the global compact. The president of the World Bank Jim Yong Kim described the international agreement as "an example of how multilateralism can and should work."
Kim highlighted that 52 per cent of refugees worldwide are under the age of 18 and "the consequences of inaction will haunt us for generations."
"We need to remember that this is about people, about refugees who dream of better lives for themselves and their families," he added. "And host communities, the vast majority of which are in low and middle-income countries that themselves aspire to grow and thrive."
The process leading to the global compact on refugees was set in September 2016 when the UN General Assembly adopted the New York Declaration for Refugees and Migrants, which gave UNHCR the task to develop a 'compact' based on those consultations.
The new international agreement also builds on important lessons from applying a new vision for a more comprehensive response to these crises, known as the Comprehensive Refugee Response Framework (CRRF), in 15 countries.
See also: For refugees and locals in Uganda, the internet is changing lives
The experiences shared in the ECOSOC chamber suggested huge promise in the new comprehensive approach underpinning the global compact to be considered by the General Assembly before the end of the year.
"Inaction is not an option," said the president of the UN General Assembly Maria Fernanda Espinosa.
Grandi urged the country leaders in the room: "Help UNHCR say to the international community that better ways exist to deal with something many consider an intractable problem. We owe it to millions of uprooted and displaced people and we owe it to their generous hosts."
Related news and stories
Joint press release: International community shows solidarity with efforts to enhance protection and find solutions for people forced to flee and host communities in Central America and Mexico
In Tanzania, UNHCR's Grandi urges more backing for solutions as the country continues to host refugees
New York market serves up food that showcases the city's rich cultural heritage
LA Lakers' Wenyen Gabriel visits homeland of South Sudan with UNHCR, to 'speak up for refugees and displaced people everywhere'
IOM, UNHCR Call for Vigorous Investigation After Dozens Die in Abandoned Trailer in Texas
WHAT IS ASYLUM?


text:
class Nvlope::Message::Header < Nvlope::Model

  keys %w{
    content-type
    date
    dkim-signature
    domainkey-signature
    from
    reply-to
    in-reply-to
    message-id
    mime-version
    received
    sender
    subject
    to
  }

  def message_id
    raw['message-id'].first
  end

  def mime_version
    raw['mime-version'].first
  end

  def content_type
    raw['content-type'].first
  end

end

redpajama_set_name: RedPajamaGithub


The Genealogue: Did Sitting Bull Jump the Border?
Did Sitting Bull Jump the Border?
Now comes news that the bones of Sitting Bull his descendants want to move may actually be in Canada.
Jerome First, a 70-year-old Montana Sioux and the great-grandson of Sitting Bull's close friend, Chief Medicine Bear, claims the latest uproar over the bones is irrelevant because Sitting Bull was secretly buried in the Turtle Mountains of southern Manitoba.
The Turtle Mountains straddle the border between southwestern Manitoba and North Dakota.


text:
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Jade Flares Size 8
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This 90 day supply is the perfect organic solution to clear, healthy skin with four simple steps.
After cleansing with Juice Beauty's Blemish Clearing Cleanser, apply this treatment serum to entire face or just to blemished areas. Use morning and night.
First cleanse with the Juice Beauty Blemish Clearing Cleanser, then apply BLEMISH CLEARING™ Serum and finish by applying this moisturizer to your face and neck every morning and night.


text:
RoboCop reality: robots and drone patrols coming to your neighborhood
By James Gelinas, Komando.com June 28th, 2019
The future is here, and people couldn't be more excited. We have higher standards of living, instant entertainment, and even next-day delivery for almost anything you could think of. There's just one detail missing from our modern world (aside from flying cars) that would make it truly futuristic: robots walking among us!
Well, don't get too comfortable with the state of our future just yet -- robots are definitely coming! Police in Southern California have revealed their first automated patrol robot to help assist the department with monitoring parks and public spaces. Meanwhile, a major tech company is bringing the power of surveillance to the people by allowing homeowners to keep an eye on their property from the skies above.
As robots creep into our lives at a rapid pace, it won't be unusual to see artificial cops and automated drones nearly everywhere we go. This does raise the question, however: Are we truly ready to sacrifice our privacy in the name of technological progress? You won't believe what these robots are capable of.
California unleashes the HP RoboCop
The Huntington Park Police Department of Southern California recently unveiled the latest addition to its force: a chest-high, egg-shaped robot with wheels that they're affectionately calling the "HP RoboCop."
This robot isn't armed with weapons like a traditional police officer (thank goodness), but it is equipped with a 360-degree camera and high-definition microphones to observe and report about its surroundings.
The department has successfully deployed the robot in a variety of locations, including Huntington Park, crosswalks, and even sections of Los Angeles International Airport. According to police, the robot helps officers spread their supervision further than they would be able to normally, and acts as a deterrence to criminal activity in the areas it shows up in.
Thank you Huntington Park for a warm welcome, hope everyone enjoyed the presentation. Happy to be an asset to the Huntington Park Police Department. #hprobocop #cityofHP pic.twitter.com/Y4wE0QnVR2
— HPRoboCop (@HPRoboCop) June 19, 2019
As of now, there haven't been plans to permanently expand the range of HP RoboCop, but the Silicon Valley company that makes them is saying the robots are being tested in select cities across the U.S. So if you see a giant egg gliding across the sidewalk in your city, know that the police are watching and listening through its electric eyes and ears!
Amazon enters the home security game
Not to be upstaged by a mere police department, Amazon is giving its users the chance to harness robots for their own safety and protection. A new patent filed by the company proposes the use of Amazon's fleet of delivery drones as a "service," meaning the miniature flying vehicles could send snapshots of your property during a package drop-off.
The potential for this service is actually quite interesting. Imagine the drone catching a fire starting at your home when it goes to drop off a package, or maybe even snitching on a burglar trying to make his way inside. The patent is scant on specific details, but judging from the existing content available, the surveillance service could prove popular among Prime users.
Conversely, it may be worth questioning how responsible it will be with its flying camera arrays. The drones already use advanced camera systems to navigate, land, and detect obstacles. It isn't too much of a stretch to imagine Amazon snapping pictures of your home, noticing you do lawn work often, and recommending lawn care supplies when you shop.
Related: Amazon rolls out more free delivery perks for Prime members
This is pure speculation, of course, but judging by the data-usage habits of other platforms, it doesn't seem outside of the realm of possibility. Whether people are comfortable with this kind of "personalization" is another matter entirely.
In the battle between HP RoboCop and Amazon delivery drones, however, I would personally side with Amazon. Its primary goal is to sell us things, and the surveillance options it might be offering puts the power of that system in our hands. Plus, the drones are so small they're unlikely to be able to hurt us much. Unless, of course, one falls out of the sky and on to our head!
Like it or not, the robots are almost here. Are you ready to welcome them into your life?
Facebook's classified robotics division is going after Google and Apple
Work in artificial intelligence and robotics is becoming a larger part of our world with every passing day. Massive tech companies like Google and Apple have been working on advancements in those areas for some time and now for the first time, Facebook is detailing its efforts to make robots smarter through AI.
Click or tap here to find out how Facebook is using AI to train its robots faster.
Robocallers are stooping to an all-time low by attacking hospitals
Find out what Apple knows about you
7 things you should be doing with your Amazon Prime account
Load up with movies and TV shows before you travel
7 digital privacy tricks you'll wish you knew before now


Because of the scandal of Hong Kong female star Gillian Chung (Ah Jiao) and Edison Chen, her good girl image is ruined, protecting their idol this caused a stir with Wu Zun's fans, these few days China, Hong Kong and Taiwan's region websites have connected together, expressing to Wu Zun's management company for propaganda, requesting the company to not have Wu Zun and Gillian perform together in the film "Martial Arts Lovers". The fans have said, "We don't want Wu Zun's first film to be tarnished, Ah Gil is already not good enough for Wu Zun, this is filming the "Butterfly Lovers", not filming "Jin Ping Mei!"
Wu Zun is suppose to enter the big screen this year, it's scheduled to start filming Ma Chu Cheng's "Martial Arts Lover" after he finishes filming the idol drama "Hot Shot", originally the pairing of Wu Zun and Ah Gil would have been the perfect golden boy and girl, but now Ah Gil is involved in the Edison Chen scandal, the collaboration may change.
Ah Gil's management company EEG has denied Ah Gil will be changed the part, but has admitted that Nicholas Tse will not be filming in "Martial Arts Lover"; but the reason is because of the schedule conflicts.
Despite Ah Gil an artist stepping forward courageously, but most Hong Kong media still criticizes her act heavily.
The reason is because Ah Gil's image was a too deifying good girl image, this included her expressing in public of a kissing scene that made her feel gross, and not having pre-marriage sexual relationship, after the picture scandal this shows her contradicting. The Hong Kong media's comments have heavily criticized, "Not despised of her debauchery, but because of her hypocrisy."
Because of Ah Gil's current image Wu Zun's fans thinks she's not good enough to match him, they're also worried that after Wu Zun accepts this film, the focus on the news will be all fuzzy. Wu Zun's manager Chen Hui Ying yesterday says, not only expressing comments online, the company has also received many letters from fans or have called regarding the similar request, as for the criticism of Ah Gil, she says: "Everyone has the right to express their comments, really appreciate the support for Wu Zun, and we also respect all voices of expression."
As for the decision to resign from filming? She says, "We have been talking with this film for a long time, but until today we have still not made a decision, this is because lately the weather hasn't been good, the filming of "Hot Shot" has been delayed, so there could be a possibility of schedule conflict to not able to film."
Hong Kong producer of "Martial Arts Lovers" Xuan Jiazhen yesterday said he respects the suggestions from the fans, but hopes to have the fans to give them some time, so that we can find a solution that will be acceptable for everyone.
you mean Gillian Chung or wu zun??
I love Calvin only and Calvin alone forever. Srry if the pic takes up too much space.
the person in teh 2nd picture... LOL, is that a girl?? oh crackers... my bad.. hehe><"
tht's good gr8 gramps cuz if u say wuzun looks scary I'm gonna hide ur teeth again...heh!
oh really, never heard of her.
gosh i gotta get thicker lenses for my glasses, i can't seem to tell the diffrence from boys and girls ><"
Her image was ruined because of the sexual pictures of her and edison chen.
" they dont think she's good enough for her "
but yeah you can search it up.
idk they did end up working together !?!?!?
i think they did.. they look similar in the advertising.. but im not sure.. im waiting to c the reviews to decide whether to watch it or not..


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Q: Give an example of a sequence $(x_n)^{\infty}_{n=1}$ such that $x_{nk}$ converges as $n \rightarrow \infty$ for all $k \leq 2$ Give an example of a sequence $(x_n)^{\infty}_{n=1}$ such that $x_{nk}$ converges as $n \rightarrow \infty$ for all $k \leq 2$, whereas $x_n$ does not converge as $n \rightarrow \infty$.
How am I go about thinking of this? Or does that sequence even exist?


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Today is the annual Day of Prayer for the Legal Protection of Unborn Children, a day on which the U.S. Bishops have instructed us to pray for the protection of the unborn and to do penance in reparation for acts of abortion.
According to the General Instruction of the Roman Missal, no. 373:
In all the Dioceses of the United States of America, January 22 (or January 23, when January 22 falls on a Sunday) shall be observed as a particular day of prayer for the full restoration of the legal guarantee of the right to life and of penance for violations to the dignity of the human person committed through acts of abortion.
Let us join with the Bishops in prayer and penance, imploring God's mercy upon our nation and the world for this terrible crime, and invoking Our Lady's aid in bringing an end to abortion and the culture of death and restoring a culture of life.
Our Lady of Guadalupe, Patroness of the Unborn, pray for us!
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Death of Pope Emeritus Benedict XVI
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Focus on Radiation Oncology
Novel Combinations
Research Articles: Therapeutics, Targets, and Development
AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies
Sonya D. Zabludoff, Chun Deng, Michael R. Grondine, Adam M. Sheehy, Susan Ashwell, Benjamin L. Caleb, Stephen Green, Heather R. Haye, Candice L. Horn, James W. Janetka, Dongfang Liu, Elizabeth Mouchet, Shannon Ready, Judith L. Rosenthal, Christophe Queva, Gary K. Schwartz, Karen J. Taylor, Archie N. Tse, Graeme E. Walker and Anne M. White
Sonya D. Zabludoff
Chun Deng
Michael R. Grondine
Adam M. Sheehy
Susan Ashwell
Benjamin L. Caleb
Stephen Green
Heather R. Haye
Candice L. Horn
James W. Janetka
Dongfang Liu
Elizabeth Mouchet
Shannon Ready
Judith L. Rosenthal
Christophe Queva
Gary K. Schwartz
Karen J. Taylor
Archie N. Tse
Graeme E. Walker
Anne M. White
DOI: 10.1158/1535-7163.MCT-08-0492 Published September 2008
Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. [Mol Cancer Ther 2008;7(9):2955–66]
AZD7762
checkpoint kinase inhibitor
DNA-targeted therapy
combination chemotherapy
Conventional DNA-targeting therapies such as chemotherapy and radiation are among the most common cancer treatments and have produced significant increases in patient survival, particularly when used in combination with drugs having different mechanisms of action. Due to their efficacy, DNA-targeting agents are likely to remain a standard of care for the treatment of many cancers for the foreseeable future. Although effective, this mechanism also leads to significant side effects as the majority of these agents are used at the maximum tolerated dose (MTD). Toxicities to the hematologic, gastrointestinal, and other organ systems are commonly observed and limit the degree of tumor control that can be achieved. Another limitation of DNA-damaging agents is that many patients develop resistance and therefore become refractory to treatment. Resistance can arise from multiple mechanisms including modulation of cellular levels of the drug, defects in apoptosis, or DNA repair.
DNA damage induced by chemotherapy or radiation therapy occurs through multiple mechanisms (e.g., alkylating agents, topoisomerase poisons, and antimetabolites). Although different mechanisms are required to repair each type of DNA damage, common to all is the cellular response of cell cycle arrest regulated by signaling cascades that are generally referred to as cell cycle checkpoint pathways.
Checkpoint signaling is initiated following genotoxic insult by the proximal kinases, ATR and ATM, two phosphatidylinositol 3-kinase family members. Activation of these kinases leads to activation of the effector kinases, checkpoint kinases 1 and 2 (Chk1 and Chk2; serine/threonine kinases). The activated effector kinases are then able to transiently delay cell cycle progression through the G1, S, or the G2 phases so that DNA can be efficiently repaired. The ATM/Chk2 pathway predominantly regulates the G1 checkpoint and the ATR/Chk1 pathway the S and G2 checkpoints. However, there is cross-talk between the pathways implying a role for both ATR and ATM pathways in all cell cycle checkpoints. In addition to directly regulating the cell cycle, the pathways also affect DNA repair, transcription, chromatin regulation, and cell death.
The hypothesis that tumors may be selectivity sensitized to DNA-damaging agents through abrogation of the G2-DNA damage checkpoint was first proposed in the 1980s followed by further support in the 1990s (1–3). The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway or other components of checkpoint signaling and response. For example, high p53 mutation rates result in reliance on S and G2 checkpoints to repair DNA damage and promote cell survival. Therefore, abrogation of these remaining intact checkpoints should lead to enhanced tumor cell death compared with normal tissue. Inhibition of Chk1 signaling using small-molecule inhibitors, dominant-negative enzymes, interference RNA, and ribozymes leads to abrogation of the S and G2 checkpoints, impaired DNA repair, and increased tumor cell death (4–12).
The present studies describe the identification and characterization of AZD7762, a novel, ATP-competitive and selective checkpoint kinase inhibitor in clinical trials. AZD7762 potently inhibits Chk1 and Chk2, abrogates DNA damage-induced S and G2 checkpoints, enhances the efficacy of gemcitabine and topotecan, and modulates downstream checkpoint pathway proteins as predicted. Furthermore, AZD7762 potently abrogates these same checkpoints in vivo and significantly enhances the antitumor efficacy of gemcitabine and irinotecan in rodent tumor models at well-tolerated doses.
AZD7762 [3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-3-piperidyl]thiophene-2-carboxamide] (Fig. 1 ) was synthesized at AstraZeneca R&D. Gemcitabine, irinotecan, and SN-38 were obtained from CG International.
Flow diagram of high-throughput screening and follow-up. Selection criteria and number of compounds progressing to the next step are listed. Three prioritized series were chosen as the lead series for continued characterization and SAR development. AZD7762, a thiophene carboxamide urea, was chosen as the clinical candidate due to an overall superior profile (efficacy, absorption, distribution, metabolism and excretion, and safety).
High-Throughput Screen and Chk1 Kinase Assay
Recombinant human Chk1 was expressed as a glutathione S-transferase fusion in insect cells using a baculovirus vector and purified by glutathione affinity chromatography. A synthetic peptide substrate for Chk1 was synthesized by Bachem (N-biotinylaminohexanoyl-KKVSRSGLYRSPMPENLNRPR). Compounds were screened at a single concentration (10 μmol/L) for the primary assay using a standard scintillation proximity assay protocol. For follow-up, a dose response was determined (IC50). Final assay concentrations of peptide and ATP (cold + 40 nCi [33P]ATP) were 0.8 and 1 μmol/L, respectively. In brief, compound, buffer containing peptide and kinase and ATP, were added sequentially to a 384-well assay plate. The plate was incubated for 2 h, reaction was stopped by the addition of buffer containing EDTA and scintillation proximity assay beads (Amersham), and plates were read using a TopCount reader (Packard). Data analysis was carried out using proprietary software.
For kinetic analysis, a filter binding assay was used. The assay reaction contained the reagents listed above, but with the following concentrations of ATP [0-600 μmol/L (cold + Ci [33P]ATP), determined Km], Chk1 (0.5 nmol/L), and AZD7762 (0, 1.5, 5, 10 nmol/L). The reaction was incubated at room temperature for 20 min, stopped by the addition of EDTA, transferred to Streptavidin Flashplate (Perkin-Elmer), incubated at room temperature for 1 h, aspirated out, and wells washed with PBS. Plates were counted using a TopCount reader and data were analyzed using proprietary software.
Cells were obtained from the American Type Culture Collection unless otherwise indicated. HT29 and HCT116 cells were cultured in McCoy's medium (Invitrogen) containing 10% FCS; SW620, H460-DNp53, HCT116 (p53+/+ and p53−/−, a generous gift from Dr. Bert Vogelstein, Johns Hopkins University, to G.K. Schwartz) and MDA-MB-231 cells were cultured in RPMI containing 10% FCS. H460-DNp53 was made by retroviral infection of a dominant-negative p53 construct. Individual clones were selected and characterized by evaluating endogenous p53 up-regulation, lack of p21 up-regulation, and G1 arrest following treatment with actinomycin D.
Anti-cdc25A was obtained from Santa Cruz; anti-cdc2 and anti-phospho-cdc2(T15) were from Cell Signaling Technology; anti-phospho-histone H3 (phH3; S10) and anti-cyclin B were from Upstate Biotechnology; and anti-cyclin A was from Neomarkers.
Western Blot Analysis
SW620 cells (3 × 106 per 10 cm plate) were seeded, incubated overnight, and treated with compounds solubilized in DMSO or vehicle (0.01% DMSO). Protein lysates were prepared in Phospho Safe buffer (Novagen) and protein concentrations were determined by BCA protein assay kit (Pierce). Standard Western blot analyses were conducted using the antibodies listed above.
Checkpoint Abrogation Assay
HT29 cells (3 × 105) were seeded in 96-well plates and incubated overnight. Cells were treated for 2 h with camptothecin (topoisomerase I inhibitor; 0.07 μg/mL) to induce the G2 checkpoint. Cells were then treated for 20 h with vehicle (0.5% DMSO) or caffeine (4 mmol/L; positive control) plus nocodazole (1 μg/mL) or a 12-point titration of AZD7762 (12.5 μmol/L to 6 nmol/L) plus nocodazole. Nocodazole alone-treated cells with no camptothecin pretreatment were used to determine the maximum mitotic index. Cells were fixed with 3.7% formaldehyde for 1 h, permeabilized with PBS containing 0.05% Triton X, and incubated with anti-phH3 antibody for 1 h followed by Alexa Fluor 488 anti-rabbit (Molecular Probes) and Hoechst stain for 1 h. Mitotic index was determined on the ArrayScan and expressed as the percentage of cells undergoing mitosis. The EC50 was calculated by concentration-response curve fitting using three-variable logistical equations within XLfit (model 205) with the curve bottom constrained to 0 and the top constrained to 100 by nocodazole alone treatment.
Potentiation Assays
SW620 (5.5 × 103 per well) or MDA-MB-231 (5 × 103 per well) cells were seeded in 96-well plates and incubated overnight. Cells were dosed for 24 h with a 9-point titration of gemcitabine ranging from 0.01 to 100 nmol/L with or without a constant dose of AZD7762 (300 nmol/L). Control wells were dosed with vehicle alone (0.1% DMSO) or 300 nmol/L AZD7762. After 24 h, medium was removed and AZD7762 alone was added back to the wells treated previously with AZD7762 for an additional 24 h. Cells were then incubated in drug-free medium for an additional 72 h. The effect on cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethophenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay as recommended by the supplier (Promega). The same experimental procedure was used for topotecan combinations (topoisomerase I inhibitor, analogue of camptothecin) except an 11-point titration of topotecan ranging from 0.1 nmol/L to 30 μmol/L was used. Net growth was calculated (AT120 - AT0 / predose) × 100 and plotted versus concentration of chemotherapy in the presence and absence of AZD7762. IC50 values were calculated by concentration-response fitting using four-variable logistical equations (Sigmoidal fit) within Origin Pro.
Clonogenicity Assays
Log-phase parental and p53-null HCT116 cells were plated, in triplicate, onto 100-mm dishes at 1,000 to 5,000 per dish. Cells were treated with increasing concentrations of gemcitabine alone for 2 h, gemcitabine followed immediately by 100 nmol/L AZD7762 for 24 h, or gemcitabine followed by drug-free medium for 24 h before AZD7762. At the end of treatment, cells were cultured in drug-free medium for 10 to 14 days. The resulting colonies were scored after staining with 0.01% crystal violet.
In vivo Studies
Animals. Male NCr mice were purchased from Taconic Laboratories and male rnu rats were purchased from Charles River. All procedures were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and within the protocols approved by the Institute of Animal Care and Use Committee at AstraZeneca.
Hollow Fiber Assay. Fibers made from polyvinylidene difluoride (molecular weight cutoff, 500 kDa; Spectrum) were immersed in 70% ethanol for 72 h, flushed with distilled water, and autoclaved. HCT116 cells were suspended in McCoy's medium with 20% fetal bovine serum and loaded into the fibers at 3 × 106/mL. HCT116 cells have a strong G2 arrest and thus it is an ideal cell line to measure abrogation of G2 arrest. Following overnight incubation in McCoy's medium, topotecan (30 nmol/L) was added to the appropriate groups and allowed to incubate for 18 h before fibers were rinsed and implanted into the mice. Animals were dosed i.v. with AZD7762. The fibers were recovered 30 h later; cells were flushed out, fixed in 70% ethanol, and stained with propidium iodide. Samples were analyzed by flow cytometry. Modfit was used to calculate detailed cell cycle profiles and results were reported as odds ratio values. The odds ratio is defined as the ratio of G2-to-G1 populations for topotecan alone versus in combination with AZD7762.
Xenograft Models in Mice. Tumor cells were harvested, pelleted by centrifugation for 5 min, and resuspended in sterile PBS. Cells (3 × 106 - 6 × 106) were implanted s.c. into the right flank of the mice in a volume of 0.1 to 0.2 mL using a 25-gauge needle. Tumors were allowed to grow to the designated size of ∼100 to 200 mm3 before the administration of compound.
Xenograft Models in Rats. Cells were harvested, pelleted by centrifugation for 5 min, and resuspended in 50% sterile PBS and 50% Matrigel. Rats received a 5 Gy whole-body radiation dose 5 days before cell implantation to improve tumor growth. H460-DNp53 cells (1 × 107) were implanted s.c., under anesthesia with isoflurane, into the right flank of the rats in a volume of 0.2 mL using a 25-gauge needle. Tumors were allowed to grow to the designated size of ∼100 to 200 mm3 before the administration of compound.
Efficacy studies. Compounds were administered by i.v. injection via the tail vein. AZD7762 was formulated in 11.3% hydroxyproplyl-β-cyclodextrin. Cyclic schedules were used and treatment ranged from three to five cycles. Each cycle included administration of a standard agent [gemcitabine or irinotecan (topoisomerase I inhibitor, camptothecin analogue)] every 3 days followed by delivery of AZD7762 as described in the figure legends. Tumor volumes were measured with electronic calipers and calculated using the formula [length × (width × width)] × 0.5. log cell kill = GD / (3.32 × TD), where GD is the growth delay and TD is the tumor doubling time of the control.
High-Throughput Screening Led to the Identification of Thiophene Carboxamide Ureas as Potent Chk1 Inhibitors
The AstraZeneca compound library was screened using the Chk1 enzyme assay with an overall hit rate of 2.2%. Six thousand compounds were profiled in dose response and ∼30% of these had IC50 <30 μmol/L. The screen performance was robust and reproducible with consistent Z′ values between 0.7 and 0.8. The flow diagram in Fig. 1 shows the high-throughput screen process, statistics for the screen, and early follow-up.
Cluster analysis revealed several structural series, three of which were selected for further optimization. From these studies, the thiophene carboxamide urea series emerged as the most promising as this structural class provided not only potent Chk1 inhibitors but also excellent solubility and drug metabolism and pharmacokinetic properties.
AZD7762 Is a Potent and Selective Inhibitor of Chk1 Kinase
AZD7762 was discovered as a potent and selective Chk1 kinase inhibitor through multiple rounds of structure-activity relationship-driven chemistry (Fig. 1). The activity of AZD7762 was evaluated against human recombinant Chk1 and a panel of protein kinases in vitro. AZD7762 potently inhibited Chk1 phosphorylation of a cdc25C peptide with an IC50 of 5 nmol/L as measured by a scintillation proximity assay. Due to the determined high Km (80 μmol/L) of Chk1 for ATP, the scintillation proximity assay could not be run at Km. However, data generated by a filter capture assay, which contained ATP concentrations at Km, yielded an IC50 of 5 nmol/L. The Ki for AZD7762 was determined to be 3.6 nmol/L. Kinetic characterization suggested that AZD7762 binds in the ATP-binding site of Chk1 and is thought to compete directly for ATP binding in a reversible manner.
In selectivity screens, AZD7762 showed significantly less inhibition against unrelated protein kinases. In general, kinases with less than 10-fold selectivity were from the same family of kinases as Chk1, CAM kinases, or Src-like kinases from the tyrosine kinase family although not Src itself (Yes, Fyn, Lyn, Hck, and Lck). Following preliminary profiling, expanded screening was biased toward CAM and tyrosine kinase family members. Data generated from in-house and Upstate screening indicated good selectivity (>10-fold) of Chk1 kinase versus 164 kinases (Supplementary Tables S1 and S2).4 Importantly, selectivity was shown in the cyclin-dependent kinase (cdk) 1/cyclin B1 scintillation proximity assay (>1,000-fold) and other cdks tested. Greater than 100-fold selectivity against multiple protein kinase C isoforms, cdks, p38 and MAPKAP kinase 2 clearly differentiates the activity profile of AZD7762 from UCN-01 (13–15). AZD7762 is equally potent against Chk2. Low-fold selectivity was observed against phosphoinositide-dependent kinase (isolated enzyme), but in cellular assays AZD7762 did not affect the phosphoinositide-dependent kinase pathway (data not shown).
AZD7762 Abrogated Checkpoint Cell Cycle Arrest Mediated by DNA-Damaging Agents
The ability of AZD7762 to abrogate the G2 checkpoint was determined in the checkpoint abrogation assay. Cells were pretreated with camptothecin for 2 h followed by AZD7762 or vehicle treatment and nocodazole for 20 h. Cells were then analyzed for phH3. Cells treated with camptothecin arrest in G2 and did not stain for phH3. Cells treated with camptothecin and AZD7762 did not arrest in G2, moved into mitosis, and were trapped by nocodazole. These cells stained for phH3 indicating abrogation of the G2 checkpoint. AZD7762 was shown to abrogate the G2 arrest induced by camptothecin with an average EC50 of 10 nmol/L (n = 12) and maximal abrogation in the range of 100 nmol/L. In the absence of camptothecin, AZD7762 alone induced cell arrest with an EC50 0.620 μmol/L, >50-fold higher than the concentrations required to abrogate the damage induced phenotype.
Combination Treatments Led to Anticipated Effects on Cell Cycle Proteins
Gemcitabine and SN-38 (active metabolite of irinotecan) lead to cell cycle arrest at the S and G2 checkpoints, respectively. Therefore, different downstream cell cycle proteins and checkpoint kinase substrates must be evaluated for each combination. The effect on cell cycle proteins was evaluated at 8 h (continuous exposure to agents) to evaluate the more immediate effects and 30 h (8 h drug treatment followed by 22 h washout) to evaluate the cell cycle proteins following the release from checkpoint kinase inhibition (Fig. 2A and B ). Treatment with gemcitabine alone led to the predicted destabilization of cdc25A, a direct substrate of Chk1, which on Chk1 activation is phosphorylated and consequently degraded. AZD7762 treatment stabilized cdc25A. Importantly, the combination led to a dose-dependent increase in cdc25A. At 30 h, the significant effect of Chk1 inhibition on the stabilization of cdc25A was no longer observed due to the reversibility of AZD7762. Cyclin A levels, in contrast, were decreased for the combination, consistent with the abrogation of the S-phase checkpoint and the effect on the cell cycle (Fig. 2A and B).
Effect of AZD7762 on cell cycle proteins following treatment of cells with DNA-damaging agents. SW620 cells were treated with gemcitabine (A and B) or SN38 (irinotecan active metabolite; C and D) with and without AZD7762 at the doses indicated. Following 8 h simultaneous treatment, cells were harvested for Western blot analysis (A and C) or drug-containing medium was removed and replaced with drug-free medium for an additional 22 h before harvesting (B and D). Western blot analyses were done on cell lysate as described in Materials and Methods.
On activation of the G2 checkpoint by SN-38, cdc25C, a checkpoint kinase substrate, is phosphorylated and inactivated. Subsequently, inactivation of cdc25C leads to an increase in phosphorylation of cdc2 (Tyr15). A high-quality cdc25C antibody was not identified, so the effect on cdc25C could not be evaluated directly. However, consistent with checkpoint kinase inhibition, combination treatment with SN-38 decreased phospho-cdc2 (Tyr15) at 8 and 30 h. Cyclin B levels also decreased, which is consistent with abrogation of the G2 checkpoint. phH3, a marker for mitotic cells, decreased on treatment with irinotecan, indicating blocked mitotic entry. In combination, phH3 increased, again consistent with abrogation of the G2 checkpoint (Fig. 2C and D).
AZD7762 Enhanced the Activity of DNA-Damaging Agents
AZD7762 showed robust gemcitabine potentiation and enhanced activity of topotecan-mediated inhibition of cell growth. Potentiation was observed in SW620 colon cancer cells (p53 mutant) with gemcitabine (Fig. 3A ). It is interesting to note that stasis was observed only in the SW620 cells at the highest dose of gemcitabine tested, whereas cell death occurred in the combination treatment because the curves dropped below 0% net growth. Additional cell lines were tested with the most consistent and profound potentiation observed in combination with gemcitabine (data not shown). Enhanced activity was observed in MDA-MB-231 breast carcinoma cells (p53 mutant; Fig. 3B) with topotecan. MDA-MB231 cells compared with SW620 cells showed a slight reduction in percent net growth from AZD7762 alone.
AZD7762 potentiated gemcitabine and topotecan. A, SW620 cells were treated with a titration of gemcitabine with AZD7762 (300 nmol/L) for 24 h. Following removal of drug-containing medium, cells were treated with AZD7762 for an additional 24 h or drug-free medium. Cells were treated with vehicle, gemcitabine, or AZD7762. Medium was removed and drug-free medium was added to all cells for an additional 72 h. Effect on cell growth and survival was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethophenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay. The percentage net growth was calculated and plotted against gemcitabine concentration. Open shapes, controls; filled shapes, combination treatment. GI50 and GI100 values were determined for gemcitabine alone and gemcitabine plus AZD7762. Standard error bars are plotted. B, MDA-MB-231 cells were treated with a titration of topotecan with AZD7762 (300 nmol/L) for 24 h. Cells were treated as described in A. The percentage of net growth was calculated and plotted against topotecan concentration. Open shapes, controls; filled shapes, combination treatment. GI50 values were determined for topotecan alone and topotecan plus AZD7762. Standard error bars are plotted. C, HCT116 (p53+/+ and p53−/−) were treated with a titration of gemcitabine for 2 h followed by AZD7762 (100 nmol/L) or drug-free medium for 24 h. Cells were then cultured for 10 to 14 d and colonies were counted. Percent survival was calculated and plotted against gemcitabine concentration. Lines, p53+/+; broken lines, p53−/−. Experiment was repeated three times with similar results.
Enhanced Cytotoxicity with AZD7762 Is Observed in p53-Mutant Tumor Cell Lines
To examine the effect of p53 loss on tumor sensitivity to combined treatment with chemotherapy and checkpoint inhibitor, we studied clonogenic inhibition by gemcitabine and AZD7762 in an isogenic pair of colon cancer cell lines. Parental and p53-null HCT116 cells were equally sensitive to a 2-h treatment of single-agent gemcitabine (Fig. 3C). Sequential treatment with gemcitabine followed immediately by 100 nmol/L AZD7762 for 24 h (Gem→AZD; Fig. 3C) or by drug-free medium for 24 h before AZD7762 (Gem→ND→AZD; Fig. 3C) resulted in a marked potentiation of clonogenic loss, more selectively in cells that lack p53. These data are consistent with the overall hypothesis that checkpoint inhibitors specifically enhanced the cytotoxicity of DNA-damaging agents in checkpoint-defective p53-mutant tumors.
AZD7762 Abrogated the G2 Checkpoint In vivo
A hollow fiber pharmacodynamic model was developed as shown in Fig. 4 . The assay was used to measure the in vivo pharmacodynamic activity of checkpoint kinase inhibitors in nude mice and rats for both compound selection and to determine the pharmacokinetic/pharmacodynamic relationship. As shown in Fig. 4B, topotecan treatment typically arrested 70% to 90% of cells in the G2 phase, and this arrest was relatively stable for up to 30 h post-implantation. Checkpoint kinase inhibition abrogated the G2 checkpoint causing a reduction in the number of cells in G2, with a concomitant increase in the number of cells reentering the G1 phase (Fig. 4B). The odds ratio is defined as the ratio of G2-to-G1 populations for topotecan alone versus in combination with AZD7762. Thus, the greater the odds ratio, the higher the degree of checkpoint abrogation. This analysis allows pharmacodynamic activity to be quantitatively assessed and statistical significance to be determined. As seen in Fig. 4C, AZD7762 treatment resulted in a dose-dependent increase in abrogation of the G2 checkpoint.
AZD7762 abrogated the G2 checkpoint in a novel hollow fiber assay. A, schematic diagram illustrating the hollow fiber assay. HCT116 cells were loaded into prepared fibers, incubated in medium, and then incubated in topotecan-containing medium for 18 h. Fibers were implanted into mice and AZD7762 was delivered i.v. Thirty hours later, fibers were removed from the mice and cells from the fibers were fixed with ethanol and stained with propidium iodide. Cell cycle profiles were determined by fluorescence-activated cell sorting analysis. B, fluorescence-activated cell sorting profiles of cells treated with vehicle control, topotecan alone, or topotecan followed by AZD7762. The percentage of cells in the G1 and G2 phase and calculated odds ratio values are listed. C, dose-dependent increase in the odds ratio following treatment with AZD7762.
AZD7762 Potentiated the Tumor Efficacy of DNA-Damaging Chemotherapy
The ability of AZD7762 to potentiate gemcitabine and irinotecan was evaluated in mouse and rat xenograft models. Mouse xenograft studies were done using either the H460-DNp53 mutant human lung cancer cell line or the SW620 colorectal tumor cell line. The H460-DNp53 cell line is relatively resistant to gemcitabine single-agent treatment, whereas SW620 cells are more sensitive. Established tumors were treated with vehicle, gemcitabine alone, AZD7762 alone, or gemcitabine in combination with AZD7762 (Fig. 5 ). In the H460-DNp53 xenograft study, neither gemcitabine nor AZD7762 alone showed significant antitumor activity (Fig. 5A). In contrast, AZD7762 significantly potentiated gemcitabine in the combination group resulting in a log cell kill of 0.9 or percent treated/control (%T/C) of 26. There was a slight but not statistically significant body weight loss (7%) in the combination group compared with the gemcitabine alone control group (no loss). In the mouse SW620 xenograft study, AZD7762 in combination with gemcitabine showed significant antitumor activity compared with either agent alone. Similar efficacy was achieved within the low- and high-dose combination group (Fig. 5A). No significant increase in body weight loss was observed in the combination group over the single-agent gemcitabine treatment group. In summary, AZD7762 significantly enhanced the activity of gemcitabine without significantly affecting the gross toxicology associated with gemcitabine therapy in the nude mouse.
AZD7762 potentiated gemcitabine in rodent xenograft efficacy models. A, athymic mice bearing established H460-DNp53 or SW620 tumors were treated with multiple cycles of therapy every 3 d (arrows). Each cycle consisted of gemcitabine alone (60 mg/kg) or gemcitabine (60 mg/kg) followed by two doses of AZD7762 (4 and 16 h after gemcitabine dose). Calculated percent inhibition is listed. Percentage inhibition was calculated as (geometric means of control - geometric means of treatment) / (geometric means of control - 1) × 100. *, P ≤ 0.001, median tumor volumes were significantly less in the treated versus the control group as determined by Student's t test. B, rnu rats bearing established H460-DNp53 tumors were treated with three cycles of therapy every 4 d (arrows). Each cycle consisted of gemcitabine alone (10 mg/kg) or gemcitabine (10 mg/kg) followed by a single dose of 10 or 20 mg/kg AZD7762 (4 h after gemcitabine dose). Tumor volume was measured using electronic calipers on the indicated days with the median ± SE tumor volumes indicated for groups of 10 animals.
In the rat H460-DNp53 xenograft study, AZD7762 potentiated the antitumor activity of gemcitabine in a dose-dependent manner (Fig. 5B) as evidenced by a decrease in %T/C with increasing dose (48% and 32%, 10 and 20 mg/kg AZD7762, respectively; Fig. 5B). In the rat model (unlike the mouse), gemcitabine was delivered at the MTD (10 mg/kg), and AZD7762 was still able to potentiate gemcitabine. In this xenograft study, gemcitabine alone caused severe toxicity (deaths or moribund) in 4 of 10 rats a few days after the third dose. Additional single-agent gemcitabine-related toxicities at this dose included skin rash and hemorrhage. In contrast, none of the rats in the low-dose combination group exhibited severe toxicity, and only one rat was sacrificed in the high-dose combination group due to body weight loss. No skin rash was observed in any of the rats in the combination groups. These data indicate that the addition of AZD7762 does not lead to significant toxicity beyond that induced by gemcitabine and that the combination can be delivered at the MTD of gemcitabine.
In the mouse xenograft study in combination with irinotecan, SW620 established tumors were treated with vehicle, irinotecan alone, AZD7762 alone, or AZD7762 in combination with irinotecan. AZD7762 dosed alone showed insignificant antitumor activity, whereas irinotecan alone displayed striking and significant activity (%T/C with increasing dose was 9 and 1, respectively; Fig. 6 ). In combination with AZD7762, %T/C increased significantly to -66% and -67%, respectively. Because tumors were not detected, animals were observed for an extended period and tumors and body weights measured for a total of 94 days post-tumor implantation to monitor tumor regrowth. At the end of the study, tumor-free survival was observed in the low- and high-dose irinotecan combinations (5/9 and 8/9 tumor-free survival, respectively). No statistically significant body weight loss was observed in any treatment group.
AZD7762 treatment resulted in tumor-free survival in combination with irinotecan in the mouse xenograft efficacy model. A, athymic mice bearing established SW620 tumors were treated with four cycles of therapy every 3 d (arrows). Each cycle consisted of irinotecan alone (25 or 50 mg/kg) or irinotecan (25 or 50 mg/kg) followed by two doses of AZD7762 (2 and 14 h after irinotecan dose). Tumor volume was measured using calipers on the indicated days with the median ± SE tumor volumes indicated for groups of 10 animals. B, %T/C was calculated as (T2 - T1) / (C2 - C1) where T is the treated group, C is the vehicle group, 1 is the start date, and 2 is the end day as defined as the day the control group is euthanized. Percentage inhibition was calculated as (geometric means of control - geometric means of treatment) / (geometric means of control - 1) × 100. Complete regression is defined as tumors measuring <63 mm3. Tumor-free survival is defined as complete regressions for a minimum of 14 d.
In the present studies, we describe AZD7762, a novel ATP-competitive inhibitor of checkpoint kinases and the consequences of inhibiting checkpoint pathways in tumor cell lines and models. AZD7762 is greater than 100-fold selective for checkpoint kinases over the majority of a panel of protein tyrosine and serine/threonine kinases. Because greater than 100-fold selectivity was observed against cdk, protein kinase C isoforms, and MAPKAP kinase 2, AZD7762 is clearly differentiated from the UCN-01 kinase selectivity profile (13–15).
AZD7762 has been shown in multiple assays to abrogate DNA damage-induced cell cycle checkpoints. Additionally, fluorescence-activated cell sorting analysis has shown abrogation of the S checkpoints induced by gemcitabine.5 Profiling of cell cycle proteins gave results that are consistent with mechanism of action and checkpoint abrogation. Gemcitabine treatment of cells leads to the phosphorylation and activation of Chk1 and Chk2 and predominantly S-phase arrest (16). Activated Chk1 then directly phosphorylates cdc25A leading to proteolytic degradation (11, 17–22) and downstream to increased phosphorylation of cdk2 (Tyr15). As a consequence, cyclin E/cdk2 and cyclin A/cdk2 complexes are inactivated, and cells arrest in the S phase. Concurrent treatment of AZD7762 and gemcitabine led to a dose-dependent increase in cdc25A levels at early time points when compound was still available to inhibit Chk1 and a delayed decrease in cyclin A levels as predicted by cell cycle progression.
SN-38 treatment of cells leads to phosphorylation of Chk1 and Chk2 within 1 h of treatment (23) and eventually results in G2 arrest. Activated checkpoint kinase proteins phosphorylate cdc25C, which is relocalized by 14-3-3 to the cytoplasm. Relocalization results in inactivation of cdc25c and leads to increased phosphorylation of cdc2 (Tyr15), which remains inactive and prevents progress through the G2 checkpoint (18, 24, 25). As expected, combination treatment leads to dose-dependent decreases in phospho-cdc2 and cyclin B1. phH3 increases, showing abrogation of the G2 checkpoint as cells have moved into mitosis.
AZD7762 has been profiled extensively (in vitro) and has been shown to increase the response to multiple DNA-damaging agents in several different cancer cell lines. As described above, gemcitabine and SN-38 have very different mechanisms of action with gemcitabine arresting cells predominantly in S phase and irinotecan in G2. Nevertheless, enhanced efficacy of both agents is clearly achieved in combination with AZD7762 although the most striking seen with gemcitabine. This might in part be due to the role of Chk1 in maintenance of replication forks, which are stalled by gemcitabine through multiple mechanisms (26, 27). Alternatively, better combination efficacy in vitro could be related to the ability of gemcitabine to activate both ATM- and ATR-driven checkpoints (16) compared with SN-38, which primarily activates ATR-driven checkpoints (23).
Given that AZD7762 is an equally potent inhibitor of Chk1 and Chk2 in vitro, the relative contributions of Chk1 and Chk2 inhibition to the observed phenotypic outcome cannot be conclusively determined from the present studies. However, there is strong literature precedent indicating a major role for Chk1 versus Chk2 when both kinases were assessed simultaneously. For example, gemcitabine treatment activates both Chk1 and Chk2, but Chk1 to a greater extent as measured by phosphospecific antibodies (16, 17). Additionally, both Chk1 and Chk2 can phosphorylate cdc25A, but in gemcitabine-treated cells only Chk1 siRNA knockdown resulted in stabilization of cdc25A. Chk2 depletion in gemcitabine-treated cells had no effect on entry into mitosis, in contrast to the premature entry into mitosis observed with Chk1 depletion (17). Finally, siRNA knockdown of ATR, ATM, or Chk1 led to sensitization of gemcitabine-treated cells, whereas Chk2 depletion had no effect (16).
In studies evaluating topoisomerase I poisons such as camptothecin (28), SN-38 (23), and BNP1350 (29), activation of both Chk1 and Chk2 was observed, but the Chk1/ATR pathway was again determined to play a more prominent role in enhancing cytotoxicity. Interestingly, simultaneous knockdown of Chk1 and Chk2 does not improve efficacy over Chk1 alone (28). A similarly prominent role of Chk1 has been shown for other chemotherapies including cytosine arabinoside (30) and platins (31). Finally, VRX046617, a Chk2 inhibitor with no Chk1 activity, did not potentiate doxorubicin or cisplatin (32).
Other recently described checkpoint kinase inhibitors such as XL-844 and PF-477736 are potent inhibitors of Chk1 with differential activities against Chk2. Whereas XL-844 is equally potent against Chk2 (33), PF-477736 is ∼100-fold selective for Chk1 (34). Both inhibitors have been shown to potentiate the effects of DNA-damaging agents such as gemcitabine. Because Chk2 does have a role in checkpoint signaling especially in response to radiation, a potential role of Chk2 inhibition in driving the cellular response to AZD7762, XL-844, or PF-477736 cannot be formally dismissed. Furthermore, it is currently unclear whether the differential profiles against Chk1 and Chk2 of these agents will lead to different outcomes or what balance of Chk1 and/or Chk2 inhibition will result in the maximum clinical response.
AZD7762 has also been profiled in vivo by monitoring abrogation of the G2 checkpoint and efficacy in combination with gemcitabine and irinotecan. Using a novel pharmacodynamic model, a dose-dependent increase in G2 abrogation was observed following i.v. administration of AZD7762. This model was extremely useful because it allowed for potency ranking of multiple compounds in vivo in a highly efficient manner (35–37). Reflecting our in vitro findings, dose-dependent potentiation of antitumor activity in combination with DNA-damaging agents was shown in several in vivo models. Potentiation was determined not to be due to increased exposures to chemotherapy in the combination setting (data not shown). Efficacy is enhanced in gemcitabine-sensitive (SW620) and relatively insensitive (H460-DNp53) human tumor models in combination with AZD7762. To test the ability of AZD7762 to potentiate gemcitabine delivered at MTD, a rat efficacy model was used and showed that increased efficacy was achieved without affecting the tolerability or MTD of gemcitabine. In combination with irinotecan delivered at MTD, AZD7762 was able to convert the growth stasis observed with irinotecan alone to sustainable tumor-free survival in eight of nine mice. This study was compelling not only because the combination treatment converted stasis to regression but also because tumors were unable to regrow in contrast to the rapid regrowth observed in the irinotecan-treated group.
The present preclinical studies show the ability of AZD7762 to potentiate gemcitabine and irinotecan in a tolerated regimen. AZD7762 and other checkpoint kinase inhibitors have the potential not only to potentiate these agents but also to enhance additional therapies that induce DNA damage. Clearly, there are many therapeutic opportunities for a checkpoint kinase inhibitor such as AZD7762. Current and future clinical trials with AZD7762 and other checkpoint kinase inhibitors will reveal which combinations provide the most effective and safe cancer treatment.
Disclosure of Potential Conflicts of Interest
S.D. Zabludoff, C. Deng, M.R. Grondine, A.M. Sheehy, S. Ashwell, B.L. Caleb, S. Green, H.R. Haye, C.L. Horn, J.W. Janetka, D. Liu, E. Mouchet, S. Ready, J.L. Rosenthal, C. Queva, K.J. Taylor, G.E. Walker, and A.M. White: employees of AstraZeneca. The work described was part of the AstraZeneca AZD7762 development program. No other potential conflicts of interest were disclosed.
↵4 Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
↵5 Tse and Schwarzt, in preparation.
Accepted June 13, 2008.
Revision received May 21, 2008.
American Association for Cancer Research
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Mol Cancer Ther September 1 2008 (7) (9) 2955-2966; DOI: 10.1158/1535-7163.MCT-08-0492
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