text:
is still very sexy even at her age. She got divorced long ago and George is the
only her son. He just turned 18. They are great friends even though many teenagers
at his age have problems with parents. The only thing that bothered him in his
relations with mothers is that she was extremely jealous to his girlfriends. He
couldn�t understand why till one night she came to his bedroom is short robe to
talk about it. As you may see their conversation ended with really wild and messy
Amanda�s sons grew up it was very painful for her to watch them having fun at
parties and clubs and dating young girls. She did her best to stay # 1 for them.
Sons often joked that she�s she only woman in the world they will ever love. Once
they had pretty frank conversation about sex and Amanda said desperately she�s
not able to explain everything they ask her about but she�s able to show! That
was nasty but her sons took her right. It was their first incest experience so
they recorded it on video.
grew up without mother. She died when Helen was a just small kid. Her father Artur
took care of her, protected and cherished her. Sure, for Helen loved him not like
a father but like a man. But she couldn�t confessed herself in it till the day
he kissed her lips while she was sleeping. She woke up and finally realized how
happy she is. She never enjoyed sex with guys from her college as much as she
enjoyed it with Artur. She got her cunt eaten out as never before and then stuffed
with her father�s strong cock.
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Unilife Corporation (UNIS) Posts Wider Q3 Loss
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Unilife Corporation (NASDAQ: UNIS) reported Q3 loss of ($0.15), versus ($0.10) reported last year. Revenue for the quarter came in at $650 thousand, versus $2.42 million reported last year.
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subset: commoncrawl


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It only takes a minute to get started. Fill out our simple sign up form and login to the site.
This step is up to you but will seriously multiply your chances of getting interest from other over 50s members on the site. Write about yourself, your hobbies and atach your nicest photo of yourself.
Use our website's sophisticated search to discover over 50s members who meet your criteria. Or just leaf through listings and discover daters that take your fancy.
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text:
We're very pleased to announce the first full release of our [Where Does My Money Go?](http://www.wheredoesmymoneygo.org/) prototype. This is now online at:
[Tom Watson MP](http://www.tom-watson.co.uk/), commented on the new release:
> Where Does My Money Go represents another milestone in the UK's transparency movement. We know that transparency changes individual and institutional behaviour and this new tool will have a big impact on the way the public sector is held to account by UK citizens.
> As well as being a great public benefit, Where Does My Money Go is also an immensely complicated tool to code and design. I applaud the team behind the project for their commitment and hard work. They're leading the way in transparency and making a difference for the country.
Our press release below contains more background information on the new prototype. For all you microbloggers out there, here is a 136 character version of the project announce:
**RT @[jwyg](http://www.twitter.com/jwyg): New visualisations of [#ukgov](http://search.twitter.com/search?q=%23ukgov) spending! See @[okfn](http://www.twitter.com/okfn)'s Where Does My Money Go? [#wdmmg](http://search.twitter.com/search?q=%23wdmmg)**
## Press Release
Now more than ever, UK taxpayers will be wondering where public funds are being spent – not least because of the long shadow cast by the financial crisis and last week's announcements of an estimated £850 billion price tag for bailing out UK banks. Yesterday's pre-budget report also raises questions about spending cutbacks and how public money is being allocated across different key areas.
However, closing the loop between ordinary citizens and the paper-trail of government receipts is no mean feat. Relevant documents and datasets are scattered around numerous government websites – and, once located, spending figures often require background knowledge to interpret and can be hard put into context. In the UK there is no equivalent to the US [Federal Funding Accountability and Transparency Act](http://thomas.loc.gov/cgi-bin/query/z?c109:s2590:), which requires official bodies to publish figures on spending in a single place. There were proposals for similar legislation in 2007, [but these were never approved](http://www.publicwhip.org.uk/division.php?date=2007-06-29&number=169).
On Friday 11th December the Open Knowledge Foundation will launch a free interactive online tool for showing where UK public spending
goes. The [Where Does My Money Go?](http://www.wheredoesmymoneygo.org/) project allows the public to explore data on UK public spending over the past 6 years in an intuitive way using an array of maps, timelines and graphs. By means of the tool, anyone can make sense of information on public spending in ways which were not previously possible.
For example, while playing around with the tool, we noticed:
* Total public spending as a percentage of gross domestic product this year increased to levels not seen since the recession of 1992.
* Healthcare spending in real terms under New Labour has almost doubled since they came to power in 1997. Education spending has increased by 75%.
* The UK spends more on old age than on education. The amount of money spent to support those in retirement is £87bn compared to the
£82bn on the whole of education.
* £665 was spent in Northern Ireland on housing and amenities for
every man, woman and child in 2008-9, compared to £413 in London.
Spending per capita in Britain's capital on housing, transport and
public order and safety all exceeded the national average by over 60%.
### Notes to editors
The [Open Knowledge Foundation](http://www.okfn.org/) is a not-for-profit organisation dedicated to improving the way knowledge is shared. The [Where Does My Money?](http://www.wheredoesmymoneygo.org/) project was a winner of the Cabinet Office's [Show Us A Better Way](http://www.showusabetterway.co.uk/call/2008/07/where-does-my-2.html) competition. The project benefits from an
[advisory group](http://www.wheredoesmymoneygo.org/people/) which includes leading transparency advocates and information visualisation experts. The prototype was conceived by the Open Knowledge Foundation and developed with data visualisation specialists [iconomical](http://iconomical.com/), based in Amsterdam. The Foundation is also currently working with the UK Government on the [technology behind the new data.gov.uk site](http://blog.okfn.org/2009/10/08/datagovuk-launched-and-its-using-ckan/).
Currently the Where Does My Money Go prototype is based on [data from HM Treasury](http://www.wheredoesmymoneygo.org/data/) – but the project team is working to collect, aggregate and incorporate much more fine-grained information, including on local spending. On Monday Gordon Brown [announced plans to publish much more detailed information on public spending](http://blog.okfn.org/2009/12/07/uk-government-announces-lots-of-new-open-data/) in a more systematic way as part of the [Smarter Government](http://www.hmg.gov.uk/frontlinefirst) initiative.


text:
A mock school bus crash was all make-believe Wednesday afternoon, but for the drivers who volunteered to sit inside the precariously slanted vehicle after a collision with a drunken driver it seemed all too real as they heard the sirens of approaching fire trucks and ambulances.
JACKSON - Thompson Square is coming to Jackson Rancheria.
STOCKTON - For the first time ever, famed chimpanzee research pioneer Jane Goodall will take audiences in select theaters across the country on an incredible journey through the memories of her life when she appears live to answer their pre-submitted questions during this special Fathom event.
A crash training like no other that has been held in Manteca, the staged rollover of a large school bus into a flood retention basin will take place today and Thursday afternoons near the corner of Airport Way and Louise Avenue.
Terri Candelaria can stop pulling her "hare" out.
Karen McLaughlin's honeymoon has started.
The Manteca Post Office on the southwest corner of Center Street and Maple Avenue in downtown Manteca is getting a well-deserved facelift.
The Manteca Women Federated meets Monday, Sept. 26, at Perko's Café at Button and East Yosemite avenues.
Ripon FFA shined at the California State Fair during the month of July.
RIPON - The Ripon Chamber of Commerce will present the 27th Annual Main Street Day & Car Show in Ripon on Oct. 15 from 9 a.m. to 3 p.m.
TRACY - Tracy is celebrating "25 Years of Beans" this year at the Annual Tracy Dry Bean Festival held this Saturday from 10 a.m. to 7 p.m. and Sunday 10 a.m. to 6 p.m. in Downtown Tracy.
MODESTO - The inaugural San Joaquin Valley Award Winning Olive Oils and Vinegars Festival is this Saturday, Sept. 10, on the south lawn of the McHenry Mansion in Modesto.
A half-mile extension of Atherton Drive was part of the much ballyhooed federal stimulus effort to get Americans back to work.
Thomas Edwards always knew he wanted to serve his country. So right after graduation from Manteca High School in 1997, he enlisted in the Army.
LATHROP – A Lathrop family's tragedy has metamorphosed into a powerful grassroots effort that has now become a catalyst for awareness of the many issues affecting the mentally ill.
Page 516 of 883
Next Wednesday with its forecast of a high of 100 degrees in Manteca would be an ideal day for moms to take their kids to ...
There are more than 1.7 billion Muslim people in the world today.
STOCKTON – The California Central Valley Archery Academy is right at home with the San Joaquin County Fair.
Manteca Police Chief Nick Obligacion is retiring.
Diane Quaresma had knocked on SaveMart Supermarkets' proverbial door several times in hopes of getting their buyers to consider her product, Almondée, an all-natural ...
Carl's Junior fast food restaurant on Hulsey Way was robbed at gunpoint shortly before 11 p.m. Thursday when a lone gunman entered through ...
Bill Andreetta knows how to bring home the bacon.
Dell'Osso Farms is going whole hog for the debut of Baconfest this Father's Day Weekend.
The pain for Debra Brown was so bad that she actually had to move out of Manteca.
STOCKTON – San Joaquin AgFest Junior Show and Auction continues to be about youngsters.
STOCKTON - William Burden of the Sierra High Future Farmers of America has a fondness for raising serama.
Increasing the daily bag limit for bass fishing in the Delta and reducing the fish size limit could go a long way to reducing the ...
Manteca Councilwoman Debby Moorhead believes the sky is the limit when it comes to the family entertainment zone (FEZ).
STOCKTON - It's been two long years for Del Hoffman since the last San Joaquin County Fair.
The popular Mistlin Sports park water play feature is staying turned off - at least for now.
Page 3 of 19
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School Rejects Boy With Lesbian Moms
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'View' Sounds Off on Lesbian Love
The ladies wonder what women get from having a partner of the same sex.
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Kensington Oval, Bridgetown, Barbados ,6/26/2016, 22:30 (IST)
Harare Sports Club, Harare ,6/22/2016, 16:30 (IST)
Edgbaston, Birmingham ,6/24/2016, 18:30 (IST)
England beat Sri Lanka by 10 wickets
Kensington Oval, Bridgetown, Barbados ,6/24/2016, 22:30 (IST)
Swank is a pornographic magazine for heterosexual men published in the United States. It includes hardcore sex, such as the use of sex toys, lesbian sex, and sexual intercourse between men and women. There are also a series of DVDs and an official website produced under the Swank name.
Who is the Undisputed Item Queen of Bollywood?


text:
UPDATE on the United 50K Offer
I just had a reader confirm that the offer ends on April 30th per a CSR at Chase. I'd heard the rumor, now have confirmation.
Insurance Tip
Hi Rick,
Here's a little tip for your site. Amex offers upgraded
rental car insurance for a flat fee of $25 for the whole rental period
for rentals of up to 42 days. You just have to sign your amex card up
for the service, and the $25 will be billed every time you rent a car.
The great deal is that if you pay with a different card when you
return the car, you get the $25 refunded by contacting amex and asking
for a refund. that means in effect that you get premium insurance for
Sounds too good to be true, but I just spoke at length with an
Amex rep at 800-338-1670, and she assured me that was the case. to
enroll or get more info, check out
Best Hobby in the World
I got this email a week or so ago and was the first thing I read in the morning. My day was made. The tip came to me from one of you, and I'm sorry I can't remember who it was, but please share the joy of helping this young soldier out.
The Frugal Travel Guy blog readers should win some type of online award for being the most generous helpful people in the world. I know Mike F (above) is smiling with his free round trip ticket. Thanks again to all that contribute.
Hi Rick,
I just wanted to take the time to THANK YOU for updating us on the Amex Premier Rewards Card bonus. I was thinking about jumping in on this one, but was hesitant to commit to the $1000 spending threshold. After your post about the "popup and email" workaround, I applied and was approved. I couldn't have done it without you.
Thanks to your blog, I've already been able to take advantage of the BankDirect promotion, I've learned a ton about properly managing my credit, and how to leverage it for free travel. I'm only 22, and still in the military, but I'm already well on my way to becoming my own, self-styled, "frugal travel guy".
Thank you for all you do. You really do make a difference in the lives of us "little guys".
Keep up the good work,
Mike F
Don't you think Mike should be Frugal Travel Guy of the Day for what he is doing for all of us?
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text:
Award Abstract #1255807
Improving Understandings of Student Engagement in STEM to Enhance Teacher
Researchers from the U.S. and from Finland are collaborating in this Science Across Virtual Institutions (SAVI) project to investigate how teachers use the information about student engagement they obtain through the use of mobile technology to make formative decisions about classroom instruction. The members of the U.S. team for this project are from Michigan State University while the members for the Finnish team are from the University of Helsinki. The project involves three face-to-face workshops, during the first of which the researchers identify how the current instruments and processes, including the Experience Sampling Method (ESM), are used to define and operationalize concepts of engagement to improve teacher practice. The second workshop finalizes the specifics of the design of the research and analysis plans, including interpretive frameworks that integrate the different methodologies currently used by the researchers, to conduct the research in classrooms in both the U.S. and Finland. The third workshop focuses on the discussion of the findings from the study and development of prospects for the usability and scalability of the tools investigated in the study. The research builds on an existing international postdoctoral research collaboration (Pathways to Adulthood) and extends that research to include the involvement of doctoral students, teacher educators and mentors as part of the research teams. The researchers are extending the approach used in prior work, the Experience Sampling Method (ESM) and its related cell phone technology, to connect to the methodology used by the Finnish researchers. The researchers are constructing a theoretical base that improves knowledge around students' sense of self-efficacy and the impact of self-efficacy on learning. The cell phone technology holds real promise as an approach that teachers could use to gain richer information on students' perspectives. The researchers are extending the concept of engagement as it is currently characterized as "flow" to include measures of student interest, challenge and skill. Motivation as a factor in student achievement is of growing importance. There is evidence that engagement as self-efficacy is related to achievement in science, and determining ways to enhance motivation and to connect it to teacher actions is important. This project is contributing to the knowledge of how to measure engagement in the environment of teachers' work in the classroom. In addition, it supports cross-national collaboration and learning by developing a cadre of young researchers who actively contribute to this collaborative research agenda.
Note: When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external site maintained by the publisher. Some full text articles may not yet be available without a charge during the embargo (administrative interval).
Some links on this page may take you to non-federal websites. Their policies may differ from this site.
Linnansaari, J., Viljaranta, J., Lavonen, J., Schneider, B., & Salmela-Aro, K.. "Finnish students' engagement in science lessons," NorDiNa, v.11, 2015, p. 192.
Moeller, J., Salmela-Aro, K., Lavonen, J., & Schneider, B.. "Does anxiety in math and science classrooms impair motivation? Gender differences beyond the mean level," International Journal of Gender, Science, and Technology, v.7, 2015, p. 229.
Please report errors in award information by writing to: firstname.lastname@example.com.


text:
34, Las Vegas, NVHello I am Javier, I am originally from Las Vegas, Nevada, and I also lived in Miami Florida for awhile. I am currently working full time, and I am working a bachelors over at...
35, Las Vegas, NVI am random, often the funniest guy in the room, humble but once in a while come off as conceited to those who are experiencing Robert from afar, talented but overly judgmenta...
38, Las Vegas, NVMy name is John, I am 5ft 11in. Well about myself. I was born with a birth defect called Aperts Syndrome it is the webbing of the fingers and toes. I have had numerous operati...
38, Las Vegas, NVOccupation: Education / Teacher / Academic Research; Height: 5' 5"; Eye Color: Brown; Hair Color: Black; &nbs...
23, Las Vegas, NVHello my name is Chyson. I'm currently 22 years old and currently a student at Nevada State College. I'm majoring in finance and I plan to be a financial advisor to help peopl...
53, Las Vegas, NVI'm a hard working, honest, fun loving, single Man, With a Son, looking for the Woman of my dreams to share all the good things in life. I've given up on the bar scene, b...
33, Las Vegas, NVI want to feel her warmth, With every touch of her hand, No secrets will be kept from her, Because she will understand. We will engrave our problems, Upon the ocean sand, So n...
48, Las Vegas, NVI am passionate about yoga, Pilates, golf, hiking, biking and maintaining close relationships with family and friends. I am an attorney and enjoy being an advocate for client...
38, Las Vegas, NVI might have the info people need to have a good time in Vegas, but Im an Idaho guy at heart. My values and morals are more Midwestern than strip, and Im open to moving away f...
50, Las Vegas, NVOccupation: Computers / Software / Hardware; Height: 5' 11"; Eye Color: Brown; Hair Color: Brown; Polit...
46, Las Vegas, NVOccupation: Construction / Craftsman; Height: 6' 2"; Eye Color: Hazel; Hair Color: Blonde; Political Vi...
52, Henderson, NVI enjoy intelligent conversations, but I enjoy partying just the same. I try to be an active person... I workout alot, but NOT to the extent of me being a vascular bubble. I'v...
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text:
How ordinary women can be transformed into cover girls with a touch of clever make-up (and a little help from the airbrush)
- Vadim Andreev, 29, from Saint Petersburg is a make-up artist
- Has been in the business since he was 16 and works with many celebrities
- Started taking before-and-after photos to show how faces can be altered
A Russian make-up artist is on a mission to prove women don't need to resort to surgery to transform plain looks into those of a beauty queen.
Vadim Andreev says he can give any woman a cover girl transformation by simply using cosmetics.
The 29-year-old beauty guru from Saint Petersburg enhances features and covers blemishes in a bid to make his subjects more beautiful and proves his amazing skill with striking before-and-after photographs.
Scroll down for video
Russian make-up artist Vadim Andreev says he can give any woman a cover girl transformation by simply using cosmetics
He says women should use under eye concealer, blush on the cheek, and highlighter on cheekbone
Vadim has been working as a make-up artist since he was 16 years old and quickly established his reputation to become a stylist to the stars.
'It was my hobby. I really loved to transform people ever since I was I child,' he tells MailOnline.
'When I grew up I decided to go into professional make-up and hair styling.'
Vadim has worked on Russian TV shows and lists various Russian celebrities as clients, from models and actresses to popstars.
He also works with 'every day' women, charging for make-overs for special occasions and weddings, and charts some of their transformations with before-and-after looks on his website vadimandreev.com
He's also happy to pass on some of his make-up wizardry by running his own make-up workshops and masterclasses and how-to videos.
Vadim has been working as a make-up artist since he was 16 years old and quickly established his reputation
'I started taking these photos to show people they can change, be more attractive, look and feel happier,' he says.
He doesn't just work with women, but also gives make-overs to men who might want a gothic or punk look - or simply to make their skin look clearer.
Vadim said his aim was to show that anyone could look like a flawless celebrity with the right time and know-how.
He admits that a few of his professional after photos have a little bit of post-production, like erasing hair on forehead or colour correction, perhaps slight contrast enhancement or sharpening. But most of them are entirely natural.
'[The photos] don't mean women should do heavy make-up all the time. There are little make-up secrets for everyday. Everybody can use them,' he says.
Vadim believes that every common girl can become a glamorous celebrity
'My secret is in the eye, I guess. I can see the beauty inside each person and show it to everybody.
'My top tips for the everyday woman are: use a light under-eye concealer, cream blush on the top of your cheek,
and highlight on cheekbones.
'Put some bronzer under your cheekbones and along the
chin and blend well over the face.
'Put some black mascara on your lashes and apply warm pink lipgloss on lips. This will give you fresh, young look.
'Recently, we did a great makeover for a friend of mine. She lives in a small town and is kind of old fashioned. She had a terrible fringe which didn't suit her. I opened her forehead, put in some hair extensions, tweezed her eyebrows, did her make-up - and she obviously became like a celebrity. She was very happy and didn't remove the make-up for three days.'
Vadim started taking these photos to show people they can change, be more attractive, and feel happier
A bit of fake tan, fancy eyeshadow, and Vadim's magic transforms a woman completely
Vadim says some photos have a little bit of post-production, like erasing hair on forehead or colour correction
He said: 'Make-up is a very personal and individual process. I believe that every common girl can become a glamorous celebrity. I want to help everyone to see their own beauty, just sometimes it takes a little bit more time and a bit of a helping hand.'
'I have a lot of plans and dreams about my future in
make-up, such as writing a book and have my own TV show about makeovers.
'And, of course, my usual work with lovely brides, giving workshops and
Using just make-up, Vadim transforms plain women into Hollywood stars with perfect skin and features
Vadim Andreev sometimes goes for more theatrical looks
With clever lighting, lots of hairspray and a make-up palette, anything is possible
- See Kanye in bed with Kim K & Taylor Swift in racy new video
- Eagle-eyed tourist in Vienna uncovers ATM skimming scam
- Watch as a duck and dog playfully chase each other
- Gotcha! Cute Golden Retriever puppy sneakily pounces on toy
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- Donald Trump: Britain leaving the EU is 'a fantastic thing'
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- Take a look at the GLASS slide on LA's tallest building
- Obama shrugs off his humiliating failure to stop Britain...
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- Michael Jackson said he feared Prince would die prematurely...
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Domenico Stinellis, File, Associated Press
WASHINGTON — The potential White House candidates need cash.
But donors aren't eager to shell out until the hopeful prove they're credible.
Which they can't — until they have the cash lined up to start their campaigns.
This explains, in part, why the 2012 Republican primary race has yet to begin in earnest.
"It's a little sluggish. The major donor folks are sitting back a bit," said Rob Bickhart, a former Republican National Committee finance chairman who is helping former Sen. Rick Santorum of Pennsylvania.
Less than a year before the lead-off primaries and caucuses, many of the Republican Party's biggest fundraisers aren't aligned with any one candidate and many are holding back to see who emerges as a front-runner in a field that lacks one.
"I have spoken to just about everyone," said Larry Bathgate, RNC finance chairman under Presidents Ronald Reagan and George H.W. Bush. Bathgate isn't sold on a 2012 candidate yet.
Four years ago at this point in the campaign, Republicans hoping to succeed President George W. Bush were on the road in Iowa, New Hampshire and South Carolina, and their fundraisers were burning up phone lines to pay for the frequent trips.
Not this time.
All-but-certain candidates Mitt Romney and former Minnesota Gov. Tim Pawlenty have lined up pieces of their fundraising teams; others are moving more slowly. None is eager to start spending.
Even some of the donors who are already supporting candidates aren't entirely convinced.
"It's an open field," said Mel Sembler, a real estate mogul and former RNC finance chairman.
Romney "is out lining up his supporters around the country. He'll be formidable but that doesn't mean he's walking away with this thing," Sembler said days before hosting Romney at his Florida home.
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text:
Sing-A-Long in Downtown Bangor To Celebrate The Bus [VIDEO]
A group of Community Connector supporters gathered on Earth Day in Downtown Bangor for a lyric-modified sing-a-long.
A group of Community Connector supporters gathered on Earth Day in Downtown Bangor for a lyric-modified sing-a-long.
Tonight's mystery artist said he got hooked on performing because he said it was a real buzz to make a complete fool of yourself. Can you guess who our jester might be??
Chris Cornell and Pearl Jam have offered tributes to the late legend Prince in musical form.
Tonight's mystery artist now works for a company called Taxi music critiquing new musicians. You'll never guess who he is. Can you ???
In 2007, Prince delivered the Super Bowl halftime show that many are judged by, covering Foo Fighters, Queen, Jimi Hendrix and more amidst his own hits.
Chris Ross and the North are quickly becoming one of Maine's most popular acts ever, and we were very fortunate to have him show up with his guitar today here in our studio!
Pretty much everything we'll ever know about the Titanic has already been revealed, but our fascination with the doomed ocean liner endures.
For honest people there is nothing more annoying than dishonest ones. I don't know about you but I hate feeling like if I am not on my guard 24/7 someone is going to take advantage and frankly that sucks...
Comedian Krazy Jake will perform next month in Dexter as part of a benefit to help a dairy farmer who lost almost everything to fire over the winter.
Tonight's mystery artist snuck away while on tour to Vegas to marry his high school sweetheart. Any guess who our romantic guy is or which band he plays with??


text:
It's a tee. It's a dress. It's the new Graphic T-shirt Dress.
Show your Photographer love and channel your inner groupie at the same time!
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i love thisi would love to have a bummer sticker saying " photographer behind the wheel, stops on the go!"
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Love this one!
Haha, l love this! Thanks for the comments, fave and watchlist too!
I'd buy this, but without the URL under the text.
Thanks for the suggestion – I removed the URL.
Well, now I'll have to buy it (for my wife!)Not straight away, but soon.
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Oct 27, 2009
|Priority date
|Dec 10, 1999
|Fee status
|Also published as
|Publication number
|J. Milton Harris, Michael D. Bentley, Xuan Zhao, Xiaoming Shen
|Original Assignee
|Nektar Therapeutics
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (17), Non-Patent Citations (35), Referenced by (4), Classifications (16), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of U.S. application Ser. No. 11/377,973, filed Mar. 17, 2006, which is a continuation of U.S. application Ser. No. 10/412,642, filed Apr. 10, 2003, now U.S. Pat. No. 7,074,878, which is a continuation of U.S. application Ser. No. 10/076,018, filed Feb. 14, 2002, now abandoned, which is a continuation application of U.S. application Ser. No. 09/459,312, filed Dec. 10, 1999, each of which is incorporated herein by reference in its entirety.
This invention generally relates to water soluble, hydrolytically degradable, and nonpeptidic polymers, and particularly to polymers and gels therefrom that are suitable for medical devices, drug enhancement and in vivo delivery of biologically active agents.
Conjugation of agents having a known or potential therapeutic benefit to water soluble, non-immunogenic polymers can impart to the agent desirable characteristics including, among others, improved solubility, greater stability, and reduced immunogenicity. It has become increasingly important in the pharmaceutical industry to develop conjugates having these characteristics so as to increase the number of options for therapeutic benefit.
An example of a polymer that can be used to develop conjugates with therapeutic agents is poly(ethylene glycol) ("PEG"). Therapeutic agents conjugated to PEG are sometimes said to be "PEGylated." Several PEGylated therapeutics have been developed that exhibit enhanced water solubility, longer circulation lifetimes, and lower immunogenicity as compared to the unconjugated therapeutic agent. Because of the rapid motion and heavy hydration of the polymer, PEGs usually are of much higher apparent molecular weight than the therapeutics to which they are attached. Thus, they tend to mask the therapeutic agent from the immune system and to preclude excretion through kidneys.
The term PEG is commonly used to describe any of a series of polymers having the general formula HO—(CH2CH2—O)n—H, where "n" represents the number of ethylene oxide monomers in the polymer. However, the parent polymer is generally unsuitable for attachment to a therapeutic agent. Hydroxyl groups are relatively unreactive toward groups commonly present on therapeutic agents and thus PEG normally has to be "activated" by converting at least one end hydroxyl group into a more reactive form. It is also usually important to activate the PEG polymer with a terminal group that is selective in its reactions. For example, several PEG derivatives have been developed that are more likely to react with amine groups. Others have been developed that preferentially react with thiol groups.
Successful PEG derivatives may have to meet a number of requirements, depending on the specific application. For conjugation to proteins, the PEG derivative should usually have a desirable and suitably selective reactivity at physiologic conditions of temperature, pressure, and pH to preserve the activity of the unconjugated protein. In some circumstances, it is desirable to cleave the PEG polymer from the therapeutic agent at some point after the agent is delivered in vivo.
Some PEG derivatives have been used in combination with other polymers to prepare insoluble gels in which drugs can be entrapped or chemically bound. For example, Sawhney et al., Macromolecules, 26:581 (1993) describes the preparation of block copolymers of PEG with polyglycolide or polylactide blocks at both ends of the PEG chain. The copolymers are then activated by terminal substitution with acrylate groups, as shown below.
In the above formula, the glycolide blocks are the —O—CH2—CO— units. The addition of a methyl group to the methylene gives a lactide block; n can be multiples of 2. Vinyl polymerization of the acrylate groups produces an insoluble, crosslinked gel with a polyethylene backbone.
The polylactide or polyglycolide segments of the polymer backbone shown above, which are ester groups, are susceptible to slow hydrolytic breakdown, with the result that the crosslinked gel undergoes slow degradation and dissolution. The hydrogel degrades in vivo and can result in non-PEG components being released into the blood stream, which can be undesirable.
It is desirable to develop improved polymers providing additional choices for use in drug delivery and other applications.
This invention provides a water soluble, nonpeptidic polymer having two or more oligomers linked together by hydrolytically degradable carbonate linkages. The polymer can be hydrolytically degraded into small oligomers in an aqueous environment, including in vivo conditions. The polymer is easy to prepare and the molecular weight of the oligomers resulting from polymer degradation can be easily controlled, which can be desirable for some applications. The polymer can be conjugated to a biologically active agent such as a protein or peptide. The polymer can impart desirable characteristics to the conjugates of improved water solubility and reduced immunogenicity. The polymer is useful for preparing insoluble cross linked structures, including hydrogels, that are hydrolytically degradable into soluble polymers of predetermined molecular weight.
The oligomers are alkylene oxide oligomers. Typically, the oligomers are ethylene oxide oligomers, and the polymer is a poly(ether carbonate) having the formula of:
where n is from about 1 to 2,000, normally from 2 to 2,000, and m is from about 2 to 200. Since carbonate linkages are hydrolytically degradable under mild conditions, the polymer will hydrolyze to produce oligomer fragments of much lower molecular weight than the starting polymer:
HO—[(—CH2CH2O)n—CO2]m—(—CH2CH2—O)nH+(m+1)H2O→(m+1) HO—(—CH2CH2—O—)n—H+mCO2
In addition to providing many of the desirable features of other polymers, including poly (ethylene glycol) as described above, this new polymer can degrade in the body and thus facilitates removal of the polymer from the body. The degradation products are themselves normally nontoxic small PEGs that typically are rapidly cleared from the body.
The polymer can be prepared in a number of ways. In one embodiment of this invention, the poly(ether carbonate) is prepared by polymerizing an activated oligomer having the formula of:
where n can be from about 2 to 2000 and Z is a reactive leaving group such as N-succinimidyl, 1-benzotriazolyl, or p-nitrophenyl.
The polymer can be prepared by polymerizing ethylene oxide oligomers of the formula:
where n can be from about 2 to 2000 with an activating molecule of Z-O—CO2-Z, where Z is as described.
Alternatively, the ethylene oxide oligomer
can be polymerized with a bifunctional ethylene oxide oligomer:
where n and Z are as described above, to form the poly(ether carbonate).
The polymerization reactions may be conducted either in an organic solvent or in a melt, in the presence of an organic base. Examples of suitable solvents include acetonitrile, THF, dimethylformamide, dimethylsulfoxide, benzene, toluene, the xylenes, chloroform, and methylene chloride. Examples of suitable organic bases include triethylamine, pyridine, quinoline, 4,4-dimethylaminopyridine and triethylamine. The polymerization reactions can be conducted at a temperature of from about 37� C. to 100� C., typically from about 45� C. to 100� C., and advantageously from about 70� C. to 90� C.
The polymer of this invention can be modified at one termius with alkyl or aryl groups to make one end of the polymer inert. The polymer can be activated at one or more of its termini to form a terminal reactive moiety. Thus, a modified or activated poly(ether carbonate) of this invention can be represented as:
where m and n are as defined above, and where X and Y can independently be H, alkyl, alkenyl, aryl, and reactive terminal moieties, including N-succinimidyloxycarbonyl, 1-benzotriazolyloxycarbonyl, p-nitrophenyloxycarbonyl, or others. Alternatively, X and Y can include linker groups terminating in active groups such as aldehyde N-maleimidyl or —S—S-ortho-pyridyl. A wide variety of activating groups and linkers can be used.
The activated polymer of this invention can be reacted with an active group on a biologically active agent, such as a protein or peptide, to form a conjugate. For example, N-succinimiayloxy, 1-benzotriazolyloxy, and p-nitrophenyloxy are leaving groups suitable for the formation of a carbamate linkage between the polymer and a biologically active agent having an amino group. Thus proteins, peptides, amino drugs or amino carbohydrates can be linked to such activated polymers. For example, when X is H, and Y is N-succinimidyloxycarbonyl, a conjugate can be formed of the following formula:
When a protein to be conjugated has an accessible thiol group, the polymer of this invention can be activated to contain a terminal reactive moiety that is reactive with thiol, including, for example, iodoacetamide, vinylsulfone, maleimide, or S—S-ortho-pyridyl, which moiety is then reacted with the thiol group to form a thiol site-specific conjugate of the protein.
When the polymer of this invention is activated at two termini, it can be used as a crosslinking agent to crosslink a multifunctional molecule to form a hydrolytically degradable hydrogel. Examples of multifunctional molecules suitable as "backbones" in formation of hydrogels include proteins such as collagen, aminocarbohydrates such as chitosan, polyamines such as polylysine and poly(vinylamine), and multi-armed or branched poly(ethylene glycol) amines. The hydrogels of this invention are useful in many biomedical applications such as drug delivery, surgical adhesion prevention, wound and scar healing, bioadhesives and surgical implants.
In another embodiment, the polymer of this invention can be activated by attachment of terminal vinyl groups. This activated polymer can be self-polymerized in the presence of a conventional vinyl polymerization catalyst to form a hydrolytically degradable hydrogel.
Thus, this invention provides a versatile polymer that is especially suited for conjugating to a biologically active agent and for forming a hydrogel. The polymer is easy to prepare and can be synthesized in large quantities. The polymer can be formed in a single reaction with multiple degradable carbonate linkages in the backbone. The conjugate and hydrogel of this invention can be degraded under physiological conditions. The degradation generates oligomers of predetermined molecular weight that can be easily cleared from the body.
The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples, which illustrate preferred and exemplary embodiments.
The invention will now be described more fully hereinafter with reference to the accompanying examples, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
As used herein, the term "polymer" means a molecule, formed by the chemical union of two or more oligomer units. The chemical units are normally linked together by covalent linkages. The two or more combining units in a polymer can be all the same, in which case the polymer is referred to as a homopolymer. They can be also be different and, thus, the polymer will be a combination of the different units. These polymers are referred to as copolymers.
The term "oligomer" is used herein to mean a molecule, typically an organic molecule, which in itself is formed by the chemical union of two or more monomer units. The monomer units of an oligomer may be different or all the same. An oligomer is capable of reacting with another oligomer which is same or different, in a polymerization reaction to form a polymer. As used herein, the term oligomer by no means limits the size of the molecule or the number of combining units or monomers in the oligomer. Rather, "oligomer" is used to indicate a unit for forming a polymer of the invention. The structure of an oligomer in a polymer may be somewhat different in chemical structure from the oligomer prior to polymerization because of the polymerization reaction and the formation of covalent linkages.
The term "carbonate linkage" is used herein to mean a linkage that includes the group —O—CO2—. It is to be understood that a carbonate linkage is distinct from a carboxylate linkage which typically has a structure of R—CO2— (where n is at least 1 and R=alkyl or aryl) and has different chemical and physical properties.
The terms "group," and "moiety," are all used herein interchangeably to refer to a distinct, definable portion or unit of a molecule. Sometimes, the structure of a group or moiety may include another smaller group or moiety. For example, a functional group of —O—CO2-Z includes Z which may be a reactive group or moiety including N-succinimidyl or 1-benzotriazolyl.
The terms "active," "reactive," and "functional" are used herein interchangeably to mean that a molecule or a group or moiety is reactive with other molecules or groups or moieties of molecules.
The term "biologically active agent" when used herein means any substance that can impact any physical or biochemical properties of a biological organism including, but not limited to, viruses, bacteria, fungi, plants, animals and humans. In particular, as used herein, a biologically active agent can be any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of a disease in humans or other animals, or to otherwise enhance the physical or mental well being of humans or animals. Examples of biologically active agents include, but are not limited to, organic and inorganic compounds, proteins, peptides, lipids, polysaccharides, nucleotides, DNAs, RNAs, other polymers, and derivatives thereof. Examples of biologically active agents include antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like. Biologically active agents include microorganisms such as bacteria and yeast cells, viral particles, plant or animal or human cells or tissues, and the like, in their native or modified forms.
The oligomers used in the practice of the invention should be selected so that they and the degradation products of the polymer of this invention are water soluble and can easily be excreted from animal bodies under natural physiological conditions. They should be non-toxic, or at least of acceptable low toxicity, and should not cause a substantial adverse effect in human or animal bodies.
Many different types of alkylene oxide oligomers are useful in forming the polymer of this invention. In its simplest form, an alkylene oxide "homo-oligomer" is used having the formula of HO—(—CHRCH2—O)n—H, where R is H or an alkyl, including methyl. Suitable oligomers also include alkylene oxide "co-oligomers," which are composed of different alkylene oxide monomers in which R is varied independently along the chain. An example of a co-oligomer is a structure in which two different R groups are present in a block such as HO—(—CHR1CH2—O—)n—(—CHR2CH2—O—)m—H, where n and m can be varied independently. An example of this type of block structure is the case in which R1 is hydrogen and R2 is methyl.
Block oligomers can exhibit surface activity. Degradable surfactants are useful in drug delivery systems and can be used to form micelles and thermally reversible gels. Random oligomers in which R1 and R2 are randomly placed along the oligomer chain are also useful. The oligomers can be branched, as in the formula
where R3 is a core branching moiety, including glycerol or pentaerythritol, R1 is alkyl n can range from 2 to hundreds, and m is at least 3.
A suitable oligomer can be formed from about 2 to 2000 monomers. However, the size of the oligomers used can vary so long as the above requirements are met. Normally an oligomer has from about 5 to 500 monomers. Advantageously, an oligomer has from about 10 to 50 monomers. An oligomer can be prepared by polymerizing or co-polymerizing monomers, and the size or molecular weight of the oligomer can be controlled by limiting the extent of this polymerization reaction.
As noted above, the oligomers contained in the polymer of this invention can be the same or different types of oligomers, or oligomers of the same type but different sizes. Therefore, the polymer of this invention can be either a homopolymer or heteropolymer.
The polymer of this invention typically has one single linear polymer backbone with two termini. However, branched polymers and star polymers are also contemplated that have two or more linear polymers, at least one of which is the linear polymer backbone of this invention, covalently linked to a central branching core.
The polymer of this invention can have an inert terminal moiety, typically H, alkyl, and aryl. The alkyl and aryl groups can be substituted or unsubstituted, and normally are methyl, ethyl, phenyl, etc. The polymer can also have one or more reactive moieties capable of reacting with a moiety in another molecule, such as an amino group or a thiol group on a protein. Examples of such reactive moieties include, but are not limited to, acryloyl, alkenyl, tresyl, N-succinimidyloxycarbonyl, 1-benzotriazolyloxycarbonyl, p-nitrophenyloxycarbonyl, N-maleimidyl, aldehydes, acetals, 1-imidazolylcarbonyl, vinylsulfone, iodoacetamide, o-pyridyldithiyl, and the like.
In one embodiment of this invention, the hydrolytically degradable polymer is a poly(ether carbonate) composed of two or more alkylene oxide oligomers and/or alkylene oxide monomers covalently linked together by carbonate linkages. For example, the poly(ether carbonate) can have the formula of
In a preferred embodiment, the poly(ether carbonate) of this invention has the formula of:
wherein n is an integer of from about 2 to 2000, normally from about 5 to 500, and advantageously from about 10 to 50; m is an integer of from about 2 to 100, typically from about to 100, and advantageously from about 10 to 50, and wherein X and Y can be same or different and each independently is H, alkyl, alkenyl, aryl, or a reactive moiety, acryloyl, tresyl, N-succinimidyloxycarbonyl, 1-benzotriazolyloxycarbonyl, p-nitrophenyloxycarbonyl, N-maleimidyl, aldehydes, acetals, 1-imidazolylcarbonyl, vinylsulfone, iodoacetamide, and o-pyridyldithiyl.
This specific form of poly(ether carbonate) contains repeating ethylene oxide oligomers linked by carbonate linkages that can be hydrolytically cleaved. Such hydrolytic cleavage leads to ethylene oxide oligomers and carbon dioxide. Therefore, the poly(ether carbonate) differs substantially from poly(ethylene glycol) or PEG in having multiple degradable backbone carbonate linkages that allow the polymer to be broken down into many smaller oligomers. Since the rate of degradation of the polymer is proportional to the number of degradable carbonate linkages present and since the size and number of the oligomers can be predetermined, substantial control over both degradation rate and the size of the degradation products is thus possible.
To prepare polymers of the invention, in one example, one or more oligomers as described above are provided each having a hydroxyl group at one terminus and a functional group of —O—CO2-Z at another terminus. The oligomers are then polymerized or co-polymerized in a condensation polymerization reaction under conditions sufficient to form a hydrolytically degradable polymer.
The functional group —O—CO2-Z is capable of reacting with a hydroxyl group to form a carbonate linkage. Typically, Z can be any reactive leaving groups so long as the functional group can react with a hydroxyl group to form a carbonate linkage. Examples of suitable leaving groups include N-succinimidyl, 1-benzotriazolyl, and p-nitrophenyl. Methods for preparing an oligomer having a functional group —O—CO2-Z as described above are well known in the art, and are disclosed in U.S. Pat. Nos. 5,650,234, 5,281,698 and 5,468,478; Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (1991), all of which are incorporated herein by reference.
As discussed above, one or more types of oligomers can be polymerized together. In addition, if desired, monomers having a hydroxyl terminus and a functional group —O—CO2-Z at another terminus, can also be included in the polymerization mixture.
Thus, to give an example, the poly(ether carbonate)
as described above can be prepared in accordance with this method by the following steps. First, an ethylene oxide oligomer is provided having a formula of HO—(—CH2CH2—O—)n—O—CO2-Z, where Z is a reactive leaving group such as N-succinimidyl, 1-benzotriazolyl, and p-nitrophenyl. This oligomer is then polymerized under controlled conditions to provide the above poly(ether carbonate).
In another example of this invention, a first oligomer is provided, which is a bifunctional oligomer having a first functional group of —O—CO2-W at one terminus and, at another terminus, a second functional group of —O—CO2-Z. Both functional groups are capable of reacting with a hydroxyl group to form a carbonate linkage. Z and W are reactive leaving groups, and can be any leaving groups known in the art so long as the functional groups containing them, as stated above, are capable of reacting with a hydroxyl group to form a carbonate linkage. Z and W can be same or different. The preferred Z and W are N-succinimidyl, 1-benzotriazolyl, and p-nitrophenyl. Two or more different bifunctional oligomers can also be used in the same polymerization reaction.
Methods for preparing such bifunctional oligomers are similar to those for making the monofunctional oligomers described above. Preferably, Z and W are the same, and the bifunctional molecule Z-oligomer-Z can be provided by activating an oligomer having two hydroxyl terminal groups, with an activating molecule having the formula of Z-O—CO2-Z. Suitable examples of the activating molecule include, disuccinimidylcarbonate, bis(1-benzotriazolyl) carbonate and bis(p-nitrophenyl) carbonate. See, e.g., U.S. Pat. Nos. 5,281,698; 5,650,234; Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (1991), all of which are incorporated herein by reference.
In addition to the first oligomer that is bifunctional, a second oligomer is also provided having two terminal hydroxyl groups. This second oligomer is then polymerized with the first oligomer to form the polymer of this invention.
Two or more types of bifunctional oligomers can be used in the same polymerization reaction. In addition, two or more types of oligomers having two terminal hydroxyl groups can also be used in a polymerization reaction. As will be apparent to a skilled artisan, when only one type of bifunctional oligomer and one type of dihydroxyl oligomer are used, and when the two oligomers are the same except for the terminal groups, the polymer formed therefrom will be a homopolymer having a single type of repeating unit or oligomer linked with hydrolytically degradable linkages. Otherwise, a heteropolymer or block polymer or terpolymer will be made containing different types of oligomers in the polymer backbone.
To give an example of the method of this embodiment, the poly(ether carbonate)
can be prepared by co-polymerizing a first ethylene oxide oligomer having a formula of
and a second ethylene oxide oligomer HO—(—CH2CH2—O—)n—OH, under suitable polymerization conditions. Z is preferably N-succinimidyl, 1-benzotriazolyl, or p-nitrophenyl. The oligomer
can be prepared by reacting Z-O—CO2-Z with HO—(—CH2CH2—O—)n—OH, under the conditions disclosed in U.S. Pat. No. 5,281,698.
In yet another method, one or more oligomers having two hydroxyl terminal groups are polymerized directly with an activating molecule having the formula of Z-O—CO2-Z to form the hydrolytically degradable polymer. Thus, for example, the poly(ether carbonate)
can also be prepared by co-polymerizing an activating molecule Z-O—CO2-Z and the oligomer HO—(—CH2CH2—O—)n—OH, under conditions sufficient to form the poly(ether carbonate).
Again, in this method, either one oligomer is used to generate a homopolymer, or two or more different oligomers can be used in the same polymerization reaction to produce a heteropolymer, or block polymer or terpolymer.
The polymerization reactions are conducted under conditions sufficient to form the hydrolytically degradable polymer of this invention. The polymerization reaction in each of the above-described methods is a condensation reaction. Many different known reaction conditions can be used. Typically, a catalyst is included in the polymerization reaction mixture. Examples of suitable catalysts are organic bases, including triethylamine, pyridine, quinoline, and 4,4-dimethylaminopyridine. Amine bases such as 4,4-dimethylaminopyridine and triethylamine are preferred.
The polymerization can be conducted in either melt or solvent. Suitable solvents include, but are not limited to, acetonitrile, THF, dimethylformamide, dimethylsulfoxide, benzene, toluene, xylenes, chloroform, and methylene chloride. The polymerization reaction rate and the extent of polymerization, which determines the average molecule weight of the final hydrolytically degradable polymer product can be partly controlled by the reaction temperature and the reaction time. Suitable reaction temperature can vary from about 0� C. to 100� C. Higher reaction temperatures lead to greater reaction speed. Preferably, the polymerization reaction is conducted at a temperature of from about 37� C. to 100� C., typically from about 45� C. to 100� C. and advantageously from about 70� C. to 90� C. When the reaction is conducted in a melt, the temperature needs be maintained at a certain minimum temperature in order to keep the reaction mixture at a melt state.
In the above described three embodiments of the method of this invention, the polymerization reactions would be predicted to lead to polymers with an activated carbonate terminal group. In practice, however, NMR analysis of the polymer products indicates that the terminal groups of the hydrolytically degradable polymer prepared therefrom often are hydroxyl groups. While not wishing to be bound by any theory, it is believed that this is caused by reaction with a small amount of water present as an impurity in the reaction. Any small amount of remaining terminally activated carbonate may be removed by hydrolyis in water for a short period or near a neutral pH. The terminal activating groups are much more sensitive to water than are the degradable carbonate linkages.
The polymer of this invention can optionally be activated at one or all termini, thus providing an activated polymer capable of being covalently linked to another molecule, including, for example, a protein, to form a conjugate. The polymer can also be capped at one terminus by an inert group and at another terminus by a reactive moiety.
The polymer of this invention can be activated at its terminus to form a terminal reactive moiety by methods well known to those familiar with the art of organic or polymer chemistry. The well established methods in the broad field of poly(ethylene glycol) chemistry are generally useful, and such methods should be apparent to a skilled artisan. The polymer can be activated at one terminus, or all termini, in which case, the reactive moieties at different termini can be same or different.
For example, the polymer may be activated to form a terminal moiety of N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379 (1981), Zaplipsky et al Eur. Polym. J. 19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry & Biological Applications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. Nos. 5,824,784, 5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). In addition, two molecules of the polymer of this invention can also be linked to the amino acid lysine to form a di-substituted lysine, which can then be further activated with N-hydroxysuccinimide to form an active of N-succinimidyl moiety (see, e.g., U.S. Pat. No. 5,932,462). All of the above references are incorporated herein by reference.
To give an example, the polymer of this invention may be activated to form a terminal reactive moiety of N-succinimidyl carbonate or 1-benzotriazolyl carbonate by reacting the polymer with di-N-succinimidyl carbonate or di-1-benzotriazolyl carbonate respectively. To give another example, terminal reactive moieties such as N-maleimidyl and o-pyridyldithiyl may be prepared by reacting the polymer with activated carbonates connected to N-maleimidyl or o-pyridyldithiyl by linker groups. Terminal aldehyde and acetal moieties can be attached by linking groups. Terminal acid groups can be attached by reaction of the above active carbonates with amino acids or other acid linkers. These acids can then be activated by formation of active esters such as succinimidyl active esters.
The polymer of this invention, activated or not, as prepared by the above methods. can be easily purified from the reaction mixture. Many methods known in the art can be used. A preferred method for purifying the polymer and its derivatives is by precipitation from a solvent in which they are essentially insoluble while the reactants are soluble. Suitable solvents include ethyl ether or isopropanol. As is apparent to a skilled artisan, other methods such as ion exchange, size exclusion, silica gel, and reverse phase chromatography can also be useful.
In accordance with another aspect of this invention, the activated polymer is used to form a conjugate with a biologically active agent. The conjugate comprises the polymer of this invention covalently linked to a biologically active agent. Because the polymer of this invention is hydrolytically degradable in vivo and can be cleaved at the carbonate linkages, thus forming smaller oligomers, the conjugate is especially useful for controlled delivery of the bound agent into animal bodies. Since the polymer in the conjugate is susceptible to breakdown into smaller oligomers, the polymer typically is excreted from animal bodies faster than a polymer of similar size that is not degradable. Thus, potential adverse effects that may be caused by a large polymer's long period of stay in the body is avoided. Therefore, conjugation of the polymer of this invention to a biologically active agent can provide for a sufficient circulation period for the agent while minimizing any adverse effects of the polymer.
In the conjugates of this invention, the linkage between the biologically active agent and the polymer of this invention can be stable or hydrolytically degradable. When it is degradable, substantially all of the polymer can be cleaved off the biologically active agent under physiological conditions, releasing the agent substantially in its native form inside the body. Methods for forming a hydrolytically degradable linkage between a biologically active agent and a water soluble polymer are well known in the art and should be apparent to a skilled artisan. For example, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997), which is incorporated herein by reference.); phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrozone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, e.g., at the end of a polymer, and a 5′ hydroxyl group an oligonucleotide.
Methods for conjugating the polymer of this invention to a biologically active agent should be apparent based on the above discussion. Typically, the polymer of this invention must be activated to form the activated polymer of this invention as described above, having at least one terminal reactive moiety. The terminal reactive moiety may vary depending on the reactivity of a target moiety on the biologically active agent to be conjugated. Examples of reactive groups on proteins are thiols and amines, while on small molecule drugs, amines, alcohols, thiols, and carboxylic acids are common reactive groups. The conjugate is then formed by reacting the terminal reactive moiety of the activated polymer with the target moiety on the biologically active agent. Such methods are well known in the art, and are discussed in the patents and publications referred to above in the context of forming terminal reactive moieties.
In a preferred embodiment, the polymer of this invention used for forming a conjugate is a poly(ether carbonate) composed of alkylene oxide oligomers, more preferably ethylene oxide oligomers, linked together by carbonate linkages. Ethylene oxide oligomers are poly(ethylene glycol)s with a predetermined molecular weight, typically from about 88 to about 8000, preferably from about 88 to about 2000. Thus, in this embodiment of the invention, the polymer behaves in a similar manner as polyethylene glycol. However, when delivered in vivo, the polymer in the conjugate will break down into a number of smaller oligomer fragments. If the linkage between the polymer and the biologically active agent is stable, then, after degradation, one oligomer is linked to the agent.
In accordance with another aspect of this invention, a hydrolytically degradable hydrogel and method of making thereof are also provided. As is known in the art, a hydrogel typically is a polymeric network formed by crosslinking one or more multifunctional backbone molecules or polymers. The resulting polymeric network is hydrophilic and swells in an aqueous environment thus forming a gel-like material, i.e., hydrogel. Hydrogels are useful for drug delivery as they can be implanted or injected into animal bodies. Typically a hydrogel comprises a backbone bonded to a crosslinking agent.
In accordance with this invention, the polymer of this invention is used as the crosslinking agent in the hydrogel. The polymer must be activated so that it has at least two terminal reactive moieties that are capable of reacting with multiple moieties on the backbone to form covalent linkages.
Alternatively, two or more types of activated polymer are used as crosslinking agents. Each activated polymer has one terminal reactive moiety capable of reacting with a moiety on the backbone, and another terminal reactive moiety capable of reacting with the corresponding terminal reactive moiety on the other type of activated polymer. An example of this other moiety is, for example, a vinyl-containing group such as an acrylate group that can participate in chain polymerization among the different types of activated polymers. When the polymer of this invention is activated so that it has two terminal vinyl groups, the polymer itself may act as both crosslinking agent and backbone, and self-polymerize into a hydrolytically degradable hydrogel through a chain polymerization reaction.
The backbone of the hydrogel is a nontoxic biocompatible macromolecule or small molecule, having at least two or preferably more active groups available to react with the terminal reactive moieties of the crosslinking agent to form covalent linkages. By "biocompatible" it is intended that the molecule used as backbone would not substantially adversely affect the body and tissue of the living subject into which the hydrogel is to be implanted or injected. More particularly, the material does not substantially adversely affect the growth and any other desired characteristics of the tissue cells surrounding the implanted hydrogel. It is also intended that the material used does not cause any substantially medically undesirable effect in any other parts of the living subject. In addition, if the molecule is degradable inside the body, the degradation products should also be substantially biocompatible as defined above. Generally, the methods for testing the biocompatibility of a material well known in the art.
Examples of suitable backbones include, but are not limited to, proteins, modified proteins such as glycoproteins, phosphorylated proteins, acylated proteins, and chemically modified proteins, peptides, aminocarbohydrates, glycosaminoglycans, aminolipids, polyols, polythiols, polycarboxylic acids, polyamines such as dilysine, poly(vinylamine) and polylysine, poly(ethylene glycol) amines, pharmaceutical agents having at least two active groups, etc. Specific examples of the backbone include, but are not limited to, branched PEG amines, fibrin, fibrinogen, thrombin, albumins, globulins, collagens, fibronectin, chitosan, and the like. In addition, the backbone may also be microorganisms such as viral particles, bacterial or yeast cells, animal or human cells or tissues.
The activated polymer of this invention used as a crosslinking agent can be in a linear, branched or star form. In branched or star forms, three or more linear polymers are covalently linked, at one terminus, to a central, branched core moiety. The central branch core moiety can be derived from the amino acid lysine, or polyols such as glycerol, pentaerythritol and sorbitol. Branched PEGs are known in the art. These branched PEGs can be incorporated as components of the poly(ether carbonate)s of this invention.
As will be apparent, because of the carbonate linkages incorporated in the crosslinking agent, the hydrogel of this invention is hydrolytically degradable. In addition, the linkages between the backbones and the crosslinking agents formed from the crosslinking reactions can also be made hydrolyzable, by methods described in the context of the conjugate of this invention. Thus, the hydrogel of this invention can gradually break down or degrade in the body as a result of the hydrolysis of the hydrolytically degradable linkages.
Therefore, the hydrogel of this invention is suitable as a biomedical material and a carrier for the delivery of biologically active agents. For example, the hydrogel can carry therapeutic drugs and can be implanted or injected in the target area of the body. The hydrogel may also carry other agents such as nutrients or labeling agents for imaging analysis. A hydrogel containing a biologically active agent is termed herein as "a delivery system".
In the various applications of the hydrogel of this invention, the biologically active agents to be delivered can be used as the backbone, or part of the backbone of the hydrogel. Alternatively, biologically active agents can be "hinged" to the hydrogel through a polymer of this invention or a linker molecule with one terminus of the polymer or the linker linked to the biologically active agent, and the other being connected through a covalent linkage to the hydrogel. In addition, biologically active agents or other substances to be delivered can also be loaded into the hydrogel during the formation of the hydrogel, or afterwards by, for example, diffusion into the matrix of the hydrogel without being covalently bonded to the hydrogel structure.
Because the crosslinking agents (i.e., the polymers of this invention) in the hydrogel are water soluble, the hydrogel can be substantially water swellable. The degradation or breakdown of the hydrogel in the body is gradual in nature and subject to control because of the hydrolytically degradable carbonate linkages in the crosslinking agents. Thus, the hydrogels of the invention are particularly useful for sustained release of a biologically active agent or other substance in the body. The hydrogels have potential utility for adhesion prevention, bioadhesives, surgical sealants, and related surgical applications.
The present invention is further illustrated in the following examples which are given to illustrate the invention, but should not be considered in limitation of the invention.
HO—(—CH2CH2—O—)nCO2Bt (nave=23) (5 g) and 1.2 g of dimethylaminopyridine were dissolved in 10 ml of anhydrous acetonitrile. The reaction was stirred at 70� C. for 22 hours. The product was precipitated with 200 ml of isopropanol, stirred for one-half hour, then collected by vacuum filtration. The product was washed with isopropanol (100 ml�2), and dried in vacuo overnight. Yield: 4.1 g (82%). The resulting polymer was 99% pure by gpc analysis and had a molecular weight of 13,800 Da with a polydispersity of 1.47.
1. Preparation of Bt-O2C—(O—CH2CH2—)n—OCO2-Bt (nave=23)
HO—(—CH2CH2—O—)n—H (nave=23) (15 g, Aldrich) was dissolved in 100 ml of anhydrous acetonitrile, and solvent was removed by distillation. The residue was dissolved in 100 ml of anhydrous acetonitrile, and 13.3 g (1.5 equivalent) of di(1-benzotriazolyl carbonate) and 3.1 ml (1.3 equiv) of anhydrous pyridine were added. The reaction was stirred at room temperature under Ar overnight. The solvent was evaporated to dryness under reduced pressure. The product was precipitated by addition of 300 ml of cold diethyl ether. The precipitate was stirred under Ar for 30 min., collected by vacuum filtration and dried in vacuo overnight. There was 100% substitution by nmr analysis.
2. Preparation of HO-PEC7K-OH
HO—(—CH2CH2—O—)n—H (nave=23) (Aldrich, 10 g) was dissolved in 50 ml of anhydrous acetonitrile and the solvent removed by distillation. The residue was dissolved in 20 ml of anhydrous acetonitrile and 10 g (1.0 equiv) of Bt-O2C—(—O—CH2CH2—)nOCO2-Bt (nave=23) and 2.5 g of dimethyaminopyridine were added. The reaction was stirred at 70� C. for about 22 hours. The product was precipitated with 500 ml of isopropanol, stirred for half hour, then collected by vacuum filtration. The collected product was washed with isopropanol 200 ml�2), and dried in vacuo overnight. The resulting polymer was about 97% pure by gpc and nmr and had a molecular weight of 7000 Da.
15.0 g of HO-PEC7KDa-OH (prepared in Example 2) was dissolved in 50 ml of anhydrous acetonitrile, and solvent was distilled off. This process was repeated once. The residue was dissolved in 50 ml of anhydrous acetonitrile, and 1.8 g of bis(1-benzotriazolyl carbonate) and 0.5 ml of anhydrous pyridine were added. The reaction was stirred at room temperature under Ar overnight. The solvent was evaporated to dryness at reduced pressure and the product was precipitated by addition of 500 ml of isopropanol. The precipitate was stirred under Ar for 30 min., collected by vacuum filtration, and dried in vacuo overnight. Substitution was greater than 95% by nmr. Yield: 14.3 g.
HO(—CH2CH2O—)nCO2NS (nave=45)(5 g) was dissolved in acetonitrile (10 ml), dimethylaminopyridine (0.6 g) was added, and the resulting mixture was stirred at 70-80� C. overnight. Methylene chloride (20 ml) was added and the resulting mixture added to 600 ml of isopropyl alcohol. The resulting precipitate was collected by vacuum filtration and dried under vacuum overnight at room temperature. Nmr and gpc analysis indicated the polymer to have a molecular weight of 38,000 Da.
HO(—CH2CH2O—)nCO2NS (nave=45)(5 g) and dimethylaminopyridine (0.6 g) were mixed and the resulting mixture was stirred at 80-90� C. overnight. Methylene chloride (20 ml) was added and the resulting mixture added to 600 ml of isopropyl alcohol. The resulting precipitate was collected by vacuum filtration and dried under vacuum overnight at room temperature. Nmr and gpc analysis indicated the polymer to have a molecular weight of 35,000 Da.
HO—(—CH2CH2—O—)n—H (nave=14) (10.0 g) was dissolved in 100 ml of acetonitrile and the solution was dried by azeotropic distillation. After removal of acetonitrile, 80 ml of methylene chloride and 20 ml of acetonitrile, 2.7 ml of pyridine, and 4.3 g of di-(N-succinimidyl) carbonate were added. After stirring overnight, the solvent was removed under reduced pressure and 10 ml of acetonitrile and 4 g of DMAP were added. The mixture was stirred at 80-90� C. for 5 h and the product precipitated by addition of isopropanol. The product was collected by vacuum filtration and dried under vacuum at room temperature to obtain the product as a white powder (4.5 g). NMR and GPC analysis indicated that the polymer has a molecular weight of 9400 Da.
In a test tube, 110 mg of Bt-O2C—O-PEC7KDa-O—CO2-Bt was dissolved in 1 ml of phosphate buffer (0.1 M, pH 7.0). To it was added 0.36 ml of 8-arm-PEG-amine (110 mg/ml in the buffer). After rapid shaking, it was allowed to sit. A hydrogel formed within 2 hours.
Degradation of the Hydrogel (Example 7) Prepared from PEC and 8-arm-PEG Amine (10 kDa)
The hydrogel (0.5 g.) was placed in 3 ml of PBS buffer or rat serum at 37� C. Rat serum was replaced with fresh serum every 10 to 12 hours. Gel disappearance was monitored visually. The gel prepared from PEC7KDa disappeared in PBS buffer within 3.5 days but in 1.5 days in rat serum, while the similar gel prepared from PEC3Kda disappeared in PBS buffer in approximately 10 days.
HO-PEC7KDa-OH (10 g)(prepared in Example 2) in toluene (200 ml) was dried by azeotropic distillation and triethylamine (1.75 ml) in CH2Cl2 was added. The resulting solution was cooled to 0� C. and acryloyl chloride (2.5 ml) was added under argon. After the addition was complete, the mixture was stirred overnight at room temperature. The resulting solution was concentrated, filtered, and the product precipitated from the filtrate by addition of ethyl ether (300 ml). The precipitate was collected by filtration, dissolved in 100 ml of methylene chloride, and the resulting solution stirred with 10 g of sodium carbonate overnight. The mixture was filtered and the product was precipitated with 500 ml of ethyl ether. The product was collected by filtration and dried under vacuum. NMR comparison of acrylate and backbone absorptions indicated approximately 100% of the terminal groups were acryloylated.
PEC7KDa diacrylate (100 mg) was dissolved in 1 ml of pH 7 buffer and 30 ul of potassium persulfate was added. To the resulting solution was added 30.mu.1 of 100 mM iron(II) sulfate solution. The solution was shaken and a hydrogel formed in several minutes.
NS—O2C—O-PEC7KDa-O—CO2—NS (300 mg) was mixed with 3 ml of a 1 wt. % solution of chitosan at pH 4. The resulting solution formed a clear, firm hydrogel within 2 hours.
Azeotropically dried benzyl-PEG1KDa-OH (40.6 g, 40.6 mmoles), Bt-O2C—O-PEG1KDa-O—CO2-BT (25 g, 20.3 mmoles) and dimethylaminopyridine (5 g, 81.2 mmoles) was mixed in 300 ml of acetonitrile. The reaction was stirred at 75-80� C. overnight. The solvent was removed by rotary evaporation and the product was precipitated using diethyl ether/isopropyl alcohol (500 ml, 50/50) in an ice bath. The product was collected by filtration and dried under vacuum. The dry product was placed in a 500 ml Parr bottle and dissolved in 100 ml of anhydrous 1,4-dioxane. 11.0 g of Pd/C (10% pd by wt.) was added and the bottle pressurized with 45 psi of hydrogen and shaken for 20.5 hours. The mixture was filtered to remove the Pd/C and the solvent removed by rotary evaporation. The crude solid was precipitated by adding 500 ml of isopropyl alcohol. The product was collected by filtration and dried under vacuum. The product was demonstrated to be greater than 98% pure with a polydispersity of 1.034 by GPC.
The dried HO-PEC3KDa-OH was dissolved in anhydrous CH3CN (150 ml) and stirred at room temperature under argon. Di-N-succinimidyl carbonate (2.6 g 10 mmoles) was added and allowed to dissolve before pyridine (0.81 ml, 10 mmoles) was added. The reaction mixture was stirred overnight (15 hrs.) The CH3CN was removed under vacuum. The crude solid was dissolved in CH2Cl2 (100 ml) and washed once with buffer solution (100 ml). The aqueous layer was extracted with an additional 50 ml of CH2Cl2 and the combined organic layers were dried using Na2SO4. The solvent was removed under vacuum until a viscous residue remained. To this was added IPA (500 ml) to precipitate the product. The product was collected by filtration and dried under vacuum. The product was demonstrated by 1H nmr to have greater than 95% purity.
CH3O—(CH2CH2O)n—CO2(CH2CH2O)n—OCH3 (nave=13) (1 wt. % in buffer, pH 7.4) was thermostated at 37� C. and the hydrolysis rate of the carbonate linkage was measured by analysis of the product using GPC. The half-life for hydrolysis under these conditions was found to be 44 days.
The foregoing description is to be considered illustrative rather than descriptive of the invention. Therefore, it should be understood that the specific embodiments described herein are illustrative of how the invention may be practiced and that modifications and other embodiments are intended to be included within the scope of the appended claims.
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|Citing Patent
|Filing date
|Publication date
|Mar 22, 2011
|Nektar Therapeutics
|Hydrolytically degradable polymers and hydrogels made therefrom
|Feb 14, 2011
|Feb 21, 2012
|Nektar Therapeutics
|Hydrolytically degradable polymers and hydrogels made therefrom
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|Nektar Therapeutics
|Hydrolytically degradable polymers and hydrogels made therefrom
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|Hydrolytically Degradable Polymers and Hydrogels Made Therefrom
|U.S. Classification
|528/196, 528/198, 525/329.9, 424/78.32, 424/94.63, 424/78.27, 526/286, 525/326.7
|International Classification
|Cooperative Classification
|European Classification
|Aug 31, 2009
Owner name: NEKTAR THERAPEUTICS, CALIFORNIA
Free format text: MERGER;ASSIGNOR:NEKTAR THERAPEUTICS AL, CORPORATION;REEL/FRAME:023196/0394
Effective date: 20090731
Owner name: NEKTAR THERAPEUTICS,CALIFORNIA
Free format text: MERGER;ASSIGNOR:NEKTAR THERAPEUTICS AL, CORPORATION;REEL/FRAME:023196/0394
Effective date: 20090731
|Jul 17, 2012
Owner name: WELLS FARGO BANK, NATIONAL ASSOCIATION, AS COLLATE
Free format text: GRANT OF SECURITY INTEREST;ASSIGNOR:NEKTAR THERAPEUTICS;REEL/FRAME:028571/0141
Effective date: 20120711
|Mar 18, 2013
|Fee payment
Year of fee payment: 4
|Oct 6, 2015
Owner name: TC LENDING, LLC, AS COLLATERAL AGENT, TEXAS
Free format text: GRANT OF SECURITY INTEREST IN UNITED STATES PATENTS;ASSIGNOR:NEKTAR THERAPEUTICS;REEL/FRAME:036796/0562
Effective date: 20151005
|Oct 14, 2015
Owner name: NEKTAR THERAPEUTICS, CALIFORNIA
Free format text: RELEASE OF SECURITY INTEREST RECORDED AT REEL 28571, FRAME 0141;ASSIGNOR:WELLS FARGO BANK, NATIONALASSOCIATION, AS COLLATERAL AGENT;REEL/FRAME:036866/0700
Effective date: 20151005


text:
Dolly is a solidly built black and white paint mare. She stands 15.3 hh and is a good solidly built horse. Dolly is sound and healthy. We have owned her…SOLD
Reg. Gelding 17 hands coming 5 yr old.. HE is so gentle and easy to handle, catch, shoe, bath, ect. he is excellent on trails and in the mountains. HE…SOLD
Pauls Purtty AKA "Fabio" is a 14 year old 16hh bay registered Quarter Horse gelding (Appendix). Fabio lunges, round pens, loads, clips, stands for the…SOLD
Great starter horse. Really sweet and loves to get a lot of attention. Both of her parents are fabulous polo horses. Please call Jennifer 602-397-0879…SOLD
Handsome, tall (16.2 barefoot with short toe) 3yo gelding for sale. Sensible and willing, yet game and very forward. Flat walks and works in the double…SOLD
This colt is an old soul!!! He seems like he was borned trained and willing to learn. He is super cute, lots of suspension and has been free jumped 3…SOLD
This 16.3 chestnut mare, with flaxen mane and tail, is a mare of highest quality. She missed Premium by 1 point, due to a injury from that same day. She…SOLD
Royal Fortune, aka Rohan, is an amazing colt. I have waited for him my whole life. He is so sweet, quiet and kind. He is by Rubignon, (Rubinstein X…SOLD