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We just wanted to let everyone know that our thoughts are with the people of Texas and Florida in the wakes of Hurricane Harvey and Irma. The MWPBA will be holding a fund raiser to help support our piping and drumming friends that have been affected. Full details will follow after things settle down.
In the meantime, we have started a campaign to collect donations on your behalf that will be distributed to bands and related organizations in the areas. Any and all donations of any amount would be greatly appreciated. Distribution will be overseen by the MWPBA Board.
We are seeing tremendous support so far, and thank everybody most sincerely!
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subset: commoncrawl


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|Reviews for Tripping
| TheDowntonLady chapter 35 . 9/23/2016
I start to read your story, and I couldn't stop. That's one of the best Remus/Tonks story I read. And you write very well.
I know it's been a long time since you update, and you'll probably never read this review, but I still hope you will update someday.
Thank you for your story
| nightwindow chapter 34 . 4/4/2015
And just now I noticed that it is full moon tonight. This cannot be a coincidence ;)
I (and others as well as I just read) would be really glad if you found a way / time / your muse to continue this awesome piece of fanfiction!
| nightwindow chapter 35 . 4/4/2015
Seriously, I read this almost straight through all in one day today, and there was not one bit I didn't love! I cannot tell you what I like most: your great sense of humor, the awesome characterizations, the perfect pace and following of the original story, the varied and creative language, or the fantastic inclusion of the other characters (I really like your Snape!).
And although I have suspected all along while reading that this story is far from finished (now well in the middle of the 6th book, I assume) I am so, SO sad that this stops here. This is by far the best Remus/Tonks FF I have read and also one of the BEST I have read in general.
I know that your last update was 4 years ago and it seems like you have not been active here on FF that much in the past years (life goes on for all of us I guess). Hell, I don't even know if you'll ever notice my review, as there have barely been any in the past years. But please be assured that would be endlessly grateful if you decided to continue & finish this story, because it is truly incredibly entertaining, well written and feels like a true contribution to the canon.
THANK YOU & Take care
| faerieMagic07 chapter 7 . 3/26/2015
Man, you really captured Remus' reserved character.
I was told that an author should surprise the readers with new and out of the line things from the character in a convincing way. But since I'm too dry to write stuff myself, I'm reading. Kudos, kudos for your awesome characterization.
| faerieMagic07 chapter 2 . 3/26/2015
I like this. Remus is reserved and not proud, always down to earth and kind.
I specially liked your version of their meeting, with Tonks tripping and being caught by Remus.
| sdgh chapter 35 . 3/19/2015
update dammit!
| animeluva chapter 35 . 2/9/2014
Awesome story! Just please finish it!
| JKStorm501 chapter 35 . 7/14/2013
Why did you abandon this story? :cccccc It would be awesome if you started it again!
| bkaddictjk chapter 35 . 1/17/2013
You have got to be kidding me... I read 35 chapters, and I meet a cliffhanger, to see that this hasn't been updated for 2/3 years? :(
| bkaddictjk chapter 7 . 1/17/2013
Oh... Merlin's Pants... that was the best awkward apology I've ever read... and I've read a lot of fanfiction...
| queenofserendipity chapter 35 . 10/31/2012
OMG! I just read all these 35 chapters- this story is AMAZING! I noticed you haven't updated in AGES- PLEASEPLEASEPLEASEPLEASEPLEA SEPLEASE DO!
| JamSack chapter 35 . 10/9/2012
Oh this is so good! I hadn't been on fanfic on ages and I was reading HBP last night and I remembered this fic! I love it! I like the the sort of frienship that Tonks has with Snape and the characters are in character which is wonderful! I loved that moment at the Hogshead and Snape knowing exactly how it feels to love someone you can never be angst, you are not my best friend in real life but I love it in fics! This one has my vote for finishing it up please!
| marauder68 chapter 35 . 8/12/2012
NOOOO! I must know what happens! This is an AMAZING story!
| iluvcoffee123 chapter 35 . 2/19/2012
| LexxiLex chapter 35 . 2/17/2012
Will you pllllleeeeaaasssseeeeeeee continue this?


text:
- Living room
- Cot on demand
- Extra beds on demand
- Room size (sqm): 32
- Number of bedrooms: 1
Mirage New Hawaii Resort And Spa Ex Marmara Club
- 50m to beach
- 10Km to town centre
- Sun terrace
- Private beach area
- Outdoor Pool
- Free WiFi in public areas
- Low Deposit Options Available
- Interest FREE Payment Plans
Mirage New Hawaii Resort And Spa Ex Marmara Club in Hurghada South has 462 rooms, spread over 5 floors. The hotel is located 50 m from the sandy beach. To make your stay more ... Continue reading
Mirage New Hawaii Resort And Spa Ex Marmara Club in Hurghada South has 462 rooms, spread over 5 floors. The hotel is located 50 m from the sandy beach. To make your stay more comfortable 6 elevators, a hairdresser, a reception, air conditioning and a lobby are available. The staff in this 5 storey hotel speaks Russian, German, French and English. An entertainment program suited for children is available for young guests. 3 fresh water swimming pools with a separate children's pool are available. A poolside bar is on site at the hotel where guests can unwind with a drink. Sunshades and sunbeds are available free of charge at the beach and at the pool. Distance from the hotel to: airport RMF (220 km), airport HRG (17 km) and tourist information (10 km). For your entertainment the hotel features a disco.
- Entertainment staff
- Nightclub/DJ (Additional charge)
Other facilities
- 24hr Reception
- Concierge service
- Dry cleaning (Additional charge)
- Garden
- Ironing service (Additional charge)
- Laundry (Additional charge)
- Luggage storage
- Meeting/banquet facilities (Additional charge)
- Packed lunches
- Private beach area
- Sun terrace
- Terrace
- Vending machine (drinks)
- Air conditioning
- Free WiFi in public areas
- Lift
- Safety deposit box
- Shops (on site)
Food and Drink
The breakfast type is Buffet and it is provided from 07:00 - 10:00. The hotel has 5 restaurants. Evenings can be spent at the lobby bar. All-Inclusive: All-Inclusive includes local spirits, late breakfast (Service Hours: 10:30 - 11:00), coffee and tea (Service Hours: 10:00 - 00:00), midnight snacks (Service Hours: 22:00 - 00:00), softdrinks, wine, snacks (Service Hours: 11:00 - 12:00) and beer.
Other Information
Payment facilities include Visa Card and Euro/Master Card. Additional fees may apply for certain facilities, amenities or activities.
To make booking a holiday at Mirage New Hawaii Resort And Spa Ex Marmara Club in Hurghada easier for you, we provide a range of different payment options. To find out if you can book Mirage New Hawaii Resort And Spa Ex Marmara Club with a low deposit holiday option, please click the "Price and Availability" button above.
Hurghada, known locally as Ghardaga, has grown from a small fishing village to an internationally renowned holiday resort town on the Red Sea coast of Egypt, famous for its underwater life and diving and snorkelling opportunities. Popular as a European package holiday destination, the town of Hurghada boasts first class hotels and all-inclusive resorts, as well as a highly vaunted nightlife. South of the town is the resort strip with hotels packed along a 12-mile (20km) stretch of beach, while within the town there are many bars, restaurants, shops and a variety of accommodation to suit all types of budget. The town's central location on Egypt's coast provides a gateway to prime diving sites throughout the Red Sea.
Resort Rating
The holiday resort of Hurghada is renowned for its variety of watersports that includes sailing, fishing, kitesurfing and windsurfing. Offshore underwater gardens and coral reefs make this an internationally recognised location among divers and snorkellers, and for the less active many hotels offer glass-bottomed boat excursions to admire the corals and marine life. Hotels and resorts also offer water activities such as banana rides, pedal boats and canoeing, as well as trips to the Giftun Island Marine Park. On land there are beautiful beaches, golf courses, horse riding, and desert excursions, which include camel rides, hiking, 4x4 or quad safaris.
Hurghada has a variety of restaurants for those on holiday to enjoy, serving food from around the world, from Indian or French to Thai, Mexican and Japanese. El Dahar (downtown) offers a wide choice of authentic Egyptian eateries, including street stalls and inexpensive restaurants, and fast food chains such as KFC, Pizza Hut and McDonalds are also available. Most local places don't serve alcohol. Hotels all have their own restaurants, which serve up a variety of local and international cuisine.
With its growing reputation as a holiday party town 'par excellence', Hurghada boasts a lively nightlife with numerous clubs, discos, pubs and trendy lounges, and a variety of entertainment that includes beach barbeques, karaoke evenings, foam parties and divers' parties. Popular are Papas Beach Club and Calypso, while Alf Leila Wa Leila is famous for belly dancing and Arabic folklore.
El Dahar, the old part of Hurghada town, has a traditional Egyptian bazaar with a variety of shops selling tourist souvenirs and holidaymakers can splurge to their hearts' content. Bargaining is expected and prices will generally be better than in the resort village shops. The best buys are papyrus scrolls or traditional 'shisha pipes'.
The beach in Hurghada is made of dark red grainy sand interspersed with rocks and boulders; sandals should be worn.
Essential Information
Time Zone
Plug TypeVoltage 220V 50Hz
This week's weather forecast
Hotel address : El Kora Road, Hurghada, Red Sea, Egypt
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|Publication number
|Publication type
|Application number
|Publication date
|Dec 27, 2005
|Filing date
|Nov 28, 2000
|Priority date
|Dec 23, 1998
|Fee status
|Also published as
|Publication number
|Merrill Goldenberg, Daxian Shan, Alice Beekman, Tiansheng Li
|Original Assignee
|Amgen Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (50), Non-Patent Citations (6), Referenced by (18), Classifications (17), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation in part of U.S. patent application Ser. No. 09/221,181, filed Dec. 23, 1998 now U.S. Pat. No. 6,245,740, which is incorporated by reference herein.
Due to recent advances in genetic and cell engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of being produced in large amounts for pharmaceutical applications. Such pharmaceutical proteins include erythropoietin (EPO), novel erythropoiesis stimulating protein (NESP), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth differentiation factor (MGDF), osteoprotegerin (OPG), glial cell line derived neurotrophic factor (GDNF), somatotrophins and obesity protein (OB protein). OB protein may also be referred to herein as leptin.
Many illnesses or conditions treated with pharmaceutical proteins require sustained protein levels to achieve the most effective therapeutic result. However, as with most protein pharmaceuticals, the generally short biological half-life requires frequent administration. These repeated injections are given at various intervals which result in fluctuating medication levels at a significant physical and monetary burden on the patients. Since many conditions respond better to controlled levels of a pharmaceutical, a need exists for controlled release of a medicament to provide longer periods of consistent release. Such sustained-release medicaments would provide a means of controlling blood levels of the active ingredient, thus providing the patient with enhanced prophylactic, therapeutic or diagnostic effects, as well as greater safety, patient convenience and patient compliance. Also such sustained release compositions can lead to dose sparing and thus lower cost of protein production. Unfortunately, the instability of most proteins (e.g. denaturation and loss of bioactivity upon exposure to heat, organic solvents, etc.) has greatly limited the development and evaluation of sustained-release formulations.
Attempts to develop sustained-release formulations have included the use of a variety of biodegradable and non-biodegradable polymer (e.g. poly(lactide-co-glycolide)) microparticles containing the active ingredient (see e.g., Wise et al., Contraception, 8:227–234 (1973); and Hutchinson et al., Biochem. Soc. Trans., 13:520–523 (1985)), and a variety of techniques are known by which active agents, e.g. proteins, can be incorporated into polymeric microspheres (see e.g., U.S. Pat. No. 4,675,189 and references cited therein). Unfortunately, some of the sustained release devices utilizing microparticles still suffer from such things as: low entrapment efficiency; active agent aggregation formation; high initial bursts of active agent with minimal release thereafter; and incomplete release of active agent.
Other drug-loaded polymeric devices have also been investigated for long term, therapeutic treatment of various diseases, again with much attention being directed to polymers derived from alpha hydroxycarboxylic acids, especially lactic acid in both its racemic and optically active form, and glycolic acid, and copolymers thereof. These polymers are commercially available and have been utilized in FDA-approved systems, e.g., the Lupron Depot™, which consists of injectable microparticles which release leuprolide acetate for about 30 days for the treatment of prostate cancer.
Various problems identified with the use of such polymers include: inability of certain macromolecules to diffuse out through the matrix; deterioration and decomposition of the drug (e.g., denaturation caused by the use of organic solvents); irritation to the organism (e.g. side effects due to use of organic solvents); low biodegradability (such as that which occurs with polycondensation of a polymer with a multifunctional alcohol or multifunctional carboxylic acid, i.e., ointments); and slow rates of degradation.
A variety of oil based formulations have been described. Welch in U.S. Pat. No. 2,491,537 discloses the use of oil suspensions (gelled vegetable oil) to provide 24 hour release of penicillin. Buckwalter in U.S. Pat. No. 2,507,193 discloses release in rabbits for up to eleven days using procaine penicillin suspended in peanut oil gelled with 5% aluminum monostearate (AIMS). Anschel in U.S. Pat. No. 2,964,448 discloses suspensions of relaxin in a vegetable oil gelled with AIMS. Anschel reports 5–7 days of relaxation and discloses longer effect (up to 23 days) by heat treating the suspension containing AIMS. Yamahira et al. in U.S. Pat. No. 4,855,134 disclose sustained-release preparations of indomethacin or interferon in admixture with a pharmaceutically acceptable biodegradable carrier, e.g., gelatin. Mitchell in U.S. Pat. No. 5,411,951 discloses compositions wherein metal-associated somatotropin is present in a biocompatible oil and it is demonstrated that the compositions can be parenterally administered for prolonged release of somatotropin in animals. Ferguson et al. in U.S. Pat. No. 4,977,140 disclose sustained release formulations comprising bovine somatotropin, a wax, and an oil. Reichert et al. in WO 96/18417 disclose pharmaceutical compositions comprising mixtures of crystalline G-CSF and vegetable oils.
There have also been a number of reports discussing efforts to develop drug delivery systems utilizing protein that are subject to aggregation. For example, Grodsky et al., U.S. Pat. No. 4,371,523, describe the use of anti-aggregation agents, e.g., glutamic acid and/or aspartic acid, to develop insulin formulations. Blackshear et al., U.S. Pat. No. 4,439,181, describe mixing glycerol or another polyol with an aqueous protein hormone solution prior to the introduction of the solution into the drug delivery system. Wigness et al., PCT Publication WO 85/02118 describe the use of glycerol to prevent precipitation of proteins within drug delivery systems; and Azain et al., EP Publication 0 374 120 A2 describe stable somatotropin compositions which utilize, inter alia, a stabilizing polyol.
Despite the advances made in the processes described above, there is still a need to develop pharmaceutical formulations which achieve a more versatile and effective means of sustained-release for clinical applications. Numerous recombinant or natural proteins could benefit from constant long term release and thereby provide more effective clinical results.
Human recombinant G-CSF selectively stimulates neutrophils, a type of white blood cell used for fighting infection. Currently, FILGRASTIM�, a recombinant G-CSF, is available for therapeutic use. The structure of G-CSF under various conditions has been extensively studied; Lu et al., J. Biol. Chem. Vol. 267, 8770–8777 (1992).
G-CSF is labile and highly susceptible to environmental factors such as temperature, humidity, oxygen and ultraviolet rays. And, because of its hydrophobic characteristics, G-CSF is difficult to formulate due to formation of dimer and higher order aggregates (macro range) during long-term storage. G-CSF has been shown to be very prone to aggregation, especially at neutral pH, elevated salt and temperatures (i.e. physiological serum conditions). This instability makes the sustained release (of a period of one week or greater) by conventional delivery systems very problematic, and in fact, such systems generally provide only a few days of release at best.
It is an object of the present invention to produce a G-CSF-containing preparation which would provide for the sustained release of G-CSF. Production of such preparations is achieved using glycerol/oil suspensions containing G-CSF, and, importantly, pharmaceutical compositions using these G-CSF/glycerol/oil suspensions are capable of providing increased bioavailability, protein protection, decreased degradation and slow release with increased protein stability and potency. Importantly, pharmaceutical compositions of the present invention provide a simple, rapid and inexpensive means of controlled recombinant protein release for effective prophylactic, therapeutic or diagnostic results.
The present invention thus relates to the preparation of a stabilized, prolonged-release injectable suspension containing a biologically active agent. The present invention stems from the observation that G-CSF is stabilized when admixed in glycerol and remains stabilized when the mixture is further suspended in a thickened oil such as sesame oil containing a low percentage of aluminum monostearate, or wax, thus providing a stabilized, prolonged-release injectable preparation. Surprisingly, and unexpectedly, it has now been determined that G-CSF in solution is soluble in high concentrations of glycerol, and still provides a stabilized, prolonged-release injectable preparation when incorporated into a thickened oil.
In one embodiment, the present invention provides sustained-release suspensions comprising an effective amount of a biologically active agent (BAA) incorporated into a polyol/thickened oil suspension, said suspension capable of providing for the sustained-release of the biologically active agent for a period of at least one week.
In another embodiment, the present invention provides a method for the parenteral administration of a BAA/glycerol/oil suspension to a warm blooded animal, wherein said suspension is administered subcutaneously, or intramuscularly and the biologically active agent is released from the suspension at a controlled rate for up to one week or more.
The present invention further relates to pharmaceutical compositions comprising the sustained release suspensions together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, anti-oxidants (e.g., ascorbic acid and Vitamin E), adjuvants and/or carriers needed for administration.
The present invention further relates to processes for preparing sustained-release injectable BAA/polyol/oil suspensions as above. The principal embodiment comprises: (a) admixing a BAA in a polyol to form a BAA/polyol mixture and (b) suspending said BAA/polyol mixture in a mixture comprising a thickened oil, or wax, to form a BAA/polyol/oil suspension.
The present invention further relates to a prefilled syringe comprising said formulation.
The present invention also relates to methods of treatment of individuals using the stabilized, prolonged-release injectable preparations described herein.
As used herein, the following terms shall have the following meaning:
"Biodegradable" is defined as meaning that the polyol/oil vehicle will erode or degrade or absorb or metabolize in vivo to form smaller non-toxic components.
"Biocompatible" is defined as meaning the oil and its thickeners and other excipients will have no intolerable adverse effect on the polypeptide, or human being treated.
"Parenteral administration" is defined as meaning any route of administration other than the alimentary canal, including, for example, subcutaneous, intramuscular, intrathecal, intraorbital, intraarticular, pulmonary, nasal, rectal and otic.
As used herein, biologically active agents refers to recombinant or naturally occurring proteins, whether human or animal, useful for prophylactic, therapeutic or diagnostic application. The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. In addition, biologically active agents of the present invention can be PEGylated or conjugated with water soluble adducts such as carbohydrates, e.g., dextran. A wide range of biologically active agents are contemplated. These include but are not limited to hormones, cytokines, hematopoietic factors, growth factors, antiobesity factors, trophic factors, anti-inflammatory factors, and enzymes (see also U.S. Pat. No. 4,695,463 for additional examples of useful biologically active agents). One skilled in the art will readily be able to adapt a desired biologically active agent to the compositions of present invention which can also include small organic or organometallic compounds.
Such proteins would include but are not limited to granulocyte-colony stimulating factors (G-CSF's) (see, U.S. Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, hereby incorporated by reference including drawings), interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 hereby incorporated by reference including drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference including drawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings), osteoprotegerin (PCT Publication No. 97/23614, hereby incorporated by reference including drawings), novel erythropoiesis stimulating protein (NESP) (PCT Publication No. 94/09257, hereby incorporated by reference including drawings) and leptin (OB protein).
Provided below is a working example using G-CSF, which, as described above, is a therapeutic protein used to treat hematopoietic disorders. In general, G-CSF useful in the practice of this invention may be a form isolated from mammalian organisms or, alternatively, a product of chemical synthetic procedures or of prokaryotic or eukaryotic host expression of exogenous DNA sequences obtained by genomic or cDNA cloning or by DNA synthesis. Suitable prokaryotic hosts include various bacteria (e.g., E. coli); suitable eukaryotic hosts include yeast (e.g., S. cerevisiae) and mammalian cells (e.g., Chinese hamster ovary cells, monkey cells). Depending upon the host employed, the G-CSF expression product may be glycosylated with mammalian or other eukaryotic carbohydrates, or it may be non-glycosylated. The G-CSF expression product may also include an initial methionine amino acid residue (at position −1). The present invention contemplates the use of any and all such forms of G-CSF, although recombinant G-CSF, especially E. coli derived, is preferred, for, among other things, greatest commercial practicality.
Certain G-CSF analogs have been reported to be biologically functional, and these may also be chemically modified, by, for example, the addition of one or more polyethylene glycol molecules. G-CSF analogs are reported in U.S. Pat. No. 4,810,643. Examples of other G-CSF analogs which have been reported to have biological activity are those set forth in AU-A-76380/91, EP 0 459 630, EP 0 272 703, EP 0 473 268 and EP 0 335 423, although no representation is made with regard to the activity of each analog reportedly disclosed. See also AU-A-10948/92, PCT 94/00913 and EP 0 243 153. Of course, if one so desires when treating non-human mammals, one may use recombinant non-human G-CSF's, such as recombinant murine, bovine, canine, etc. See PCT WO 9105798 and PCT WO 8910932, for example.
The type of G-CSF used for the present preparations may be selected from those described in PCT Publication No. 94/17185, as cited above and herein incorporated by reference in its entirety. The 174 amino acid sequence for mature, recombinant methionyl human G-CSF is presented herein as SEQ ID NO: 1, where the first amino acid of the mature protein is threonine (T) (at position 1) and a methionyl residue is located at position −1 (not included in the sequence below).
However, as with any of the present G-CSF moieties, the methionyl residue at position −1 may be absent.
Also included are those proteins as set forth above with amino acid substitutions which are "conservative" according to acidity, charge, hydrophobicity, polarity, size or any other characteristic known to those skilled in the art. These are set forth in Table 1, below. See generally, Creighton, Proteins, passim (W. H. Freeman and Company, N.Y., 1984); Ford et al., Protein Expression and Purification 2:95–107 (1991), which are herein incorporated by reference.
Conservative Amino Acid Substitutions
glutamic acid
aspartic acid
In addition, biologically active agents can also include but are not limited to insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), tumor necrosis factor-binding protein (TNF-bp), brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), insulin-like growth factors (IGFs), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte derived growth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin, platelet-derived growth factor (PGDF), colony simulating growth factors (CSFs), bone morphogenic protein (BMP), superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, somatotropins, streptokinase and kallikrein. The term proteins, as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof.
The BAA used to prepare the sustained-release compositions of the present invention can be in solution or powder form and is first admixed with a polyol, e.g., glycerol. Precise concentrations of polyol will be used, depending upon the amount of BAA used. The polyol is added in an amount sufficient to stabilize (e.g., prevent aggregation) the BAA during long-term storage of the BAA in the suspension.
Other biocompatible C-4 to C-19 polyols contemplated for use include, but are not limited to, C-4: erythritol; C-5: arabinose, xylose, ribose; C-6: inositol, fructose, galactose, glucose, mannose; C-12: maltose and sucrose. If the polyol used is in solid form, it will be first prepared as an aqueous or aqueous organic solution or fluidized by means of heat or pressure, and admixed with the BAA.
The level of polyol used to prepare the BAA/polyol mixture can range from 5%–100% (e.g., 100%=neat glycerol; 90%=90% glycerol, 10% water). When the BAA to be used is in powdered form, the resultant BAA/polyol mixture will be in the form of a suspension. When the BAA to be used is in solution form, the resultant BAA/polyol mixture will be in solution form. The polyol concentration in the final BAA/polyol/oil suspension can range from 10%–40%, more preferably 20%–30%. In a preferred embodiment wherein G-CSF powder is the biologically active agent, and glycerol is the polyol, 20% glycerol is used in the final suspension.
The oils used in the present invention are biocompatible, of low acidity and essentially free from rancidity. Such oils are selected from the group consisting of, for example, sesame seed, cannola, saffron, castor, cottonseed, olive, peanut, sunflower seed, ethyl oleate, vitamin E including α-tocopherol and its derivatives, and Miglyol 812.
The glycerol/oil suspensions will also contain a "thickener" or "gelling agent" which serves to retard hydration of the suspension, give the body of oil greater viscosity or viscoelasticity, and thereby decrease the rate of release of the BAA from the suspension following administration and also increase the stabilization of the BAA, and increase the physical stability of the suspension as a whole (i.e., prevent phase separation). Such agents include polyvalent metal salts of organic acids, e.g., aluminum, zinc, magnesium or calcium salts of lauric acid, palmitic acid, stearic acid and the like, and oleaginous materials such as waxes and high viscosity oils and organic or inorganic fillers such as polymers and salts. Aluminum monostearate and distearate and white wax are particularly preferred agents. Said agents are usually present at concentrations (based on weight of oil) of between about 0.1% and about 99%, more typically between about 0.5% and about 90% and for metal salts even more typically 0.5% to 20%. This ratio is important for purposes of assuring that the agent doesn't increase the viscosity of the suspension to the point where the suspension is no longer useful for injection through a syringe. For highly viscous formulations, implants are also contemplated.
The glycerol/oil suspensions may further comprise surface active agents or emulsifiers to stabilize the glycerol/oil suspension and prevent it from separating. This surface active agent or emulsifier can be ionic or nonionic and may be selected from the group consisting of, for example, Span 40, Span 80, PLURONICS�, and egg lecithin, or mixtures thereof, preferably with a HLB (hydrophile-lipophile balance) of 1–10, more preferably 2–8, and even more preferably 4–8. The surfactant can also help dissipate the oil in the biological environment. The surfactant is usually present at 0.1% to 50%, preferably 0.2% to 20%, and more preferably 0.5% to 10% by weight of oil. Certain materials, such as hydrogenated vegetable oil can function as both a thickener and stabilizer of the glycerol suspension.
The BAA/glycerol/oil suspensions of the present invention can be prepared by suspending a biologically active agent (in powdered form) in a subtantially pure glycerol solution (at high glycerol concentrations, e.g., 70%–100%) to form a BAA/glycerol suspension, and then suspending said BAA/glycerol suspension in a solution comprising oil alone or oil containing a "gelling agent" suspended or dissolved in the oil. The oil (containing gelling agent) may first need to be heated (with mixing) to assure that the gelling agent completely dissolves in the oil.
Alternatively, the BAA/glycerol/oil suspensions of the present invention may also be prepared by mixing a biologically active agent (in solution form) in a subtantially pure glycerol solution (at high glycerol concentrations, e.g., 70%–100%) to form a BAA/glycerol solution, and then suspending said BAA/glycerol solution in a solution comprising oil alone or oil containing a "gelling agent" suspended or dissolved in the oil.
In general, comprehended by the invention are pharmaceutical compositions comprising effective amounts of biologically active agent, or derivative products (e.g., precipitates), together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, anti-oxidants (e.g., ascorbic acid and Vitamin E), adjuvants and/or carriers needed for administration. (See PCT 97/01331 hereby incorporated by reference.) The optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending upon the route of administration, desired dosage and duration of release. Exemplary pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, Pa., pgs. 1435–1712 (1990)). The pharmaceutical compositions of the present invention are particularly attractive for parenteral administration, e.g., by injection intramuscularly, subcutaneously, or intraperitoneally.
Therapeutic uses of the compositions of the present invention depend on the biologically active agent used. One skilled in the art will readily be able to adapt a desired biologically active agent to the present invention for its intended therapeutic uses. Therapeutic uses for such agents are set forth in greater detail in the following publications hereby incorporated by reference including drawings. Therapeutic uses include but are not limited to uses for proteins like granulocyte-colony stimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT Publication No. 94/17185, hereby incorporated by reference including drawings), interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293, hereby incorporated by reference including drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by reference including drawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby incorporated by reference including drawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby incorporated by reference including drawings), OB protein (see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby incorporated by reference including figures), novel erythropoiesis stimulating protein (PCT Publication No. 94/09257, hereby incorporated by reference including drawings), and small molecule drugs. In addition, the present compositions may also be used for manufacture of one or more medicaments for treatment or amelioration of the conditions the biologically active agent is intended to treat.
As specifically relates to G-CSF, the therapeutic has been shown to be effective in treating inflammatory bowel disease. For example, it has been reported that an adolescent boy with Crohn's disease and enterocutaneous fistulas had a response to treatment with G-CSF (filgrastim) after all standard treatments failed; Vaughn and Drumm, New England Journal of Medicine, 340(3):239–240 (1999). It has also been reported that prolonged high-dose therapy with G-CSF may have anti-inflammatory effects in colitis; Hommes et al., Clin Exp. Immunol., 106:529–533 (1996). It is thus envisioned that the G-CSF-containing suspensions of the present invention will also be effective in treatment of inflammatory bowel diseases.
One skilled in the art will be able to ascertain effective dosages by administration and observing the desired therapeutic effect. Preferably, for G-CSF, the formulation of the suspension will be such that between about 0.01 μg G-CSF moiety/kg body weight/day and 10 mg G-CSF moiety/kg body weight/day will yield the desired therapeutic effect. The effective dosages may be determined using diagnostic tools over time. For example, a diagnostic for measuring the amount of G-CSF in the blood (or plasma or serum) may first be used to determine endogenous levels of G-CSF protein. Such diagnostic tool may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of endogenous G-CSF protein is quantified initially, and a baseline is determined. The therapeutic dosages are determined as the quantification of endogenous and exogenous G-CSF protein moiety (that is, protein, analog or derivative found within the body, either self-produced or administered) is continued over the course of therapy. The dosages may therefore vary over the course of therapy, with, for example, a relatively high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits. Alternatively, the levels of neutrophils are determined and monitored over the course of the therapy. The dosage is adjusted to maintain the required level of neutrophil counts with the lowest frequency of injections.
The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.
This example describes the preparation of G-CSF powder by spray-drying.
G-CSF solution (˜2.75 mg/ml, with 5% sorbitol, in 0.58 mM HCl) was placed in dialysis tubing (Spectrum Lab Inc., flat width 18�2 mm, diameter 11.5 mm, 1.0 ml/cm), and dialyzed against water (pH 3.25) at 4� C. for 24 hours. During the dialysis, the water is changed four times. Dialyzed G-CSF solution (˜1100 ml) was then placed in an ultrafiltration cell and air pressure applied on the solution. After two hours, about 300 ml of concentrated G-CSF solution was collected and filtered through a 0.2 mm filter unit. The concentration of the final G-CSF solution is 9.134 mg/ml. The spray-drying was performed on a BUCHI 190 Mini Spray Dryer (Brinkmann Institute), and all of the glassware of the spray dryer was first washed with deionized water, followed by sterile water, followed by ethanol. The spray-drying was performed with inlet air flow of 450 normal liters/hour, and the feed rate of G-CSF solution was 1.0 ml/min. G-CSF powder (2.640 grams, 82.7% G-CSF) was obtained from the 290 mL starting G-CSF solution.
This example describes the preparation of G-CSF/glycerol suspensions and the use of the G-CSF/glycerol suspensions to prepare G-CSF/glycerol/oil formulations.
Step 1. A G-CSF/glycerol suspension was first prepared by placing 105.4 milligrams G-CSF spray-dried powder (prepared as described in Example 1) and 2.401 mL neat glycerol in a mortar and grinding the mixture until no course particles were seen.
Step 2. A thickened oil suspension was then prepared by placing 45.67 grams sesame oil (Croda, Inc.) and 1.91 grams aluminum monostearate (AIMS)(Fluka) in a 125 mL erlenmeyer flask and mixing with a magnetic stirrer at room temperature for 20 minutes, followed by heating at 165� C.–170� C. under nitrogen atmosphere with stirring. The stirring is continued for two hours, and the mixture then cooled to room temperature, resulting in an opalescent gel-like thickened oil (3% AIMS).
Step 3. One mL G-CSF/glycerol suspension and 4 mL thickened oil were placed in a mortar and ground together until well mixed. The suspension (G-CSF/20% glycerol/3% AIMS/oil) was stored in a sterile sample vial at 4� C. until needed.
This example describes the preparation of a G-CSF/glycerol-containing viscous oil suspension further containing L-ascorbic acid and surfactant.
L-Ascorbic acid (50 mg) was dissolved in a 1 mL glycerol solution by heating and stirring the mixture. After being cooled to room temperature, the ascorbic acid/glycerol solution was mixed with GCSF powder (45.3 mg) and Span 80 (250 mL).
3.75 mL thickened oil (3% AIMS) prepared as described above was added to the G-CSF/ascorbic acid/glycerol mixture and ground together to give a viscous oil suspension (G-CSF/20% glycerol+ascorbic acid/Span 80/3% AIMS/oil).
This example shows the preparation of an oil thickened with 7% white wax.
The thickened 7% wax/oil was produced (using the procedure described in Example 2, Step 2) by heating a mixture of white wax (4.49 grams) and sesame oil (59.65 grams) at 160� C. under nitrogen atmosphere for 2 hours.
This example shows the preparation of various G-CSF-containing oil formulations using 7% wax as thickener and with different glycerol levels.
Preparation 1: G-CSF powder (27.6 mg) and glycerol (600 μL) were mixed in a mortar and ground until no observable coarse particles were seen. Then 2.4 mL of the thickened 7% wax/oil prepared as described in Example 4 was added to the GCSF/glycerol suspension. The mixture was ground together with mortar and pestle to give a viscous oil formulation (G-CSF/20% glycerol/7% wax).
Preparation 2: GCSF powder (45.3 mg) was mixed with 1.00 ml of ascorbic acid/glycerol solution (prepared as described in Example 3), an then 4.0 mL of thickened 7% wax/oil was added. The resulting mixture was ground together to give a viscous oil formulation (G-CSF/20% glycerol+ascorbic acid/7% wax.
Preparation 3: G-CSF powder (27.3 mg) and glycerol (450 μL) were mixed in a mortar and ground until no observable coarse particles were seen. Then 2.55 mL of the thickened 7% wax/oil prepared as described in Example 4 was added to the GCSF/glycerol suspension. The mixture was ground together with mortar and pestle to give a viscous oil formulation (G-CSF/15% glycerol/7% wax).
Preparation 4: G-CSF powder (27.5 mg) and glycerol (750 μL) were mixed in a mortar and ground until no observable coarse particles were seen. Then 2.25 mL of the thickened 7% wax/oil prepared as described in Example 4 was added to the GCSF/glycerol suspension. The mixture was ground together with mortar and pestle to give a viscous oil formulation (G-CSF/25% glycerol/7% wax).
This example shows the preparation of an G-CSF/glycerol oil thickened with 10% white wax.
The thickened 10% wax/oil was produced (using the procedure described in Example 2, Step 2) by heating a mixture of white wax (6.5 grams) and sesame oil (58.5 grams) at 160� C. under nitrogen atmosphere for 2 hours.
GCSF powder (27.4 mg) and neat glycerol (600 μl) were mixed together, and then 2.40 mL of thickened oil (10% wax) was added to the GCSF/glycerol suspension. The mixture was ground to give a viscous oil formulation (G-CSF/20% glycerol/10% wax).
This example describes the in vivo testing of the suspensions prepared in Examples 2–6.
Splenectomized mice (BDF1) were injected once (subcutaneously) with 30 mg/kg of the various G-CSF-containing suspensions, and the various controls. The mice had their blood analyzed over several days. G-CSF powder (-glycerol) in 3% AIMS oil (30 mg/kg); G-CSF powder in glycerol (30 mg/kg); G-CSF powder dissolved in water (30 mg/kg); and 1�PBS were run as controls. The data is summarized in Table 1 below.
Neutrophil Count (106/mL)
Day 3
Day 5
Day 7
G-CSF in pH 3.25 water
(+5% sorbitol)
G-CSF in glycerol
G-CSF (no glycerol)
in 3% AIMS/oil
G-CSF/20% glycerol
3% AIMS/oil
G-CSF/20% glycerol
ascorbic acid/Span 80
3% AIMS/oil
G-CSF/20% glycerol
7% wax/oil
G-CSF/15% glycerol
7% wax/oil
G-CSF/25% glycerol
7% wax/oil
G-CSF/20% glycerol
10% wax/oil
As evidenced by the data in Table 1, the polyol/thickened oil suspensions are capable of providing for the sustained release of G-CSF for periods of at least one week. Importantly, it should be noted that G-CSF could not be delivered in the oils without the addition of the polyol.
This example shows the preparation of an oils thickened with glycerin stearate.
Preparation 1: Glycerol tristearate (1.00 gram), glycerol monostearate (4.00 grams), and sesame oil (45.00 grams) were placed in a bottle and heated at 160� C. under nitrogen atmosphere for 2 hours. The mixture was then cooled to room temperature while being vortexed. A white thickened oil was obtained.
Preparation 2: Glycerol monostearate (0.80 grams) and sesame oil (9.20 grams) were placed in a bottle and heated at 160� C. under nitrogen atmosphere for 2 hours. The mixture was then cooled to room temperature while being vortexed. A white thickened oil was obtained.
This example describes the preparation of thick oil using a mixture of sesame oil and the more viscous hydrogenated vegetable oil.
Sesame oil (6.00 mL) and hydrogenated vegetable oil (34.00 mL) were placed in a bottle and the mixture heated at 160� C. under nitrogen atmosphere for 2 hours. After the mixture cooled to room temperature, a thickened oil was obtained.
This example shows the preparation of G-CSF/glycerol in oil suspensions where the oil contains a mixture of sesame and hydrogenated vegetable oil and where the hydrogenated vegetable oil thickens the mixture.
Preparation 1: GCSF powder (10.0 mg) and neat glycerol (0.20 mL) were mixed, and then an oil mixture (hydrogenated oil/sesame oil=5/3, 0.80 mL) was added. The mixture was ground together with a mortar and pestle to give a viscous suspension formulation. This formulation was filled into a syringe and was syringable.
Preparation 2: GCSF powder (10.3 mg) and glycerol (0.20 mL) were mixed, and then an oil mixture (hydrogenated oil/sesame oil=3/17, 0.8 mL)) was added. The mixture was ground together with a mortar and pestle to give a viscous suspension formulation. This formulation was filled into a syringe and was syringable.
This example shows the preparation of a thickened oils using stearic acid, stearyl alcohol, and combinations thereof, as thickeners�G-CSF/glycerol.
Preparation 1: Stearic acid (1.00 gram) and sesame oil (9.00 grams) were placed in a bottle and the mixture heated at 160� C. under nitrogen atmosphere for 2 hours. After cooling to room temperature with shaking the mixture became a viscous thickened oil.
Preparation 2: Stearyl alcohol (1.00 gram) and sesame oil (9.00 grams) were placed in a bottle and the mixture heated at 160� C. under a nitrogen atmosphere for 2 hours. After cooling to room temperature with shaking the mixture became a viscous thickened oil.
Preparation 3: Stearyl alcohol (0.50 grams), stearic acid (0.50 grams), and sesame oil (9.00 grams) are placed in a bottle and the mixture heated at 160� C. under nitrogen atmosphere for 2 hours. After cooling to room temperature with shaking the mixture became a viscous thickened oil.
Preparation 4: G-CSF powder (9.8 mg) and neat glycerol (0.20 mL) were mixed and then 0.80 mL of thickened oil (10% stearyl alcohol) was added. The mixture was ground for 10 minutes to give an oil formulation which was filled into a 1 mL syringe and was syringable.
Preparation 5: G-CSF powder (10.3 mg) and neat glycerol (0.20 mL) were mixed and then 0.80 mL of thickened oil (10% thickener, stearyl alcohol/stearic acid=3/1) was added. The mixture was ground for 10 minutes to give an oil formulation which was filled into a 1 mL syringe and was syringable.
This example shows the preparation of G-CSF/glycerol/oil emulsion formulations wherein G-CSF was first admixed with an aqueous glycerol (50% glycerol/50% water) phase.
The resultant G-CSF/glycerol phase consisted of 12.7 mg/mL G-CSF, 50% glycerol, 1%(w/v) Pluronic F68, 10 mM acetate (pH 4.0) and 0.44 mM HCl. A mixture of 1% Pluronic L101 in corn oil formed the oil phase. A 50:50 and 70:30 mixture of the two phases were homogenized with a Virtis Handishear homogenizer for 45 seconds to form the respective emulsion formulations.
This example is prepared in a similar manner to Example 2 except the G-CSF dose is approximately 10 mg/Kg. After a single injection the neutrophils were elevated for at least one week.
This example describes the preparation of various G-CSF/glycerol/oil suspensions wherein G-CSF solutions were mixed with various glycerol solutions to prepare G-CSF/glycerol mixtures that were then suspended in thickened oils.
Concentrated G-CSF solution (e.g., 200 mg/mL) was prepared by concentrating bulk G-CSF solution using a Microcon centrifugal filter device. Volumes of neat glycerol were added to prepare G-CSF/glycerol solutions of varying concentrations of G-CSF and glycerol. Excipients such as methionine were also added to a couple of the G-CSF/glycerol solutions. G-CSF/glycerol/thickened oil formulations were then prepared in a similar manner to that described in Example 6. A G-CSF/glycerol/oil emulsion formulation was also prepared in a similar manner to that described in Example 12 and comprising an aqueous phase (pH 3) consisting of 15 mg/mL G-CSF, 30% glycerol and 2.5% Pluronic F68; and an oil phase consisting of 2% aluminum monostearate and 2.5% Pluronic L101; and wherein the ratio of the oil phase to aqueous phase was 80%/20% (v/v).
Various formulations were then prepared and tested in vivo as described above. The injections (100 μL) in the splenectomized mice were at 6 mg/kg, except for the emulsion formulation, which was at 20 mg/kg. The data is summarized in Table 2 below.
TABLE 2 Neutrophil Count (106/mL) Formulation Day 3 Day 5 Day 7 Carrier, 1X PBS 1–2 1–2 1–2 G-CSF in neat glycerol 27.4 17.4 3.1 G-CSF in 90% glycerol/10% H2O 26.9 44.7 G-CSF in 75% glycerol/25% H2O 26.4 33.1 G-CSF in 60% glycerol/40% H2O 24.4 32.0 G-CSF in 10% glycerol/90% H2O similar to carrier (Note: 83% oil phase) G-CSF in 90% glycerol/10% H2O ~22 ~36 ~10 0.5 mM methionine G-CSF in 70% glycerol/30% H2O ~13.5 ~42 ~7 1.0 mM methionine G-CSF in 30% glycerol similar to vehicle control Emulsion without G-CSF
As evidenced from the Table 2 data, G-CSF solution is soluble in high concentrations of glycerol and stabilized such that suspensions which provide sustained release of G-CSF for a period of at least 5 days can be prepared. The addition of methionine as an excipient also seems to provide added stability which appears to allow for lower concentrations of glycerol to be used.
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|Monoclonal antibodies to stem cell factor receptors
|Jun 16, 1993
|Jan 6, 1994
|High and medium power radioelectric filter
|Oct 8, 1993
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|Kabushiki Kaisha Iseki Kaihatsu Koki
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|Amgen Inc.
|G-csf analog compositions and methods
|Dec 21, 1994
|Jun 29, 1995
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|Lyophilized stem cell factor formulations
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|Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
|Dec 12, 1995
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|May 30, 1996
|Dec 19, 1996
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|Method for manufacturing cobalt catalysts
|Jun 18, 1996
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|Opening arrangements and methods for closure members
|Jun 26, 1996
|Jan 16, 1997
|Takeda Chemical Industries, Ltd.
|Method of producing sustained-release preparation
|Aug 2, 1996
|Feb 27, 1997
|Amgen Inc.
|Methods of reducing or maintaining reduced levels of blood lipids using ob protein compositions
|Dec 20, 1996
|Jul 3, 1997
|Amgen Inc.
|Creighton, et al., Proteins, passim (W.H. Freeman and Company, N.Y. 1984).
|Ford, et al., "Fusion Tails for the Recovery and Purification of Recombinant Proteins", Protein Expression and Purification, vol. 2, pp. 95-107, (1991).
|Hutchinson, et al., "Biodegradable Polymers for the Sustained Release of Peptides", Biochemistry Social Trans., vol. 13, pp. 520-523, (1985).
|Lu, et al., "Folding and Oxidation of Recombinant Human Granulocyte Colony Stimulating Factor Produced in Escherichia coli", The Journal of Biological Chemistry, vol. 267 (13), pp. 8870-8777, (1992).
|Sims et al., J. American Oil Chemists' Soc., 1977, 54, No. 1, pp. 4-7.
|Wise, et al., "Polylactic Acid as a Biodegradable Carrier for Contraceptive Steroids", Contraception, vol. 8(3), pp. 227-234, (1973).
|Citing Patent
|Filing date
|Publication date
|Oct 27, 2009
|Dec 25, 2012
|Nora Therapeutics, Inc.
|Method for reducing the risk of spontaneous abortion in a human female subject
|Apr 27, 2012
|Jul 9, 2013
|Nora Therapeutics Inc.
|Compositions and methods for reducing the risk of preterm labor
|Jan 3, 2012
|Jul 23, 2013
|Nora Therapeutics, Inc.
|Methods for reducing the risk of an unsuccessful pregnancy in a subject having an immune system abberation
|Jun 24, 2013
|Jul 21, 2015
|Nora Therapeutics, Inc.
|Methods for reducing the risk of spontaneous abortion in a human female comprising administering an effective amount of G-CSF
|Jul 7, 2015
|Apr 26, 2016
|Thomas Julius Borody
|Probiotic recolonisation therapy
|Jul 7, 2015
|Aug 9, 2016
|Crestovo Llc
|Probiotic recolonisation therapy
|Jul 7, 2015
|Oct 18, 2016
|Crestovo Llc
|Probiotic recolonisation therapy
|Feb 5, 2016
|Feb 21, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Jun 9, 2016
|Feb 21, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Jun 9, 2016
|Apr 4, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Sep 7, 2016
|Apr 18, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Sep 9, 2016
|May 16, 2017
|National Institutes of Health (NIH); U.S. Department of Health and Human Services (DHHS); The United States of America, NIH Division of Extramural Inventions and Tehnology Resources (DEITR)
|Compositions and methods for transplantation of colon microbiota
|Sep 8, 2016
|Jun 20, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Feb 5, 2016
|Aug 22, 2017
|Crestovo Llc
|Probiotic recolonisation therapy
|Oct 27, 2009
|Apr 1, 2010
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|Compositions and methods for reducing the likelihood of implantation failure or spontaneous abortion in recipients of assisted reproduction
|Mar 1, 2010
|Jun 24, 2010
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|Method for reducing the likelihood of preterm labor in a subject in need thereof
|Nov 22, 2010
|Apr 28, 2011
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|Methods and kits for reducing the likelihood of implantation failure and pregnancy-related disorders in recipients of artificial insemination
|May 19, 2011
|Sep 15, 2011
|Thomas Julius Borody
|Electrolyte purgative
|U.S. Classification
|424/85.1, 424/85.4, 530/399, 514/965, 514/964, 530/350
|International Classification
|Cooperative Classification
|Nov 28, 2000
Owner name: AMGEN INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GOLDENBERG, MERRILL;BEEKMAN, ALICE;SHAN, DAXIAN;AND OTHERS;REEL/FRAME:011373/0980
Effective date: 20001128
|Jul 6, 2009
|Maintenance fee reminder mailed
|Dec 27, 2009
|Lapse for failure to pay maintenance fees
|Feb 16, 2010
|Expired due to failure to pay maintenance fee
Effective date: 20091227


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Martins was stretchered off during his debut at Aston Villa but should be fit by the time the Premiership resumes after the international matches.
"He's got a heavy dead leg," said the Magpies manager.
"That's probably the best it could have been from where I was standing because I was fearing the worst when he went off on a stretcher."
Roeder already has striker Michael Owen out with a long-term cruciate knee injury and has a shortage of strikers at the club.
He added: "I didn't know whether it was a knee injury of significance or not so to suffer a dead leg that will take 10-12 days to heal - I can handle that."
Roeder also revealed he is set to add to his squad to create much-needed competition for places after he felt certain players under-performed against Villa.
"There were periods where we played well and periods where we did not and I thought there were a few players who disappointed me," he said.
"I wasn't satisfied with two or three performances and I've never had to say that before.
"But I've got two weeks to sort that out and, hopefully, before Thursday there will be one or two more new players in at the club as well.
"You can never say it will definitely work out but we have been working very hard in the background."


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Thousands of online Visa card transactions may have gone unprocessed after the system failed at 5.10am. Customers who tried to make transactions saw the error code: '6: Bank Did Not Reply'.
Visa's backup measures in the UK enabled the system to recover three hours after it failed. The company is now carrying out diagnostics.
The credit card giant told customers in an email: "During this [system failure] time period, authorisation requests on some Visa branded cards did not receive any bank response."
Visa admitted that some transactions would not have been processed, but could not say how many. The company said that backup measures had enabled the system to recover.
Europe's 315 million Visa debit, credit and commercial cards were used to make purchases and cash withdrawals of more than £8.1bn in 2006.
Visa failure hits online shoppers
By Jane Hoskyn on Apr 17, 2007 10:06AM


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The ever-evolving fresh-meals business has given rise to strong new partnerships between retailers and manufacturers, who are working together in unprecedented ways to woo meals customers away from restaurants and into the supermarket.
Supermarket executives are recognizing the value of dealing directly with manufacturers to source fresh entrees and other meal components, and national brand manufacturers are bringing advertising money to the table.
Some retailers, trying to do


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Bill Shorten on the Labor leadership and future of the party
From joining the Labor Party at the age of 17 to competing for its leadership with Anthony Albanese, Bill Shorten reflects on his background and discusses his vision for its future.
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"Being in the Labor Party is probably the thing I've done in the longest, continuous fashion of my adult life - 29 years...I was always interested in politics, history, interested in the way things are set up."
From his childhood years and early interest in politics to his current bid for the Labor leadership, Bill Shorten joined Richard Glover in the 702 studio on Friday to discuss his past, future and that of the Labor Party.
Asked about the highlights during Labor's years in office, Mr Shorten made mention of various policies and initiatives spanning the Whitlam era to now and said his focus was on the years to come.
"I'm a middle of the road person, I'm left of centre but not greatly left of centre. What I get is that people work best when they're working together."
"I liked Keating's superannuation, the idea that we can save for our retirement and control it ourselves. I liked Keating and Hawke's expansion into Asia.
"But I also like what we've done in the Rudd-Gillard era too, the apology to the Stolen Generations, putting a price on carbon pollution, helping navigate the Global Financial Crisis.
"For me, the challenge isn't even the golden periods - be it Whitlam with higher education, be it those Hawke/Keating achievements, be it Rudd and Gillard's achievements, for me the challenge for the Labor Party is, what will this country look like in 2020 and 2030?"
Describing the ballot as "great, dynamic and exciting", Mr Shorten offered the following response to a question on policy differences and political stances between himself and Mr Albanese.
"I won't say anything bad about Anthony. If he gets elected, he'll do a good job, I have no doubt."
"What I think I bring to this proposition is not some examination of how left one is and some sort of ideological purity. What I bring to the proposition, I believe, is I'm interested in what we look like in 2020 and 2030."
Hear the full interview with Mr Shorten by locating the audio to the right.
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Lilli Thal was educated in medieval history, information technology, and multimedia before beginning her freelance writing career in 2002. That same year she won the Martin award for the best children's mystery story. She lives and works from her home in Germany.
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Senior Quantitative Research Analyst
Senior Quantitative Analyst
We are currently seeking a Senior Quantitative Analyst to join our Research Community. The Analyst position at CEB is an entry point for individuals interested in learning strategic research—the foundation for any business advisory, strategic planning or research-oriented career. Senior Quantitative Analysts enjoy exposure to the full array of strategic thought work: primary and secondary research; benchmarking and norming surveys; predictive modeling; and proprietary research exercises focused on peeling apart ideas in search of insight. Importantly, as an entry point position at CEB, the Analyst role is a key feeder to careers reaching the highest levels of the firm.
Key Responsibilities
- Analyzing large data sets to identify actionable insights for senior executives
- Assisting with large-scale data collection efforts across member organizations and with external vendors
- Employing analytical tools, such as root cause analysis, to identify the drivers of members' business problems and to create hypotheses regarding solutions
- Contributing to the design of quantitative research projects, including writing surveys to test hypotheses
- Communicating and presenting analytical insights to internal partners
- Coordinating and participating in interviews with executives, academics, and industry experts to scope quant studies
Role Qualifications
- 1-2 years of experience and an undergraduate degree with training in quantitative social science fields, such as: Economics, Econometrics, Applied Microeconomics, Public Policy, Political Science, or Sociology
- Proficiency with basic statistical concepts (an advanced degree with a significant focus on application may be considered in lieu of work experience)
- Proficiency in at least one statistical software package (SAS, Stata, SPSS, etc.) and advanced knowledge of Microsoft Excel
- Excellent oral communication, writing, and graphics skills
- Original/creative thinking and superior problem solving abilities
- Ability to work independently and as part of a team
- Strong project management and time management skills
- Detail-oriented and strong commitment to high quality results to serve needs of CEB members and internal partners
- Self-motivated/self-starter approach to projects
- Computer programming knowledge/experience a plus
CEB is an Equal Opportunity Employer committed to diversity in the workplace. All qualified applicants will receive consideration for employment without regard to sex, race, color, age, national origin, religion, disability, genetic information, marital status, sexual orientation, gender identity/assignment, citizenship, pregnancy or maternity, protected veteran status, or any other status protected by applicable national, federal, state or local law. CEB promotes a drug-free workplace. CEB will consider for employment qualified applicants with a criminal history in a manner consistent with the requirements of applicable laws regarding criminal background inquiries.
Meet Some of CEB's Employees
Andrew M.
Business Development Associate
Andrew works with his team to find and reach out to new business prospects for CEB. He interfaces with Fortune 500 companies across the world—and gets them on board with CEB's solutions.
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