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The present invention relates to a method of treating a patient with visco-surgery, particularly ocular visco-surgery, utilizing a collagen-based viscoelastic solution having a reversibly high viscosity. The invention also relates to collagen-based viscoelastic solutions which further include mucopolysaccarides or growth factors.
1. A viscoelastic solution comprising an acylated soluble collagen and a mucopolysaccharide in a saline physiologic diluent having an ionic strength of no more than 0.001μ, said collagen and mucopolysaccharide being in a weight ratio of collagen to mucopolysaccharide of between about 100:1 and about 20:1, and said solution having a kinetic viscosity ranging from 1,000 cps to 800,000 cps, said solution having an osmolarity of between about 260 to 340 milliosmoles, with about 75 to about 95% of the osmolarity of said solution provided by nonionic solutes.
2. The viscoelastic solution according to claim 1 wherein said mucopolysaccharide is selected from the group consisting of chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, salts of hyaluronic acid, keratan sulfate, heparin sulfate and mixtures thereof.
3. The viscoelastic solution according to claim 1 wherein said nonionic solute is selected from the group consisting of glycerol, sorbitol, xylitol, threitol, mannitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, heptose, hexoses, pentoses and mixtures thereof.
4. The viscoelastic solution of claim 3 wherein said nonionic solute is glycerol.
5. The viscoelastic solution according to claim 1 wherein the kinetic viscosity ranges from 45,000 cps to 80,000 cps.
6. The viscoelastic solution according to claim 1 further comprising a growth factor in a concentration ranging from 10 ng/ml to 10,000 ng/ml.
7. The viscoelastic solution according to claim 6 wherein the growth factor is selected from the group consisting of growth hormone, interleukins, epidermal growth factors, fibroblast growth factors, insulin-like growth factors and mixtures thereof.
8. A collagen-based viscoelastic solution comprising acylated soluble collagen and a growth factor dissolved in a saline physiologic diluent, said growth factor having a concentration ranging from 10 ng/ml to 10,000 ng/ml, and said diluent having an ionic strength of no more than 0.001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, wherein about 75 to about 95% of the osmolarity of said solution is provided by nonionic solutes.
9. A method of treating a patient comprising injection of a viscoelastic composition comprising an acylated soluble collagen in a saline physiologic buffer to separate and protect tissues being treated, said saline physiologic buffer having an ionic strength of no more than 0.001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, wherein about 75 to about 95% of the osmolarity of said solution is provided by nonionic solutes.
10. The method of claim 9 wherein said nonionic solute is selected from the group consisting of glycerol, sorbitol, xylitol, threitol, mannitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, heptose, hexoses, pentoses and mixtures thereof.
11. The method of claim 10 wherein said nonionic solute is glycerol.
12. The method of claim 9 wherein the kinetic viscosity of said viscoelastic composition is between about 1,000 cps and 800,000 cps.
13. The method of claim 12 wherein the kinetic viscosity of said viscoelastic composition is between about 45,000 cps and 85,000 cps.
14. The method of claim 9 wherein the kinetic viscosity of said viscoelastic composition drops at least 88% upon the addition of salt to increase the ionic strength to at least 0.0022μ.
15. The method of claim 9 wherein said viscoelastic composition further comprises a mucopolysaccharide and wherein the weight ratio of said collagen to said mucopolysaccharide ranges between about 100:1 and about 20:1.
16. The method of claim 15 wherein said mucopolysaccharide is chondroitin sulfate.
17. The method of claim 9 wherein said viscoelastic composition further comprises between about 10 ng/ml and about 10,000 ng/ml of a growth factor.
18. A method of inserting an intraocular lens into the posterior or anterior chamber of a human or animal eye comprising:
introducing into said anterior or posterior chamber an effective amount of a viscoelastic solution comprising acylated soluble collagen and a saline physiologic diluent having an ionic strength of no more than 0,001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, wherein about 75 to about 95% of the osmolarity Of said solution is provided by nonionic solutes,
said amount sufficient to reduce the trauma which normally results from inserting an intraocular lens into said chamber in the absence of a viscoelastic solution, and inserting said lens into said chamber.
19. The method of claim 18 further comprising a physiologic saline irrigation step following said insertion, said irrigation sufficient to remove substantially all of said viscoelastic solution.
20. A process of preparing a viscoelastic solution consisting essentially of reconstituting acylated soluble collagen in a saline physiologic diluent having an ionic strength of no more than 0.001μ, said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, wherein about 75 to about 95% of the osmolarity of said solution is provided by nonionic solutes.
21. A viscoelastic solution consisting essentially of acylated soluble collagen and a mucopolysaccharide in a saline physiologic diluent having an ionic strength of no more than 0.001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, and a kinetic viscosity from 45,000 cps to 80,000 cps wherein bout 75 to about 95% of the osmolarity of said solution is provided by nonionic solutes.
22. A viscoelastic solution consisting essentially of acylated soluble collagen in a saline physiologic diluent having an ionic strength of no more than 0,001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, and a kinetic viscosity ranging from 45,000 cps to 80,000 cps, wherein bout 75 to about 95% of the osmolarity of aid solution is provided by nonionic solutes.
23. A method of treating a patient with visco-surgery comprising injection of a viscoelastic composition of soluble collagen in a saline physiologic buffer to separate and protect tissues being treated, said saline physiologic buffer having an ionic strength of no more than 0.001μ and said solution having an osmolarity ranging from 200 m osmol to 400 m osmol, wherein about 75 to about 95% of the osmolarity of said solution is provided by nonionic solutes.
This is a continuation, of Ser. No. 07/939,955, filed Sep. 3, 1992, now abandoned, which is a continuation of application Ser. No. 07/547,458, filed Jul. 3, 1990, now abandoned.
The present invention relates to viscoelastic solutions of collagen suitable for use in visco-surgery, particularly ophthalmic visco-surgery. Specifically, the present invention relates to such viscoelastic solutions wherein their viscosity is dramatically reduced upon the addition of solutions containing cations, such as saline solution, balanced salt solution, etc.
Aqueous lubrication solutions have widespread use in ophthalmic surgery as protective agents for the easily damaged intraocular tissues. A major concern in ophthalmic surgery has been the frequent observation of elevated intraocular pressure associated with the surgical use of viscoelastic solutions. Elevated intraocular pressure can reduce the flow of blood-born oxygen and nutrients to the eye and to the optic nerve. Not surprisingly, such elevated pressure is strongly correlated with loss of vision (glaucoma). It is, thus, desirable that solutions for ophthalmic surgery be easily removed from eye tissues by irrigation and aspiration. It is further desirable that any residues remaining in the eye show reduced tendency to induce elevated ocular pressure.
A number of solutions have been used or proposed for use as viscoelastic solutions These include solutions of sodium hyaluronate (Healon ™, Phamacia LKB Biotechnology, Inc. and Amvisc™, IOLAB); chondroitin sulfate; mixtures with chondroitin sulfate with sodium hyaluronate (Viscoat™, Cooper-vision/ALCON); methylcellulose; carboxymethylcellulose; polyacrylamide or polymethacrylamide; and collagen derivatives. A number of these solution are reviewed by Arshinoff, Curr. Can. Ophthalmic Prac. 4:64-74, 1986.
Sodium hyaluronate solutions are the most widely used viscoelastic solutions for ophthalmic surgery Balazs (U.S. Pat. No. 4,141,973) or Pape (U.S. Pat. No. 4,328,803) describe their manufacture and/or use. These solutions, however, have been found to induce transient elevations in intraocular pressure. They have the further disadvantage of being expensive and failing to adhere to ocular prostheses and surgical instruments.
Chondroitin sulfate solutions are not pseudoplastic (and not viscoelastic) and consequently are difficult to inject through the narrow gauge needles used in ophthalmic surgery (see MacRae et al., "The Effects of Sodium Hyaluronate, Chondroitin Sulfate and Methyl Cellulose on the Corneal Endothelium and Intraocular Pressure," Amer. J. Ophthal. 95:332-341, 1983). Additionally, these solutions are less effective than sodium hyaluronate solutions in maintaining space between separated ocular tissues Finally, such solutions are associated with sharp elevations in intraocular pressure.
Unmodified collagen is usually unsuitable for ophthalmic solutions due to its low solubility at pH values close to neutral pH. However, Devore et al. (U.S. Pat. Nos. 4,713,446 and 4,851,513) and Miyata et al. (U.S. Pat. No. 4,748,152) have developed collagen-based viscoelastic solutions wherein the collagen is derivatized at lysyl amino acids to increase its solubility at neutral pH.
Miyata et al. (supra) teach partially succinylated ateleopeptide collagen dissolved in physiologic saline. The viscosities taught therein are modest (highest is 10,000 centipoise for a 3% solution). Miyata et al. have no teachings relating to a collagen solution that is readily irrigated out of eye tissues. Nor are there any disclosures in Miyata et al. of the effects of the solution on intraocular pressure.
Devore et al. (supra) teach a method of producing collagen suitable for viscoelastic solutions by acylating the lysyl amino acids of collagen with a combination of monofunctional and bifunctional reagents. The result of this acylation treatment is that some collagen lysine amino groups are modified to substitute a carboxylic acid group in place of the basic amino function. The residue of the lysine amino groups are covalently linked to lysine amino groups of the same or an adjacent collagen molecule. The viscoelastic solution is produced by reconstituting this derivatized collagen in a physiologic saline solution. These are no disclosures in the Devore et al. patents of collagen viscoelastic solutions that are readily irrigated out of ocular cavities. Neither is there any disclosure of the effects of the collagen solutions taught therein on intraocular pressure.
Desirable properties of viscoelastic solutions include: transparency; stability (i.e. they maintain their useful properties over long term storage); high viscosity; pseudoplastiticity; viscoelasticity; thixotropy; and biological compatibility.
Pseudoplastic solutions are useful in visco-surgery because their viscosity drops when they are subjected to high shear flow, such as occurs during injection through the high gauge (narrow) needles used in ophthalmic surgery. (Pseudoplastic fluids are non-Newtonian fluids wherein the kinetic viscosity (centipoise-sec or cps) decreases as the sheer rate increases.) Thus, pseudoplastic surgical solutions are injectable despite their viscosity.
Viscous solutions are useful due to their tendency to resist flowing out of the ocular cavity into which they have been injected.
Viscoelastic solutions are useful in viscosurgery because they resist having their shape deformed (i.e., they are elastic), thus helping to maintain the shape of body cavities into which the solutions are injected. (The elasticity of the solution results in the Weissenberg Effect, which describes the tendency of viscoelastic solutions to flow at right angles to an applied force.) During surgery, viscoelastic materials protect cell and tissue surfaces from mechanical trauma; create space by separating two adjacent but not adherent tissue surfaces, or by breaking normal or pathological tissue adhesions; maintain space, for instance, in the interior chambers of the eye or in the lens sack, allowing for safe surgical manipulations by permitting the insertion of implants without dislocating or touching sensitive tissues; contain hemorrhages; and also act as a "soft instrument" or "surgical tool" to move, manipulate or relocate tissues.
Following injection of a viscoelastic solution, at high shear, through a narrow gauge needle (e.g. into a chamber of the eye), it is useful for the solution to regain the majority of its viscosity. Solutions having this recovery property are "thixotropic."
A biocompatible substance, as used herein, shall be noninflammatory, nontoxic, nonimmunogenic, pH buffered and shall have osmolarity between about 200 milliosmoles and about 400 milliosmoles, preferably between about 250 milliosmoles and about 350 milliosmoles, most preferably between about 280 milliosmoles and about 330 milliosmoles.
What is needed in the art is a viscoelastic solution that has transparency, stability, high viscosity, pseudoplasticity, viscoelasticity, thixotropy, and biological compatibility, but moreover, can be used in ophthalmic surgery with reduced risk of elevated intraocular pressure.
The present inventors have unexpectedly discovered that soluble collagen, when reconstituted in low ionic strength buffers, shows markedly increased viscosity and continues to have viscoelastic properties. This increased viscosity is maintained as the osmolarity of the solution is increased using nonionic solutes.
By this invention, high viscosity solutions useful for ophthalmic surgery (or any visco-surgery involving delicate tissue in need of protection) can be prepared at lower collagen concentrations. The viscosity of the solution can, subsequently, be dramatically lowered by simply diluting or irrigating with saline solutions. Thus, the viscoelastic solutions of the present invention are easily irrigated out of ocular cavities following ophthalmic surgery. Therefore, their use reduces the risk of elevated intraocular pressure associated with residues of viscoelastic solution remaining in the eye following surgery.
It has been discovered that viscoelastic solutions according to the present invention reduce the risk of elevated ocular pressure even when they are not washed out of ocular spaces.
The addition of mucopolysaccoharides, such as chondroitin sulfate, and growth factors, such as epidermal growth factor (EGF) or growth hormone (GH), to the viscoelastic solutions used in the present invention is expected to provide further benefits.
The viscoelastic collagen solutions of the present invention have osmolarities ranging between about 260 and 340 milliosmoles, about 75% to about 95% of the osmolarity provided by nonionic solutes such as glycerol.
Such solutions have been found to have higher viscosities or the same viscosity at lower concentrations of soluble collagen then comparable collagen solutions dissolved in physiologic saline solutions. Collagen suitable for use in the present invention can be prepared by reacting collagen (at its lysyl amino acids) with an acylating agent, e.g., glutaric anhydride, succinic anhydride, heptafluorobutyric anhydride, methacrylic anhydride, phthalic anhydride, methyl glutaric anhydride, dimethyl glutaric anhydride, benzene sulfonic acid, β-styrene sulfonyl chloride, hydroquinone sulfonic acid, polyvinyl sulfonic acid, etc. For instance: ##STR1## where P is the collagen backbone. The above reaction reverses the ionic charge at physiologic pH of the reacted lysyl amino acids (plus to minus). The reaction also increases the solubility at physiologic pH of the collagen product.
Such collagen can be reconstituted (following intermediate processing steps) in sodium phosphate buffer to give a kinetic viscosity of 30,000 cps (at 1 sec-1) and dialyzed against distilled water. After 24 to 72 hours of dialysis, the viscosity is dramatically increased (e.g. 200,000 cps). The viscosity of the solution can be reduced to about 60,000 cps by dilution with distilled water. The osmolarity of such a solution of collagen can be increased to the physiologic range (205 to 405 milliosmoles) by addition of a nonionic solute, such as glycerol, without substantial loss of viscosity. However, the addition of salt to increase the ionic strength to at least 0.0022μ produces a drop in viscosity of as much as 88% (with dilution effects accounting for none of the viscosity drop). Preferably, the addition of salt to increase the ionic to 0.0043μ produces a drop in viscosity of at least 50 fold. The dialysis step is not essential to the invention. Comparable high, reversible viscosities are seen with collagen solutions that are reconstituted simply by dissolving precipitated or lyophilized collagen in low saline buffer.
It is known in the prior art that dialysis of collagen against water results in long strands of end-to-end associate collagen molecules (see Bruns et al., U.S. Pat. No. 4,581,030). Such end-to-end association results in increased effective molecular weight and some associated increase in viscosity. While not wanting to be restricted to theory, this previously observed phenomenon does not adequately explain the dramatic viscosity effects described herein. The dramatic increase in viscosity seen in the solutions of the present invention (e.g., 30,000 cps increased to 200,000 cps) suggest more complex, multiple interactions such as seen for hyaluronate solutions (see Balasz, E., Sodium Hyaluronate and Viscosurgery, Healon, Miller, D. and Stegman, R., eds. John Wiley & Sons, New York, 1983).
The viscoelastic collagen solutions of the present invention are prepared by reconstitution of collagen in a low saline physiologic buffer. "Reconstitution," as used herein, means any method of obtaining collagen dissolved in a desired buffer. This may be done by a number of methods including:
1) Dialysis to exchange the buffer dissolving collagen for the desired buffer.
2) Redissolving precipitated collagen in the desired buffer.
3) Dilution of a collagen solution with a buffer concentrate to obtain the desired buffer composition.
As used herein, "low saline" buffer shall mean buffer having ionic solutes that yield no more than about 0.001μ of the ionic strength, preferably no more than about 0.0008μ. A "low saline physiologic" buffer shall meet the above saline requirement and have nonionic solutes for a total osmolarity between about 200 and about 400 milliosmoles, preferably between about 250 and about 350 milliosmoles, most preferably between about 280 and about 330 milliosmoles.
Appropriate nonionic solutes include glycerol, sorbitol, xylitol, threitol, mannitol, arabitol, ribitol, adonitol, erythritol, dulcitol, altritol, iditol, various heptoses, hexoses and pentoses, polyglycols, etc.
Since mucopolysaccarides such as chondriotin sulfates (e.g., chondroitin sulfate A, B or C) adhere to endothelial cells, it is anticipated that the addition of mucopolysaccarides to the viscoelastic collagen solutions will also increase the adherence of the solution. This increase in adherence should enhance the protection of endothelial tissues (such as the tissues lining the ocular cavities). While the source of the affinity of such mucopolysaccharides for endothelial cells is not completely understood, it may, in part, be due to the receptors for mucopolysaccharides which are present on the endothelium (Lutjen-Drecoll, E., ARVO Annual Meeting Abstacts, Apr. 29-May 4, 1990, Association for Research in Vision and Opthamology, publisher, Vol. 31(4), p. 184). Other mucopolysaccarides which should be useful adherence promoting additives to the viscoelastic collagen solutions are salts of hyaluronic acid, dermatan sulfate (also known as chondroitin sulfate B), keratan sulfate, heparan sulfate, etc. A useful range of collagen to mucopolysaccaride weight ratios is anticipated to be between about 100:1 and about 10:1, more preferably between about 40:1 and about 20:1.
Hyaluronic acid is available from Sigma Chemical Co., St. Louis, MO (from human umbilical cord, bovine vitreous and rooster comb) and from Calbiochem Biochemicals, San Francisco, Calif. (from human umbilical cord). Chondroitin sulfate A is available from Sigma Chemical Co. (from bovine trachea) and Calbiochem (from bovine trachea). Chondroitin sulfate B is available from Sigma Chemical Co. (from bovine mucosa, porcine skin) and Calbiochem (from porcine mucosa). Chondroitin sulfate C is available from Sigma Chemical Co. (from shark cartilage) and Calbiochem (from shark cartilage). Keratan sulfate is available from Sigma Chemical Co. (from bovine cornea) as is heparan sulfate (from bovine intestinal mucosa and bovine kidney). A number of these mucopolysaccarides are also available from Seikagaku Kogoyo Co., Ltd., Tokyo, Japan.
Growth factors (such as EGF, transforming growth factors (TGFs) GH, interleukins, fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), insulin-like growth factors (IGFs), etc.) may be added to the collagen viscoelastic solution. These growth factors are anticipated to enhance the viability of the tissues manipulated and exposed during the course of visco-surgery. The amount of growth factor added to the viscoelastic solution of the present invention will vary with the specific growth factor, but will generally be in the range of 10-1,000 ng/ml. The range for EGF is anticipated to be between about 1 ng/ml and about 100,000 ng/ml; for FGFs between about 10 ng/ml and about 500 ng/ml; and for PDGFs and IGFs between about 10 ng/ml and 10,000 ng/ml.
Growth factors have been found to enhance the rate of wound healing in many tissues, including corneal tissues (see Dijke et al., Bio/Technology, 7:793-798, 1989). Accordingly, it is anticipated that growth factors will enhance the rate of recovery of corneal tissue from the trauma of viscosurgery. For example, EGF has been shown to increase corneal reepithelialization and to stimulate corneal cell-proliferation and migration. In addition, preliminary data suggests that EGF enhances corneal reinnervation and stimulates into corneal endothelial cell-proliferation.
EGF is available from Amgen, Thousand Oaks, CA and Sigma Chemical Co.; fibroblast growth factors are available from Amgen Biochemicals and R&D Systems, Oxford, England; transforming growth factors are available from R&D Systems; and PGFs are available from R&D Systems and Collaborative Research, Inc., Medford, Mass. Interleukins are available from R&D Systems and Collaborative Research, Inc.; IGFs are available from R&D Systems; and GH is available from Cambridge Medical Technologies Corp., Billerica, Mass. and Boehringer Mannheim Corp., Indianapolis, Ind.
The pH of the viscoelastic collagen solution is preferably stabilized generally by between about 5 mM and about 50 mM of buffer. Buffers that are usable with the invention are any that are biologically compatible. These include phosphate buffers, carbonate buffers, and phosphate-bicarbonate.
The saline solutions that may be used to reduce the viscosity of the viscoelastic solutions of the present invention include any of the numerous such solutions that are used in medicine or biology research for non-disruptive manipulation of tissues and cells. These include numerous phosphate buffered saline solutions (e.g., 0.84 percent by weight NaCl, 0.054 by weight KCl, 0.028 percent by weight Na2 HPO4, 0.004 percent by weight NaH2 PO4, and, optionally, 0.017 percent by weight CaCl2), Hank's balanced salt solutions (GIBCO Labs., Grand Island, New York) and Balanced Salt Solutions available from several ophthalmic pharmaceutical companies, including Alcon Laboratories (Ft. Worth, Tex.), Pharmafair (Hauppauge, N.Y.), Akorn, Inc. (Abita Springs, La.), and Allergan Pharmaceuticals (Irvine, Calif.). In general, these saline solutions will have osmolarities ranging from about 280 to about 340 milliosmoles and sodium salts in excess of potassium salts (e.g., 20.5 to 1 ratio such as for the above-described phosphate buffered saline solution).
Collagen suitable for use in the present invention is soluble at physiologic pH. Usually, this means a collagen that has been reacted with an acylating agent having two or more carboxylic acids or carboxylic acid derivatives. The degree of reaction being effective to substantially solubilize collagen at physiolic pH. For Type I collagen an effective degree of acylation is thought to be in excess of 35% of collagen lysyl amino acids acylated. Type IV collagen, when isolated from the placenta, is soluble a physiologic pH without further processing. Type II and Type III collagen are not as abundant as Type and Type IV and are for this reason alone, no anticipated to have as much utility in the practice of the present invention as Types I and IV. However, these collagens are anticipated to be solubilized by substantially the same degree of acylation as is effective for Type I collagen.
It is anticipated that collagen from unusual sources or tissues not traditionally used as a source of commercial collagen may have somewhat different solubility properties. The degree of acylation required to prepare such collagen for use in viscoelastic solutions with the viscosity properties of the present invention can be determined by routine experimentation. Such viscoelastic solutions are within the scope of the present invention.
A preferred acylating reaction for the solubilization of collagen is taught in U.S. Pat. Nos. 4,851,513 and 4,713,446. Also preferred is acylation using glutaric anhydride (reaction pH between about 7.0 and about 9.0). Another source of collagen suitable for use in the present invention is disclosed in U.S. patent application Ser. No. 157,638, filed Feb. 18, 1988now U.S. Pat. No. 4,969,912, wherein a method is taught for solubilizing telopeptide-containing collagen from comminuted animal tissue using acylating reagents. (All patents, patent publications and literature citations found herein are incorporated by reference into this disclosure.)
The acylating reagents useful for preparing the collagen used in the present invention include C3 to C8 organic compounds having two or more carboxylic acid-related functional groups, at least one of which is an amino-reactive form (e.g., acid halide, acid anhydride, ester, alkyl cyanate).
Usually, the collagen will be prepared from a nonhuman crude collagen source. The method for obtaining the collagen from the crude collagen source, e.g., tendon, hide, etc., is normally not critical, and some flexibility may be used in the selection of the particular tissue and the method applied thereto. Applicants prefer to extract collagen from a connective tissue, such as bovine hide. If the collagen is to be used for ophthalmic applications, it is preferred that it be obtained solely from the corium layer of the bovine hide, otherwise known as "split" hides. Split hides are commercially available from the Andre Manufacturing Co., Newark, N.J.
The collagen may be solubilized by any of the standard extraction methods, e.g., acid or salt extraction, enzymedigestion, or a combination of these. The acylation-dependent solubilization of U.S. patent application Ser. No. 157,638 (supra) may also be used. Preferably, dehaired and cleaned hide is solubilized with a proteolytic enzyme (pepsin, for example) and solubilized collagen is precipitated at pH 7, after inactivation and removal of the enzyme, by addition of NaCl to about 2.5M. Pepsin-treated collagen precipitates leaving behind in solution (to be discarded) the digested nonhelical terminal peptides of the collagen molecule and other non-collagenous contaminates, e.g., saccharides, mucopolysaccharides, etc. Inactivated enzymes are removed by filtration and centrifugation at 4° C. The pepsin-treated collagen is then further purified by repeated redissolution in acidic water (pH 2-4) and reprecipitation by salt treatment, e.g., by the addition of 0.8M sodium chloride solution at pH 3.
It is preferred that the chemicals used during collagen processing, particularly the final stages of processing, be free of inflammatory contaminants (e.g., pyrogens).
It is preferred that the kinetic viscosity of the viscoelastic solutions of the present invention be at least about 1,000 cps, but less than about 500,000 cps. It is particularly preferred that the viscosity be between about 45,000 cps and about 80,000 cps. It is preferred that upon addition of at least equal volumes of irrigating solution, such as Balanced Salt Solution, the kinetic viscosity should drop at least 50%. It is particularly preferred that this drop in viscosity be at least 75%.
The invention is described below with specific working examples which are intended to illustrate the invention without limiting the scope thereof.
One liter of sterile filtered, purified, atelopeptide collagen (1.5 mg/ml to 4.0 mg/ml) in 0.1M acetic acid is placed in a pyrogen-burnt glass beaker and stirred with a magnetic stir bar. A sterile solution of 10N NaOH is added to bring the pH to about 9.0. Sterile glutaric anhydride (18% by weight of the collagen weight) is added to the solution as a powder. Through the course of the acylation procedure the pH is maintained at about 9.0 by incrementally adding sterile 1N NaOH. After approximately 2 minutes, succinic dichloride is added (7% by weight of the collagen weight) via a syringe outfitted with an 0.2 μm Gelman syringe filter (Gelman Sciences Inc., Ann Arbor, Mich.). After 30 minutes, another portion of glutaric anhydride (18% by weight of collagen weight) is added. After an additional 60 minutes, the acylation is stopped by addition of 6N HCl until the pH is 4.3. Under these conditions, the acylated collagen precipitates.
After 60 minutes, the collagen precipitate is collected by centrifugation. The precipitate is washed three times by resuspension in pyrogen-free water and centrifugation. The collagen is then resuspended in approximately 80 ml of 0.005M sodium phosphate buffer the osmolarity adjusted to 305±25 milliosmoles with glycerol (pH 7.4±0.4). After an overnight incubation, the collagen is completely dissolved. The solution is then diluted by small increments until the viscosity (measured from aliquots using a Brookfield Viscometer, Brookfield Instruments, Stoughton, Mass.) is between about 45,000 cps and about 80,000 cps at a shear rate of 1 sec-1. The osmolarity and pH of the sampled alloquots are also monitored. The osmolarity is monitored using a Precision Micro Osmometer (Precision Systems, Natick, Mass.) and adjusted by dilution with glycerol-free sodium phosphate buffer (0,005M) or by addition of glycerol. The pH is monitored with a pH electrode (Orion Research, Cambridge, Mass.) and adjusted with NaOH or HCl solutions as appropriate.
The concentration of collagen is then measured based on hydroxyproline content determined using the method of Woessner (In The Methodology of Connective Tissue Research, D. Hall, ed., Joynson-Bruvvers, Ltd., Oxford, page 227). Uronic acid content, an indicator of glycosaminoglycan presence, is measured using the standard carbazole method (Bitter & Muir, Anal. Biochem., 4, page 330, 1962). As a further quality control measure, a UV scan is performed on each lot of collagen-based viscoelastic solution.
Once the viscosity, osmolarity and pH are adjusted to the specification values (see Table I), the solution is filtered through a sterile 5.0 μm filter.
TABLE I______________________________________Viscoelastic Collagen Solution SpecificationsCharacteristic Acceptable Preferred______________________________________Osmolarity 305 ± 100 m Osmol 305 ± 25 m OsmolpH 7.4 ± 0.6 7.4 ± 0.4Color -- ColorlessViscosity 1,000-500,000 cpsa 45,000-80,000 cpsEndotoxin <500 Eu/mlb <5 Eu/mlCollagen <10% (w/v) <2.0% (w/v)ConcentrationMelt Temperature 5-100° C. 30-40° C.Sterility Sterile SterileIonic Strength ≦0.001μ ≦0.0008μ______________________________________ a At a shear rate of 1 sec-2. b Eu = endotoxin units.
Lot A and comparative Lot A' collagen solutions were acylated as outlined in EXAMPLE 1, but were redissolved after the washing step as follows:
Lot A:
Collagen was redissolved in 0.1N sodium phosphate, pH 7.69, with glycerol added to adjust the osmolarity to 326 milliosmoles.
Viscosity was 69,200 cps (at 1 sec-1) at a collagen concentration of 1.13% (w/v).
Lot A':
Collagen was redissolved in 1N sodium phosphate buffer, pH 7.0, with the final osmolarity adjusted by dilution with sterile water to 268 milliosmoles.
Viscosity was 43,800 cps (at 1 sec-1) at a collagen concentration of 3.9% (w/v).
Two more lots (B and C) were prepared by acylating atelopeptide collagen with glutaric anhydride alone. These lots were redissolved, following three water wash steps (comparable to those outlined in EXAMPLE 1), in 1N sodium phosphate buffer. These lots were then dialyzed against deionized water for two days. Lot B was adjusted to a protein concentration of 1.55% (w/v), a pH of 6.8, and a osmolarity (adjusted with glycerol) of 275 milliosmoles. The kinetic viscosity was 228,000 cps (at 1 sec-1).
FIG. 1 shows the kinetic viscosity profile (log (shear rate) vs. log (poise.sec)) for Lot C before (open symbols) and after dialysis (closed symbols). The collagen concentration was 1.4% (w/v) and the pH was 6.8. Notice the dramatic increase in viscosity obtained after dialysis.
Adult female, New Zealand white rabbits (5-6 lbs.) were used for an ocular vitreous replacement study. One day prior to the study, by slit lamp evaluation both eyes of each animal were examined using fluorescein dye to determine the condition of corneal epithelium. The corneal epithelium was evaluated to determine if any corneal abrasion, ulceration or irregularities were present. On the day of the study, each animal was again evaluated prior to the injection of the test material. Fluorescein dye was not used in this pre-injection evaluation. Animals exhibiting pre-existing corneal, lens or conjunctival injury, irritation or irregularity were not used in this study.
General anesthesia was achieved by intramuscular injection of Rompun (10 mg/kg body weight) and Ketamine HCl (50 mg/kg body weight). The pupil was dilated with 0.05 ml of 1% Cyclopentolate HCl and 2.5% Phenylephrine HCl administered topically. One drop of Proparacaine 0.5% was administered topically prior to injection of the vitreous replacement solution. All injected solutions were sterile and administered undiluted. The effects on intraocular pressure of Lot A and Lot A' (Example 2), Viscoat™ and Healon™ were compared. Viscoat™ was purchased from Alcon-Cooper/Cilco. Healon™ was purchased from Pharmacia (Uppsula, Sweden). All viscoelastic solutions were refrigerated at 6° C. until the time of use.
One rabbit eye was prepared for surgery and a 25-gauge butterfly infusion needle was inserted at the 10 o'clock position, 0.2 mm anterior to the limbus. To prevent collapse of the anterior chamber, the outflow tubing remained capped until the injection of the viscoelastic solution was initiated. A 1 cc syringe with a 27 gauge needle was filled with the viscoelastic solution to be injected into the eye to replace the vitreous and inserted at the 2 o'clock position, 0.2 mm anterior to the limbus. The beveled edges of the needles were positioned away from the corneal endothelium. The anterior chamber of the eye was filled with 0.15 cc of the viscoelastic solution. After replacement of the aqueous humor with the test material, the outflow and the inflow needles were removed. In the other eye, no viscoelastic solution was injected into the anterior chamber. This eye served as a baseline for intraocular pressure measurements. The intraocular pressure was monitored with a pneumotonometer. Eyes were evaluated prior to injection and at 0, 1, 3, 5, 24 and 48 hours post-injection.
The results obtained, using three rabbits for each measurement, are shown in TABLES II and III.
TABLE II__________________________________________________________________________Intraocular Pressure/mm Hg Pre- Injection 1 hour 3 hours 5 hours 24 hours 48 hours__________________________________________________________________________LOT ARabbit 1: 19.8 10.1 25.0 21.7 11.8 21.0Rabbit 2: 21.3 7.9 10.8 14.3 11.6 31.0Rabbit 3: 19.7 11.0 21.9 22.3 29.8 22.2Mean ± SD: 20.2 ± .7 9.7 ± 1.6 19.2 ± 7.5 19.4 ± 4.5 17.7 ± 10.5 24.7 ± 5.5VISCOAT ™Rabbit 1: 15.5 9.1 41.1 31.1 10.7 24.0Rabbit 2: 23.1 36.8 9.6 8.5 8.4 31.1Rabbit 3: 11.9 8.1 40.1 14.6 14.0 24.3Mean ± SD: 16.8 ± 5.7 18.0 ± 16.3 30.3 ± 18.0 18.1 ± 11.7 11.0 ± 2.8 26.5 ± 4.0__________________________________________________________________________
TABLE III__________________________________________________________________________Intraocular Pressure/mm Hg Pre- Injection 1 hour 3 hours 5 hours 24 hours 48 hours__________________________________________________________________________LOT A'Rabbit 1: 32.9 9.4 12.1 13.8 19.2 3.4Rabbit 2: 20.7 20.6 38.5 34.8 33.5 30.7Rabbit 3: 23.3 30.3 32.8 26.5 37.8 19.4Mean ± SD: 25.6 ± 6.4 20.1 ± 10.5 27.8 ± 18.9 25.0 ± 10.6 30.2 ± 9.7 28.0 ± 7.7HEALON ™Rabbit 1: 16.0 7.9 6.1 7.8 20.1 10.8Rabbit 2: 15.3 13.7 16.2 26.0 34.5 16.3Rabbit 3: 19.0 6.4 15.3 25.2 15.5 18.6Mean ± SD: 16.7 ± 2 9.3 ± 3.9 12.5 ± 5.6 19.7 ± 10.3 23.4 ± 9.9 15.2 ± 4.0__________________________________________________________________________
The data of TABLES II and III demonstrate that collagen viscoelastic solutions according to the present invention, LOT A, are equal to or superior to prior art solutions LOT A' (collagen solution according to U.S. Pat. Nos. 4,713,446 and 4,851,513), Healon™ (sodium hyaluronate), or Viscoat™ (a mixture of sodium hyaluronate and chondroitin sulfate) when left in the anterior chamber of the eye. Viscoelastic solutions according to the present invention are easier to irrigate out of the intraocular cavities. They are anticipated to be substantially removed from the eye following ocular surgery and, thus, result in still fewer incidents of elevated intraocular pressure relative to prior art solutions.
In view of the present specification and appended claims, various additions, modifications, and omissions will be obvious to those skilled in the art and are within the invention as claimed below.
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text:
Brett Wiese went at the things he wanted in life with pure determination — much like his idol, Edmonton Oilers' rising star Taylor Hall.
Sadly, the 21-year-old Calgary victim will be there in spirit alone when his beloved Oilers drops the puck on their home opener Tuesday night; however, the team has taken the time to ensure his memory is not forgotten.
After learning Wiese, a high-achieving University of Calgary student, had been fatally stabbed while trying to break up house-party altercation Jan. 12, the Oilers sent a jersey with Hall's autograph along with a card from team general manager Steve Tambellini expressing his condolences.
The gesture was overwhelming for Wiese's father Jody, who shared a love for the Oil with his son and had purchased tickets for the two of them to attend Tuesday's affair against the San Jose Sharks.
"For them to take time to recognize our son, I was very proud of them and I'm proud of my son," Jody said Sunday, before adding he still plans to attend the game alongside Brett's oldest cousin, who will likely don the Hall jersey.
An Oilers team spokesperson confirmed efforts to honour Wiese Sunday, but no one was not available for further comment.
Brett Wiese's funeral was held Friday in his hometown of Ponoka and his young sister Morgan wore the Hall jersey, said uncle Kip Wiese. All of the pallbearers also wore Oilers jerseys.
Now, the Wiese family attempts to move forward, but Jody admits Tuesday's game will be an emotional one.
"Brett would have wanted us to be there," he said. "We'll be up there cheering loudly and probably crying a little bit too."

subset: commoncrawl


text:
Seared Salmon Salad with Curry
This sweet and spicy salad makes a satisfying lunch and offers plenty of protein, carbohydrates, and nutritious vegetables.
|12 small new or fingerling potatoes, halved
|2 teaspoons salt, divided
|2 tablespoons extra virgin olive oil, divided
|1 teaspoon curry powder
|Salt and pepper to taste
|1/2 teaspoon cayenne
|2 pounds boneless, skinless salmon filet*
|1/2 teaspoon sugar
|2 large shallots, diced finely
|2 cups plain organic Greek yogurt
|2 teaspoons cider vinegar
|6 cups arugula
Preheat oven to 350°F. Toss potatoes in 1 tablespoon of olive oil, some salt, and pepper. Place in a roasting dish and cook for 20 minutes until tender. Cut salmon filet into 6 even-sized pieces and rub with remaining olive oil, salt, and pepper.
Spray non-stick skillet with cooking spray and heat to medium-high. Sear salmon on each side for 2 minutes. Transfer to baking sheet. Bake in the oven with potatoes for 5 minutes or to desired doneness (medium to medium rare is best). If you prefer well done, cook for 8 minutes.
In a small bowl, combine shallots, cider vinegar, salt, curry, cayenne, sugar, and yogurt.
Divide arugula evenly between 6 plates. Place a piece of salmon and two potatoes over arugula and drizzle generously with yogurt curry mixture. Makes 6 servings.
* Read Fish: Landing the Healthiest Choices to learn about what fish is safe to eat and to look up your safest options for buying salmon filets.


text:
This is my personal blog. I'm the founder and CEO of FutureKids.co. I've worked in startups and tech my entire career.
I was most recently VP marketing for Klout. Prior to Klout, I worked for Automattic, the makers of WordPress.com, where I served as VP of user growth and was responsible for marketing and communications. I joined Automattic after several years at Mozilla, where I was VP marketing and launched four major versions of Firefox. I started my career in technology marketing at Adobe Systems where I led product marketing for Creative Suite, InDesign and PageMaker.
My first job out of college was as a writer and product manager at Spectrum HoloByte, which I left to join the Milarepa Fund as a web producer.
I received an MBA from the Haas School of Business at UC Berkeley and an AB in English from Stanford. I live in Oakland, California, with my wife and our daughters.
Photos of mostly work adventures on my Flickr.
Here are a few articles about the work I've done:
- How Firefox Gets Grassroots Marketing Right at Advertising Age
- Mozilla Firefox Goes 'Organic' at Treehugger
- Firefox 3 and community at ZDNet
This blog is hosted on WordPress.com. They rule, try them out! The name of this blog is an homage to PKD.


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Cisse had been expected to take up his place in the Magpies squad to face Swansea on Saturday after recovering from a back problem sustained during the 1-0 defeat by West Ham last week.
But as the 27-year-old was withdrawn from international duty with Senegal in midweek, the country's football association are threatening to invoke FIFA's five-day rule.
That rule would prevent Cisse playing for his club within five days of the international game from which he was withdrawn.
Newcastle boss Alan Pardew claims Senegal were informed of the situation and his medical records were sent off, but he admits there is a problem.
"We have a bit of an issue going on at the moment with the federation," said Pardew.
"We feel we have covered all the bases to give them the information, but they seem not to have received it, so we are in discussions with Senegal and the FA about his availability for Saturday.
"But we've got enough squad quality to put a good side out for Saturday, for sure."


text:
So with my bias openly stated, I'd like to counterbalance it with this: As a veteran MMO player, I also won't put up with things I think are absolute crap for very long. Much like everyone else, I have a life to attend to outside of MMOs, I had to shell out for a copy of the game, and I pay my own subscription each month. I get just as irked when I feel as if I'm not getting value for that dollar as anyone else does. With that in mind, let's take a look at DC Universe Online from launch to current day, and I'll toss out my thoughts on picking up the game afterward.
The UI, however, was nothing short of a hot mess. Voice chat was screwed up more often than not. If you turned voice chat off to give yourself a respite after listening to other people mouth-breathing, eating, etc., the text chat would drive you to reconsider that decision by virtue of its somehow being even buggier and problematic. Trying to send tells worked only sporadically, grouping was difficult, and the League functions were akin to smashing your head against a wall repeatedly: only fun if you like headaches and frustration.
Some players also felt that the game lacked enough content, an opinion Chris Cao largely rebuffed when we asked him about it. The point, he'd said, was to design a game that is geared for a casual player, targeting at five to seven hours per week.
By contrast, the recent Update 2 addition and the patch that just came out this last Thursday (April 14th) were both significantly better in terms of giving players new content that fits in with the feel of DC's world and bug fixes that didn't break many other things in the process. As Cao mentioned the last time I had a chance to speak with him, the developers are tracking what types of content people spend their time on while logged in and designing new content based on that. Considering the reception from the community for the last two patches, I'd say that's a tremendously wise choice as there haven't been nearly as many complaints this round compared to post-Update 1.
Currently, the major issue faced by the game is two-fold. First, there are a lot of people who get to endgame having never grouped only to find that they're not able to get instance groups as easily since they don't know as many people and aren't trained for roles. Casual players who do log in only five hours a week (Cao's target market above) are not going to be into sitting around roleplaying or doing heavy community building. They want to log in, play, have fun, and log out. As such, if you only have an hour per day to log in and you spend 15 minutes of that time waiting for a group to pop (because you don't have time to develop social connections), you're going to be driven away from the game.
The second thing that I find problematic is that if you don't happen to have a life that allows you to log in during peak hours (currently approximately 6-12 p.m. EDT on my home server Zero Hour), then you're going to have a miserable time finding a group, because even the most populated servers are quiet on off-hours. Mind you, I'm speaking for PC-side. I understand the PS3 players don't have nearly as many issues with server population, and I'm thrilled for them. Logging in and playing for a couple of hours before you lay eyes on another person's avatar is really kind of sad.
As for what could be done, I have a few ideas, but the single most useful thing that Sony Online Entertainment could do is either to consider a server merge on the PC side or to create a cross-server queue system to make getting groups faster. While there is always the issue of collision of names when you're talking about a server merge, that beats the pants off of being unable to enjoy the game because you can't find a group. Some might also argue that a cross-server queue system is detrimental to a community, but if you've built your game to cater to people who only log in five to seven hours per week, you've already set it up to lack a large in-game community from the word go. You also have to consider that by making it more difficult for those casual players to get groups when they do hit endgame, SOE creates a much larger possibility that those players won't stick around, which is detrimental to those who do want a community since they help pay the bills.
For someone who has the time and social nature to dedicate to getting involved with an active League, who likes running raids, and who does most of his gaming during prime time, I'd recommend it if you're on the fence. If, however, you tend to prefer PUGs, you want instance queues to match you up, you don't want to mess with a League, and you aren't available during that prime-time window, then I'd suggest you wait for a little while, or at least realize that endgame will require a bit more from you than leveling 1-30 in a casual manner will. Something simply needs to be done to allow the current endgame population in off-peak hours to play together more readily.
Hopefully that helps to clear things up on my opinion about whether or not the game is a good investment. Mind you, this is based entirely on how the game is currently. In six months, this will likely change as we see more content added to the game and the population grows, is merged, or dwindles. There's a lot of fun to be had in the game. It's up to SOE to make sure that all DC Universe Online players, regardless of schedule or time constraints, can take advantage of it.
Every week on Saturday, strip off the mask of your Alter-Ego and soar through the world of DC Universe Online with Krystalle, then catch up with Larry on Wednesdays as he showcases the superhero game on the Alter-Ego livestream. Send up a bat-signal to ping Krystalle or Larry with your burning questions. (Packages wrapped in green with a purple bow will be returned unopened.)


text:
Vivian McPeak
Vivian McPeak is a Pacific Northwest based musician and social justice activist. He is also the executive director of the world's largest cannabis policy reform rally, Seattle Hempfest.
Monthly Archive for February, 2014
Why Pot Prohibition Has Failed
After decades of being disregarded as a viable political movement, cannabis policy reform—often referred to as the Legalization Movement—is at last finding validity as a social force to be reckoned with. Due to the tireless advocacy of community-based shoe-leather activists everywhere, combined with a government policy that has created more damage than the herb it […] [Read More]
Marijuana Superbowl B.S.
It has been estimated that 50 million cases of beer will be sold to Americans intending to watch the Superbowl. Certainly, some of the people consuming those millions of cans and bottles of beer (and all the other forms of liquor that will be consumed by Superbowl revelers) will end up driving impaired. There will […] [Read More]


text:
There is still no deal between Galesburg's teachers' union and district leaders, following nearly 12 hours of contract negotiations Monday, August 11, 2014.
Talks continued Tuesday morning, August 12, 2014 though, as both teachers and district leaders in Galesburg, Illinois, say they remain optimistic that a strike can be avoided.
Related: Galesburg teachers and district battle over salary schedules and security
Representatives from the Galesburg Education Association and District 205 met with mediators Monday. Around 10:30 p.m. that night, the chief negotiator for the GEA said they had received a proposal from the Board of Education.
"It's late, obviously. We're gonna sleep on it, and come back at 10 tomorrow," said Luan Statham. "We're trying to stay positive and still hoping to avoid a strike."
Both sides started negotiating again at 10 a.m. Tuesday. They will negotiate until 1 p.m. Tuesday. That evening, GEA teachers will meet to either ratify a new contract or assign strike duties.
Classes are scheduled to begin on Thursday, August 14, 2014.
"We want to be with our kids, and so, that's always in the back of our heads, that we want to get back to school and be with our kids. So, it is very emotional for us. It's coming down to the wire, and we're trying to stay positive and get this done," said Statham.


text:
Wisconsin man among four presumed dead off Washington coast
SEATTLE - The Coast Guard has released the names of four people presumed dead after their fishing trawler disappeared off the Washington coast after sending a distress signal.
Twenty-five year old Chris Langel of Kaukauna, Wisconsin is among the missing.
The other three men are identified as 42-year-old Dave Nickels and 38-year-old Jason Bjaranson of Warrenton, Oregon and 19-year-old Luke Jensen of Ilwaco, Washington.
The Coast Guard suspended its search for the men Sunday.
Petty Officer Shawn Eggert says crews in Coast Guard cutters and a helicopter covered more than 1,300 square miles since the 70-foot Lady Cecelia sent a distress signal early Saturday. He says they've now searched longer than anyone might have survived in the water.
Rescue crews searched through the night after finding debris, an empty lifeboat and an oil sheen several miles off the coast.
The search was called off before midday Sunday.
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text:
|Kids Craft: Love Birds
Event Type: Children's Program
Date: 2/13/2014
Start Time: 3:30 PM
End Time: 4:30 PM
Love is in the air...literally! Join us as we celebrate Valentine's Day by making festive love birds. Best for ages 3+.Library: Oak Park Public Library Dole Branch
Location: Meeting Room - Dole Branch
Contact: Dole Branch Library
Contact Number: 708.386.9032
Presenter: Jenny


text:
Clue: Some kitchen waste
We have 2 answers for the clue Some kitchen waste. See the results below.
Possible Answers:
Related Clues:
- Hides
- D.C. team, for short
- Drums, in jazz lingo
- Scrapes
- Drums, slangily
- One side in an informal game
- Shucks
- Dollars, slangily
- Outer surfaces
- Shirts' opponents
Last Seen In:
- New York Times - August 07, 2010
- New York Times - October 04, 2008
Found an answer for the clue Some kitchen waste that we don't have? Then please submit it to us so we can make the clue database even better!


text:
As if it wasn't bad enough paying for lawyers in half the countries of the world Apple is now being threatened with a suit by the Justice Department here at home in the US. This time it's over the pricing of ebooks in the company's online store.
As we know, Apple's suing Samsung, Samsung's suing Apple in courts around the world (Germany and Australia particularly come to mind, about various design patents), Proview is having a go in Mainland China and California (over the rights to the iPad name) and who can remember how many handset makers are having at each other over patents?
Just to add to Apple's joy the Justice Department has said that it intends to sue over ebook pricing:
The Justice Department has warned Apple Inc. and five of the biggest U.S. publishers that it plans to sue them for allegedly colluding to raise the price of electronic books, according to people familiar with the matter.
There are slight differences between this and the European Union suit against Apple and the publishers on the same issue. For the laws about what you can and cannot do are slightly different in the two jurisdictions. But back in December the EU did indeed start the process of investigating the very same issue.
That point at issue is really quite simple. There are, conceptually, two ways that a book can be priced. The publisher decides what that price should be and then negotiates a discount to that for the retailer. Or the publisher decides upon a wholesale price and the retailer then decides what they'd like to sell it for.
There are problems with either model: in the first, the so called "agency model", this gives the publisher absolute power over the retail price of the book. On the face of it European law certainly (and US maybe) states that it's not legal for a producer to have such power. It restricts the competition that the retailers might have, that competition lowering prices to consumers.
On the other hand, the second model, this allows companies such as Amazon to run loss leaders. Selling new titles, or highly desired ones, for less than what they are paying the publishers for them. This is great for consumers in the short term of course but might lead to Amazon's financial resources curtailing retail competition in the long term.
Within the economics of it all there's not really a "right" answer. You can create models where either system has the potential to be abused against the interests of the consumer. So which is better for said consumer is really a matter of judgement about how things are at any one time in any one market.
But it is true that the basic assumption of EU law is that the agency model is potentially abusive of the consumer and thus should not be used. Guess we're going to find out whether the Justice Department thinks the same way.


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Michaell Packer is a faculaty member of the LA Music Academy.
He graduated from the University of Northern Colorado with a Bachelor of Arts in Music.
Michael has studied privately with Ralph Humphrey and Jeff Hamilton.
Performance credits include Free Flight, Nils Lofgren, Carl Anderson, Ben Vereen, Steve Allen, Michael Bolton (CableAce Awards) and Cirque Du Soleil (CableAce Awards).
Television credits include 16th, 17th, 18th and 19th Annual CableAce Awards (TNT), The EasterSeal Telethon (National Broadcast), KTLA Morning Show with Leeanza Cornet, World Cup Closing Ceremonies (World Wide Broadcast) and the New York New York Casino & Hotel, Las Vegas, NV. (In-house recording).
His teaching experience consists of 15 years of private instruction and several clinics in the USA and Europe.
Michael is the author of "Feet Don't Fail Me Now" (Hal Leonard Publications) and "Feet First" (Michael Packer Publications).
2009: new DVD "Bass Drum and Hi-Hat Technique: Applying the Moeller Technique to the Bass Drum"
Michael has also written for Modern Drummer Magazine. Please visit www.michaelpacker.com for more info.
Another innovative specialty pedal, The 5000ADH features an aerodynamic heel-less design that allows players to be less encumbered and play in a more comfortable position. As with all 5000 pedals, tension and throw are fully adjustable.


text: HTS is a place where therapists are valued and patient care comes first. Employing over 1,200 therapists and serving more than 20,000 patients, HTS is the leading provider of therapy in the Midwest Our success is attributed to our wonderful team of therapists. Many of our therapists have said HTS feels more like a family than a corporation. With 25 years of industry experience, we take pride in our clinical programming, unwavering support, and commitment to providing quality patient care


text:
KINGSTON, R.I. -- January 10, 2001 -- The University of Rhode Island
Cooperative Extension is seeking volunteers in Washington County to help
protect public drinking water supplies by participating in the Rhode Island
Source Water Assessment Program.
"Since local residents have an intimate knowledge of the local
community, and since everyone in the county relies on groundwater resources
for their drinking water, we believe that the community should be involved,"
explained Alyson McCann, coordinator for Cooperative Extension's Home*A*Syst
program, a volunteer-based residential pollution prevention program.
URI is working cooperatively with the Rhode Island Department of Health
to complete local source water assessments as required by the Environmental
Protection Agency. These assessments, which identify pollution threats
to public drinking water supplies throughout the state, are under way in
South Kingstown, Charlestown and Narragansett.
Cooperative Extension and the health department have developed a two-pronged
approach to citizen involvement in the Source Water Assessment Program.
Some volunteers will inventory potential sources of contamination to the
water supplies, while others will provide input on the assessments by identifying
local land use issues, selecting management options for analysis, and reviewing
assessment results to identify and develop future planning needs.
Inventory volunteers will be trained by the Home*A*Syst program staff
to read and update land use maps, as well as identify high-risk land use
activities within the major wellhead protection areas during a two-hour
training session on Monday, January 29 at 7 p.m. at the Cross Mills Public
Library on Old Post Road in Charlestown. Volunteers will then have one
month to complete their assigned wellhead protection area inventories.
Staff from Cooperative Extension's Municipal Training Program will lead
assessment volunteers through three, two-hour meetings during a four to
five month period to provide input on the assessments, which use computer-generated
maps to systematically study natural and land use related factors that affect
pollution risks to drinking water supplies. The first assessment work session
is Tuesday, February 6 at 4 p.m., also at the Cross Mills Public Library
in Charlestown.
Cooperative Extension and the health department will use the information
provided by each group of volunteers to complete the source water assessments
and assist communities in improving their drinking water protection efforts.
"Rhode Island is unique in its efforts to involve local citizens in
this program," noted McCann. "This is an opportunity for residents
to serve their communities and protect the quality of their drinking water."
For more information or to volunteer for this project in South Kingstown,
Charlestown or Narragansett, call Home*A*Syst Program Assistant Holly Burdett
at (401) 874-5398. For more information about the Source Water Assessment
Program contact Clay Commons, R.I. Department of Health, Office of Drinking
Water Quality at (401) 222-7769.
For Information: Alyson McCann 874-5398 , Todd McLeish 874-7892
Contact email@example.com
for more information about the page.
Copyright © 2001 University of Rhode Island. All rights reserved. Disclaimer.
Page last revised on Saturday, February 24, 2001


text:
My evidence is craniometric, scripts,cultural features and linguistic similarities. In fact it appears their survivors lived until quite recently.
Drinking, singing and dancing are expected to take place deep in the mountains of Miaoli and Hsinchu when the "Ritual of the Little Black People" (矮靈祭) is performed by the Saisiyat tribe once again this weekend.
For the past 100 years or so, the Saisiyat tribe (賽夏族) has performed the songs and rites of the festival to bring good harvests, ward off bad luck and keep alive the spirit of a race of people who are said to have preceded all others in Taiwan.
In fact, the short, black men the festival celebrates are one of the most ancient types of modern humans on this planet and their kin still survive in Asia today. They are said to be diminutive Africoids and are variously called Pygmies, Negritos and Aeta. They are found in the Philippines, northern Malaysia, Thailand, Sumatra in Indonesia and other places.
Chinese historians called them "black dwarfs" in the Three Kingdoms period (AD 220 to AD 280) and they were still to be found in China during the Qing dynasty (1644 to 1911). In Taiwan they were called the "Little Black People" and, apart from being diminutive, they were also said to be broad-nosed and dark-skinned with curly hair
One of the most eminent anthropologists K C Chang said that the Dawenkou culture,considered to be a precursor culture was largely African:K.C. Chang, The archaeology of ancient China, (Yale University Press:New Haven,1977) p.76)
Here is much later craniometrics from the China National Knowledge Infrastructure project:
Negroid skeletons dating to the early periods of Southern Chinese history have been found in Shangdong, Jiantung, Sichuan, Yunnan, Pearl River delta and Jiangxi especially at the initial sites of Chingliengang (Ching-lien-kang) and Mazhiabang (Ma chia-pang) phases
The fur-ther comparison of Neolithic Man with Modern Man in China (including inhabitants of Northern China, Fujian province and Hainan island) is made, also based on the main index values and total prognathism of skull, showing that the development of the physi-cal characteristics of Chinese is a genetic and successive course from Later Paleolithic Man to Modern Man via Neolithic Man. Thus, so-called negroid-Australoid racial traits, such as the narrow and long cranial pattern, heigh vaulted crania, lower orbit, wide nose and some projective prognathism (from the morphological observations), themsel-ves are intrinsic characteristics of Neolithic Man in China, only there is a little diffe-rentia in the degrees of display of these traits among groups.
Also the late Neolithic Australoids practiced African customs still seen today.Skull binding and teeth ablation.
Their racial type is Mongoloid and just as in the case of Ta-w(?)n-k'ou bears a certain resemblance to the Polynesian cranium type.The latter is attri-buted to a sub-division of the Southern Mongoloid.The incidence of cranial deformation among the males of Hsia-hsia-hou is only fiftyper cent as against the nearly one hundred per cent of Ta-w(?)n-k'ou.The cranial defor-mation among the females is one hundred per cent at both sites.The pre-mortem loss ofthe lateral incisors at Hsi-hsia-hou is similar to Ta-w(?)n-k'ou and probably resulted froma ritual practice in initiation rites.The incidence of ablation of teeth at Hsi-hsia-hou isthirty three per cent among the males and sixty seven per cent among the females.
The skulls from the Ye-dian neolithic site are closer to those of the Ta-wen-kou andShi-hsia-hou sites than to those of the other Chinese sites in non-metric and metricfeatures.The author believed that the inhabitants of the Ye-dian neolithic site belongedto the same racial type as those of the Ta-wen-kou and Shi-hsia-hou sites which hadbeen considered by Yen Yin as morphologically belonging to Polynesian type
This is modern African skull elongation as still practiced.
However,another study describes the Yangshao as "physically Chinese"
A careful study of the bones of 400 individuals removed from
more than 300 graves indicates that the Jiahu ethnic group may be
identi®ed with the North AsianMongolian group, and also with the
Xiawanggang and Miaodigou groups in Henan Province and the
Dawenkou, Yedian and Xixiahou groups that were later found in
Shandong Province. The range of male heights was from 170 to
180 cm. In the late Palaeolithic Zhoukoudian Cave, `unspecialized'
Mongoloids were described6. By the Yangshao period (3000 BC±
5000 BC)7, the skull measurements are `physically Chinese' and
`modern'6. The physical similarity of the Jiahu people to the later
Dawenkou (2600 BC±4300 BC) indicates that the Dawenkou might
have descended from the Jiahu, following a slow migration along the
middle and lower reaches of the Huai river and the Hanshui valley.
There are definite topological parallels with W.African and Chinese languages. The last group to be assimilated by the Han,the Qiang of N.Szechuan left extremely interesting traces of their original language which have persisted until now.
Their tonal type is African ,specifically Dagaare ,spoken in Ghana as far as Burkina Faso. The language is of the Niger-Congo family .
Contrastive levels of tone in some Niger-Congo languages H, L DyulaBambara, Maninka, Temne, Dogon, Dagbani, Gbaya, Efik, Lingala
H, M, L Yakuba, Nafaanra, Kasem, Banda, Yoruba, Jukun, Dangme, Yukuben, Akan, Anyi, Ewe, Igbo
T, H, M, L Gban, Wobe, Munzombo, Igede, Mambila, Fon
T, H, M, L, B Ashuku (Benue-Congo), Dan-Santa (Mande)
PA/S Mandinka (Senegambia), Fula, Wolof, Kimwani
none Swahili
Abbreviations used: T top, H high, M mid, L low, B bottom, PA/S pitch-accent or stress
Adapted from Williamson 1989:27
Here's the study on African tonals used by Qiang:http://sinica.academia.edu/JonathanEvans/Papers/167301/African_tone_in_the_Sinosphere
In 10 out of 12 respects such as language floating tones,toneless syllable pitches,contour structure, default tone pattern etc it is more African than Asian.The Qiang are extremely important for their famous oracle bone script they say is from the Shang.
were skilled in making oracle bones
More on the Qiang, (Remai):
Although now a minor segment of the population, the Qiang is commonly believed to be a very old people whose history can be traced to the Shang dynasty (Gu 1980; Ren, Li, & Zhou 1984). It is also believed to be a once strong and populous group whose offspring can now be found among, besides the Qiang in northwestern Sichuan, the Han, the Tibetans, and many minorities in southwestern China (Ran, Li, and Zhou 1984).
According to HKU Dagaare shares similar ideophones with Cantonese.
slide 19
More than one lexical verb may be found in the same clause
Example - I bought some water and drank it
Ngo5 maai5 seoi2 jam2
1.SG buy.PERF water drink
N da la koO nyu
1.SG buy.PERF FOC water drink
Here are examples of Proto saharan script/Shang oracle bone and Harappan script.
The discovery of Intercultural style vessels from Susa (in Iran),Sumerian, Egyptian and Indus Valley sites suggest a shared ideological identity among these people (Kohl 1978).
Some of the craniometric evidence is contrary or deliberately misinterpreted. I think the overwhelmimng weight supporting evidence supports the case for blacks in Neolithic and Paleolithic China. What say the CHFers???
Similar signs appear on Chinese, Harappan, South Indian Megalithic, Libyan and Cretan pottery (see figure 1). These signs were invented by the Proto-Saharans for purposes of communication


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J. Lo shows off her other set of twins. Har har. [DListed]
Pete and Ashlee got a new puppy, which will promptly be neglected once their baby is born. [Seriously? OMG!]
Jay-Z might be expanding his empire by buying a chunk of the third-place Yankees, topping off his list of problems at 100. [ICYDK]
Angelina Jolie is never having those babies, damn it! [WWTDD]
Shocker! Celebrities walk their dogs. We've never seen anything like it! [I'mNotObsessed]
As usual, Audrina from The Hills is hot and wearing minimal clothing. [Egotastic]


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|33 posts found
Apprentice Member
Joined: 7/09/12
Originally posted by BadOrb
Red: And this in-game rewarded speeder which has better protection against knock-offs would be ... ? ??
On the other topic, I haven't seen any actual speed stats (just it's exceptional knockoff avoidance) on the pod speeder so I don't know if what you said was true. But if the cashshop mount is the fastest then that would wreck havoc on PVP servers for those trying to flee an incoming opponent(s). Made even worse by the soon to be released Ilum revamp, if it actually includes non instanced PVP, that is.
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Apprentice Member
Joined: 7/04/11
Originally posted by ktanner3
touche' That thing was an abomination :)
Survivor of the great MMORPG Famine of 2011
Originally posted by darkhalf357x
What you described is the actual definition of Pay To WIn. If it doesn't involve PVP, it isn't pay to win. Period..
What kills PVP in games is when Devs listen to the whiners who think their special character should dominate every one on one matchup. Some builds just aren't meant to be one on one. Some are suppose to be support classes and should stay that way.
Currently Playing: Star Wars The Old Republic
Originally posted by Karteli
It's the Avalanche Heavy Tank (90% speed) and drops in the Nightmare Exposive Conflict operation , it used to be immune to knock-offs but now it's almost immune to knock-offs. Link to the drops in that Operation are below.
I think on the subject of PVP and the new speeder you might be right it might effect PVP and PVE ( to a certain extent , like getting to the node before some ninja , which sounds good in a way ). Hopefully they tested that out . One other thing they have ramped up the protection against knock-offs on all their cartel speeders ( hope that doesn't cause more arguments , but it seems that now makes a few speeders redundant , not the CE and DE ones it seems , just some NPC ones ).
PSO 4 years , EQOA 4 months , PSU 7 years , SWTOR launch ongoing , PSO2 SEA launch ongoing , Destiny 360 launch ongoing.
Yeah, I wasn't going to bring it up, but since you did, there is that new thing where if you bought a cartel speeder:
"Players who have purchased vehicles via the Cartel Market will now find that their protection against being knocked off has been scaled up to Speeder Piloting III regardless of current Speeder Piloting level."
But if you didn't buy a cartel speeder, go fish.
Is this simple convenience (not really vanity since it is functional) or P2W creep?
I'm sure different people will have different opinions on the matter.
Originally posted by tiefighter25
They are really cheap on the GTN though and many people have been complaining that they can't vendor such items , so now they can sell them.
I do believe the game is getting easier though and that is a good example of it. i know of a few others like orange moddable items as heroic mission rewards ( which are very nice ) , various other things like speeder licenses at lower level. More that than P2W creep I feel.
PSO 4 years , EQOA 4 months , PSU 7 years , SWTOR launch ongoing , PSO2 SEA launch ongoing , Destiny 360 launch ongoing.
Originally posted by tiefighter25
That's IT???? A mount that at top speed doesn't go faster than any other? We're fishing all right. Fishing for reasons to make hay about nothing.
Currently Playing: Star Wars The Old Republic
Originally posted by ktanner3
You like the game, I get it.
The reason it's called creep is because each step seems trivial, till of course you reach a point where you look back and all the incremental steps add up to quite a long way.
I stated it was a small step, you're reply is essentially, "Yeah, but is that it? It's such a samll step."
So I guess we're both right in a weird diplomatic way.
Originally posted by tiefighter25
Currently Playing: Star Wars The Old Republic
You keep ratcheting up the tone of your posts.
I'm stating that the speeders started out as pure vanity items. Rather quickly they have added quite a bit of extra functionality to the store bought speeders as opposed to the in game obtained speeders.
I'm not saying that they fit the bill of a pay-to-win item, but the certainly seem to be trending that way as opposed to a pure vanity item.
Cartel purchased speeders: The new $18 speeder is the fastest in game. It also has the second highest knock off protection in game. (The highest being an endgame raid drop). Players who buy a speeder have increased knock off protection as opposed to those who don't buy a cartel speeeder since their speed pilot level will automatically be increased from level 1 to level 3.
I don't think I'm fishing for Pay-to-Win creep. In this case, what was once pure vanity items are adding additional functionality.
With creep, the system might not even arrive at full blown pay-to-win, it describes the way the cash shop system is trending.
You may make the argument that the items I described aren't (arguably) the best in the game, and the best in hte game can still be found by traditional means. You could also ask how else are they going to sell items. YOu could also point out that other cash shops in FTP games use a similar system. The thing is, SWTOR is charging a monthly sub to the very people that it is also selling these items to which makes it a bit ethically (for lack of a better term) fuzzy.
You could also say that it's just my opinion, but that is a given.
If you want me to switch from observationalist to hater here:
The new trending to $18 pay-to-win speeder is now the only Large speeder (which seem to be quite popular) in the game. The other one, the Korrealis speeder was removed.
This was obviously done by Bioware to improve sales of their new pod-racer and also proves that they have been planning on FTP cartel sales since March 2012.
There, now you can get annoyed and tell me to take off the tin-foil hat or whatever and it will seem more reasonable.
Novice Member
Joined: 11/10/12
I´m sorry but putting a "podracer" type speeder in this game, with the whole flawed concept of wheelchair speed lazy programmed mounts even more ridiculous.
I bet this "podracer" (rofl) has the same 110% snail speed like all others.
How do you imagine "racing" this monstrosity, there is no g-force, no traction, no physical correct behavious of "speeders" whatsoever. Lame.
Here, SW Podracer, another 10 year old game besides SWG which kicks SWTOR in the balls:
Watch this first
then watch this
Secrets of Dragon´s Spine Trailer.. ! :D
Best MMOs ever played: Ultima, EvE, SW Galaxies, Age of Conan, The Secret World
Apprentice Member
Joined: 7/09/12
Originally posted by FromHell
Haha nice comparison! The 2nd video didn't seem very .. lively.
My first thought was this was a Pod Racer for grandma to ride around inside a sci-fi Walmart.
Outside of the best in game speed boost stats, 2nd best knockback only to nightmare raiding, etc .. the Pod Racer really should have been used in an actual race minigame, or not at all. It just doesn't fit the outside world, and is so wimpy to a "real" Pod Racer it's almost funny.
As for Pod Racing not existing in the Old Republic time period, EA already screwed up much of the internal lore with legacy that I don't think it would really matter. If anything it would have only attracted more people to play.
Want a nice understanding of life? Try Spirit Science: "The Human History"
Originally posted by tiefighter25
The cartel speeders speeder pilot level doesn't change automatically at all , it is the same as all speeder pilot levels , the only thing that has changed is the knck-off effect , which is now equall to the knock-off effect of speeder pilot level 3's knock-off level , phew.
We also ( as subbs ) get a stipend of 500 CC or more to spend on the cartel market per month , so not so fuzzy really.
finally I agree they have been planing the freemium conversion since about March 2012.
PSO 4 years , EQOA 4 months , PSU 7 years , SWTOR launch ongoing , PSO2 SEA launch ongoing , Destiny 360 launch ongoing.


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