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Purification and stabilization of peptide and protein pharmaceutical agents
Methods are provided for purifying peptides and proteins by incorporating the peptide or protein into a diketopiperazine or competitive complexing agent to facilitate removal one or more impurities, i.e. undesirable components, from the peptide or protein. In a preferred embodiment, a peptide, such as insulin, containing one or more impurities, e.g., zinc ions, is entrapped in diketopiperazine to form a precipitate of peptide/diketopiperazine/impurity, which is then washed with a solvent for the impurity to be removed, which is a nonsolvent for the diketopiperazine and a nonsolvent for the peptide. Formulations and methods also are provided for the improved transport of active agents across biological membranes, resulting for example in a rapid increase in blood agent concentration. The formulations include microparticles formed of (i) the active agent, which may be charged or neutral, and (ii) a transport enhancer that masks the charge of the agent and/or that forms hydrogen bonds with the target biological membrane in order to facilitate transport. In a preferred embodiment, insulin is administered via the pulmonary delivery of microparticles comprising fumaryl diketopiperazine and insulin in its biologically active form. The charge on the insulin molecule is masked by hydrogen bonding it to the diketopiperazine, thereby enabling the insulin to pass through the target membrane. This method of delivering insulin results in a rapid increase in blood insulin concentration that is comparable to the increase resulting from intravenous delivery.
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This application is a divisional of U.S. Ser. No. 09/606,468 filed Jun. 29, 2000, now U.S. Pat. No. 6,444,226, which claims the benefit of U.S. Ser. No. 60/141,433, filed Jun. 29, 1999.BACKGROUND OF THE INVENTION
The present invention is generally in the field of pharmaceutical formulations, and more particularly related to methods and compositions for purifying and stabilizing peptides and proteins, such as insulin, which are used in pharmaceutical applications.
In a normal person, the &bgr;-cells of the pancreatic islets of Langerhans produce insulin, required by the body for glucose metabolism, in response to an increase in blood glucose concentration. The insulin metabolizes incoming glucose and temporarily stops the liver's conversion of glycogen and lipids to glucose thereby allowing the body to support metabolic activity between meals. The Type I diabetic, however, has a reduced ability or absolute inability to produce insulin due to &bgr;-cell destruction and needs to replace the insulin via daily injections or an insulin pump. More common than Type I diabetes, though, is Type II diabetes, which is characterized by insulin resistance and increasingly impaired pancreatic &bgr;-cell function. Type II diabetics may still produce insulin, but they may also require insulin replacement therapy.
Type II diabetics typically exhibit a delayed response to increases in blood glucose levels. While normal persons usually release insulin within 2-3 minutes following the consumption of food, Type II diabetics may not secrete endogenous insulin for several hours after consumption. As a result, endogenous glucose production continues after consumption (Pfeiffer, Am. J. Med., 70:579-88 (1981)), and the patient experiences hyperglycemia due to elevated blood glucose levels.
Loss of glucose-induced insulin secretion is one of the earliest disturbances of &bgr;-cell function (Cerasi et al., Diabetes, 21:224-34 (1972); Polonsky et al., N. Engl. J. Med., 318:1231-39 (1988)), but the causes and degree of &bgr; cell dysfunction are unknown in most cases. While genetic factors play an important role, (Leahy, Curr. Opin. Endocrinol. Diabetes, 2:300-06 (1995)), some insulin secretory disturbances seem to be acquired and may be at least partially reversible through optimal glucose control. Optimal glucose control via insulin therapy after a meal can lead to a significant improvement in natural glucose-induced insulin release by requiring both normal tissue responsiveness to administered insulin and an abrupt increase in serum insulin concentrations. Therefore, the challenge presented in the treatment of early stage Type II diabetics, those who do not have excessive loss of &bgr;-cell function, is to restore the release of insulin following meals.
Most early stage Type II diabetics currently are treated with oral agents, but with little success. Subcutaneous injections of insulin are also rarely effective in providing insulin to Type, II diabetics and may actually worsen insulin action because of delayed, variable, and shallow onset of action. It has been shown, however, that if insulin is administered intravenously with a meal, early stage Type II diabetics experience the shutdown of hepatic glucogenesis and exhibit increased physiological glucose control. In addition, their free fatty acids levels fall at a faster rate than without insulin therapy. While possibly effective in treating Type II diabetes, intravenous administration of insulin, is not a reasonable solution, as it is not safe or feasible for patients to intravenously administer insulin at every meal.
Insulin, a polypeptide with a nominal molecular weight of 6,000 Daltons, traditionally has been produced by processing pig and cow pancreas to isolate the natural product. More recently, however, recombinant technology has been used to produce human insulin in vitro. Natural and recombinant human insulin in aqueous solution is in a hexameric configuration, that is, six molecules of recombinant insulin are noncovalently associated in a hexameric complex when dissolved in water in the presence of zinc ions. Hexameric insulin is not rapidly absorbed. In order for recombinant human insulin to be absorbed into a patient's circulation, the hexameric form must first associate into dimeric and/or monomeric forms before the material can move into the blood stream. The delay in absorption requires that the recombinant human insulin be administered approximately one half hour prior to meal time in order to produce therapeutic insulin blood level, which can be burdensome to patients who are required to accurately anticipate the times they will be eating. To overcome this delay, analogs of recombinant human insulin, such as HUMALOG™, have been developed, which rapidly disassociate into a virtually entirely monomeric form following subcutaneous administration. Clinical studies have demonstrated that HUMALOG™ is absorbed quantitatively faster than recombinant human insulin after subcutaneous administration. See, for example, U.S. Pat. No. 5,547,929 to Anderson Jr., et al.
In a effort to avoid the disadvantages associated with delivery by injection and to speed absorption, administration of monomeric analogs of insulin via the pulmonary route has been developed. For example, U.S. Pat. No. 5,888,477 to Gonda, et al. discloses having a patient inhale an aerosolized formulation of monomeric insulin to deposit particles of insulin on the patient's lung tissue. However, the monomeric formulation is unstable and rapidly loses activity, while the rate of uptake remains unaltered.
While it would be desirable to produce rapidly absorbable insulin derived from natural sources, transformation of the hexameric form into the monomeric form, such as by removing the zinc from the complex, yields an insulin that is unstable and has an undesirably short shelf life. It therefore would be desirable to provide monomeric forms of insulin, while maintaining its stability in the absence of zinc. It also would be advantageous to provide diabetic patients with monomeric insulin compositions that are suitable for pulmonary administration, provide rapid absorption, and which can be produced in ready-to-use formulations that have a commercially useful shelf-life.
These problems with impurities, metal ions that affect stability or bioavailability, occur with many other proteins and peptides.
U.S. Pat. No. 6,071,497 to Steiner, et al. discloses microparticle drug delivery systems in which the drug is encapsulated in diketopiperazine microparticles which are stable at a pH of 6.4 or less and unstable at pH of greater than 6.4, or which are stable at both acidic and basic pH, but which are unstable at pH between about 6.4 and 8. The patent does not describe monomeric insulin compositions that are suitable for pulmonary administration, provide rapid absorption, and which can be produced in ready-to-use formulations that have a commercially useful shelf-life.
It would therefore be advantageous to develop alternative insulin delivery compositions for Type II diabetics that provide more rapid elevation of insulin blood levels and are easily administered to ensure patient compliance. It also would be desirable to apply the delivery compositions and methods to other biologically active agents.
It is therefore an object of the present invention to provide improved methods for purifying peptides and proteins, especially in the preparation of compositions suitable for pulmonary administration.
It is another object of the present invention to provide stable monomeric peptide compositions suitable for pulmonary delivery.
It is a further object of the present invention to provide methods and compositions for the facilitated transport of insulin and other biologically active agents across biological membranes.
It is another object of the present invention to provide methods and compositions for the improved absorption of insulin or other biologically active agents in the bloodstream.
It is a still further object of the present invention to provide methods and compositions for the improved absorption of insulin or other biologically active agents in the bloodstream characterized by ease of administration.SUMMARY OF THE INVENTION
Methods are provided for purifying peptides and proteins by incorporating the peptide or protein into a diketopiperazine or competitive complexing agent to facilitate removal one or more impurities, i.e. undesirable components, from the peptide or protein. In a preferred embodiment, a peptide, such as insulin, containing one or more impurities, e.g., zinc ions, is entrapped in diketopiperazine to form a precipitate of peptide/diketopiperazine/impurity, which is then washed with a solvent for the impurity to be removed, which is a nonsolvent for the diketopiperazine and a nonsolvent for the peptide. Alternatively, the impurity can be removed by using complexing agents to selectively complex with and displace the impurities, for example, such as by dialysis.
Formulations and methods also are provided for the improved transport of active agents across biological membranes, resulting, for example, in a rapid increase in blood agent concentration. The formulations include microparticles formed of (i) the active agent, which may be charged or neutral, and (ii) a transport enhancer that masks the charge of the agent and/or that forms hydrogen bonds with the target biological membrane in order to facilitate transport. In a preferred embodiment, insulin is administered via pulmonary delivery of microparticles comprising fumaryl diketopiperazine and insulin in its biologically active form. The charge on the insulin molecule is masked by hydrogen bonding it to the diketopiperazine, thereby enabling the insulin to pass through the target membrane. This method of delivering insulin results in a rapid increase in blood insulin concentration that is comparable to the increase resulting from intravenous delivery.BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a is a graph of mean blood glucose values over time (minutes). FIG. 1b is a graph of mean C-peptide concentrations during experiments comparing levels of C-peptide (ng/ml) over time (minutes) when insulin was administered intravenously, subcutaneously, and by inhalation.
FIG. 2a is a graph of glucose infusion rate (mg/kg/min) over time (minutes) comparing insulin administered intravenously, subcutaneously, and by inhalation. FIG. 2b is a graph of mean insulin concentrations (&mgr;U/ml) over time (minutes) comparing insulin administered intravenously, subcutaneously, and by inhalation.DETAILED DESCRIPTION OF THE INVENTION
Encapsulation or entrapment of large polymers, such as proteins and peptides, in diketopiperazines can be used to remove impurities or contaminants such as metal ions or other small molecules. The diketopiperazines also serve both to stabilize and enhance delivery of the entrapped materials. Formulations also have been developed for the enhanced transport of active agents across biological membranes. These formulations include microparticles formed of (i) the active agent, which may be charged or neutral, and (ii) a transport enhancer that masks the charge of the agent and/or that forms hydrogen bonds with the membrane. The formulations can provide rapid increases in the concentration of active agent in the blood following administration of the formulations.
For example, it was discovered that hexameric insulin can be delivered to the lung in fumaryl diketopiperazine formulation, reaching peak blood concentrations within 3-10 minutes. In contrast, insulin administered by the pulmonary route without fumaryl diketopiperazine typically takes between 25-60 minutes to reach peak blood concentrations, while hexameric insulin takes 30-90 minutes to reach peak blood level when administered by subcutaneous injection. This feat has been successfully replicated several times and in several species, including humans.
Removing zinc from insulin typically produces unstable insulin with an undesirably short shelf life. Purification to remove zinc, stabilization and enhanced delivery of insulin is demonstrated by the examples. Formulations of insulin trapped in fumaryl diketopiperazine were found to be stable and have an acceptable shelf life. Measurement of the zinc levels demonstrated that the zinc had been largely removed during the entrapment process, yielding monomeric insulin in a stable delivery formulation.
Rapid absorption of a number of other peptides, including salmon calcitonin, parathyroid hormone 1-34, octreotide, leuprolide and RSV peptide, has been observed when the peptide is pulmonarily delivered in fumaryl diketopiperazine—providing peak blood concentrations within 3-10 minutes after pulmonary delivery.
I. Materials
A. Agent to be Delivered
The agent to be delivered is referred to herein as the active agent, or molecule to be encapsulated or entrapped. It may or may not be a charged species. Examples of classes of active agents suitable for use in the compositions and methods described herein include therapeutic, prophylactic, and diagnostic agents, as well as dietary supplements, such as vitamins.
The exact mechanism by which the diketopiperazines form a complex with the materials to be delivered is not known, but it is believed that the diketopiperazines form a complex with the material to be purified. This process is referred to herein interchangeably as entrapment or encapsulation.
These materials can be any polymer or large organic molecules, most preferably peptides and proteins. Generally speaking, any form of drug can be entrapped. Examples include synthetic inorganic and organic compounds, proteins and peptides, polysaccharides and other sugars, lipids, and nucleic acid sequences having therapeutic, prophylactic or diagnostic activities. Proteins are defined as consisting of 100 amino acid residues or more; peptide are less than 100 amino acid residues. Unless otherwise stated, the term protein refers to both proteins and peptides. The agents to be incorporated can have a variety of biological activities, such as vasoactive agents, neuroactive agents, hormones, anticoagulants, immunomodulating agents, cytotoxic agents, antibiotics, antivirals, antisense, antigens, and antibodies. In some instances, the proteins may be antibodies or antigens which otherwise would have to be administered by injection to elicit an appropriate response. Representative polymers including proteins, peptides, polysaccharides, nucleic acid molecule, and combinations thereof.
Preferred peptides and proteins include hormones, cytokines and other immunomodulatory peptides, and antigens/vaccines. In a preferred embodiment, the active agent is monomeric insulin or a stabilized form of insulin which has been purified to remove zinc. In another preferred embodiment, the active agent is glucagon.
The active agent, or drug, can be an antigen, where the molecule is intended to elicit a protective immune response, especially against an agent that preferentially infects the lungs, such as mycoplasma, bacteria causing pneumonia, and respiratory synticial virus. In these cases, it may also be useful to administer the drug in combination with an adjuvant, to increase the immune response to the antigen.
Any genes that would be useful in replacing or supplementing a desired function, or achieving a desired effect such as the inhibition of tumor growth, could be introduced using the matrices described herein. As used herein, a "gene" is an isolated nucleic acid molecule of greater than thirty nucleotides, preferably one hundred nucleotides or more, in length. Examples of genes which replace or supplement function include the genes encoding missing enzymes such as adenosine deaminase (ADA) which has been used in clinical trials to treat ADA deficiency and cofactors such as insulin and coagulation factor VIII. Genes which effect regulation can also be administered, alone or in combination with a gene supplementing or replacing a specific function. For example, a gene encoding a protein which suppresses expression of a particular protein-encoding gene, or vice versa, which induces expresses of a protein-encoding gene, can be administered in the matrix. Examples of genes which are useful in stimulation of the immune response include viral antigens and tumor antigens, as well as cytokines (tumor necrosis factor) and inducers of cytokines (endotoxin), and various pharmacological agents.
Other nucleic acid sequences that can be utilized include antisense molecules which bind to complementary DNA to inhibit transcription, ribozyme molecules, and external guide sequences used to target cleavage by RNAase P.
As used herein, vectors are agents that transport the gene into targeted cells and include a promoter yielding expression of the gene in the cells into which it is delivered. Promoters can be general promoters, yielding expression in a variety of mammalian cells, or cell specific, or even nuclear versus cytoplasmic specific. These are known to those skilled in the art and can be constructed using standard molecular biology protocols. Vectors increasing penetration, such as lipids, liposomes, lipid conjugate forming molecules, surfactants, and other membrane permeability enhancing agents are commercially available and can be delivered with the nucleic acid.
Imaging agents including metals, radioactive isotopes, radioopaque agents, fluorescent dyes, and radiolucent agents also can be incorporated. Examples of radioisotopes and radioopaque agents include gallium, technetium, indium, strontium, iodine, barium, and phosphorus.
Impurities which can be removed from the active agent composition include metal ions such as zinc, and other di- or multi-valent ions, and small inorganic molecules and solvent residuals.
B. Diketopiperazines
Diketopiperazines useful in the present compositions and methods are described, for example, in U.S. Pat. No. 6,071,497, which is incorporated herein in its entirety.
(i). General Formula
The diketopiperazines or their substitution analogs are rigid planar rings with at least six ring atoms containing heteroatoms and unbonded electron pairs. One or both of the nitrogens can be replaced with oxygen to create the substitution analogs diketomorpholine and diketodioxane, respectively. Although it is possible to replace a nitrogen with a sulfur atom, this does not yield a stable structure.
The general formula for diketopiperazine and its analogs is shown below.
Wherein n is between 0 and 7, Q is, independently, a C1-20 straight, branched or cyclic alkyl, aralkyl, alkaryl, alkenyl, alkynyl, heteroalkyl, heterocyclic, alkyl-heterocyclic, or heterocyclic-alkyl; T is —C(O)O, —OC(O), —C(O)NH, —NH, —NQ, —OQO, —O, —NHC(O), —OP(O), —P(O)O, —OP(O)2, —P(O)2O, —OS(O)2, or —S(O)3; U is an acid group, such as a carboxylic acid, phosphoric acid, phosphonic acid and sulfonic acid, or a basic group, such as primary, secondary and tertiary amines, quaternary ammonium salts, guanidine, aniline, heterocyclic derivatives, such as pyridine and morpholine, or a zwitterionic C1-20 chain containing at least one acidic group and at least one basic group, for example, those described above, wherein the side chains can be further functionalized with an alkene or alkyne group at any position, one or more of the carbons on the side chain can be replaced with an oxygen, for example, to provide short polyethylene glycol chains, one or more of the carbons can be functionalized with an acidic or basic group, as described above, and wherein the ring atoms X at positions 1 and 4 are either O or N.
As used herein, "side chains" are defined as Q-T-Q-U or Q-U, wherein Q, T, and U are defined above.
Examples of acidic side chains include, but are not limited, to cis and trans —CH═CH—CO2H, —CH(CH3)═CH(CH3)—CO2H, —(CH2)3—CO2H, —CH2CH(CH3)—CO2H, —CH(CH2CO2H)═CH2, -(tetrafluoro)benzoic acid, -benzoic acid and —CH(NHC(O)CF3)—CH2—CO2H.
Examples of basic side chains include, but are not limited to, -aniline, -phenyl-C(NH)NH2, -phenyl-C(NH)NH(alkyl), -phenyl-C(NH)N(alkyl)2 and —(CH2)4NHC(O)CH(NH2)CH(NH2)CO2H.
Examples of zwitterionic side chains include, but are not limited to, —CH(NH2)—CH2—CO2H and —NH(CH2)1-20CO2H.
The term aralkyl refers to an aryl group with an alkyl substituent.
The term heterocyclic-alkyl refers to a heterocyclic group with an alkyl substituent.
The term alkaryl refers to an alkyl group that has an aryl substituent.
The term alkyl-heterocyclic refers to an alkyl group that has a heterocyclic substituent.
The term alkene, as referred to herein, and unless otherwise specified, refers to an alkene group of C2 to C10, and specifically includes vinyl and allyl.
The term alkyne, as referred to herein, and unless otherwise specified, refers to an alkyne group of C2 to C10.
As used herein, "diketopiperazines" includes diketopiperazines and derivatives and modifications thereof falling within the scope of the above-general formula.
Fumaryl diketopiperazine is most preferred for pulmonary applications.
(ii). Synthesis
Diketopiperazines can be formed by cyclodimerization of amino acid ester derivatives, as described by Katchalski, et al., J. Amer. Chem. Soc. 68:879-80 (1946), by cyclization of dipeptide ester derivatives, or by thermal dehydration of amino acid derivatives in high-boiling solvents, as described by Kopple, et al., J. Org. Chem. 33(2):862-64 (1968), the teachings of which are incorporated herein. 2,5-diketo-3,6-di(aminobutyl)piperazine (Katchalski et al. refer to this as lysine anhydride) was prepared via cyclodimerization of N-epsilon-P-L-lysine in molten phenol, similar to the Kopple method in J. Org. Chem., followed by removal of the blocking (P)-groups with 4.3 M HBr in acetic acid. This route is preferred because it uses a commercially available starting material, it involves reaction conditions that are reported to preserve stereochemistry of the starting materials in the product and all steps can be easily scaled up for manufacture.
Diketomorpholine and diketooxetane derivatives can be prepared by stepwise cyclization in a manner similar to that disclosed in Katchalski, et al., J. Amer. Chem. Soc. 68:879-80 (1946).
Diketopiperazines can be radiolabelled. Means for attaching radiolabels are known to those skilled in the art. Radiolabelled diketopiperazines can be prepared, for example, by reacting tritium gas with those compounds listed above that contain a double or triple bond. A carbon-14 radiolabelled carbon can be incorporated into the side chain by using 14C labeled precursors which are readily available. These radiolabelled diketopiperazines can be detected in vivo after the resulting microparticles are administered to a subject.
(a) Synthesis of Symmetrical Diketopiperazine Derivatives
The diketopiperazine derivatives are symmetrical when both side chains are identical. The side chains can contain acidic groups, basic groups, or combinations thereof.
One example of a symmetrical diketopiperazine derivative is 2,5-diketo-3,6-di(4-succinylaminobutyl)piperazine. 2,5-diketo-3,6-di(aminobutyl) piperazine is exhaustively succinylated with succinic anhydride in mildly alkaline aqueous solution to yield a product which is readily soluble in weakly alkaline aqueous solution, but which is quite insoluble in acidic aqueous solutions. When concentrated solutions of the compound in weakly alkaline media are rapidly acidified under appropriate conditions, the material separates from the solution as microparticles.
Other preferred compounds can be obtained by replacing the succinyl group(s) in the above compound with glutaryl, maleyl or fumaryl groups.
(b) Synthesis of Asymmetrical Diketopiperazine Derivatives
One method for preparing unsymmetrical diketopiperazine derivatives is to protect functional groups on the side chain, selectively deprotect one of the side chains, react the deprotected functional group to form a first side chain, deprotect the second functional group, and react the deprotected functional group to form a second side chain.
Diketopiperazine derivatives with protected acidic side chains, such as cyclo-Lys(P)Lys(P), wherein P is a benzyloxycarbonyl group, or other protecting group known to those skilled in the art, can be selectively deprotected. The protecting groups can be selectively cleaved by using limiting reagents, such as HBr in the case of the benzyloxycarbonyl group, or fluoride ion in the case of silicon protecting groups, and by using controlled time intervals. In this manner, reaction mixtures which contain unprotected, monoprotected and di-protected diketopiperazine derivatives can be obtained. These compounds have different solubilities in various solvents and pH ranges, and can be separated by selective precipitation and removal. An appropriate solvent, for example, ether, can then be added to such reaction mixtures to precipitate all of these materials together. This can stop the deprotection reaction before completion by removing the diketopiperazines from the reactants used to deprotect the protecting groups. By stirring the mixed precipitate with water, both the partially and completely reacted species can be dissolved as salts in the aqueous medium. The unreacted starting material can be removed by centrifugation or filtration. By adjusting the pH of the aqueous solution to a weakly alkaline condition, the asymmetric monoprotected product containing a single protecting group precipitates from the solution, leaving the completely deprotected material in solution.
In the case of diketopiperazine derivatives with basic side chains, the basic groups can also be selectively deprotected. As described above, the deprotection step can be stopped before completion, for example, by adding a suitable solvent to the reaction. By carefully adjusting the solution pH, the deprotected derivative can be removed by filtration, leaving the partially and totally deprotected derivatives in solution. By adjusting the pH of the solution to a slightly acidic condition, the monoprotected derivative precipitates out of solution and can be isolated.
Zwitterionic diketopiperazine derivatives can also be selectively deprotected, as described above. In the last step, adjusting the pH to a slightly acidic condition precipitates the monoprotected compound with a free acidic group. Adjusting the pH to a slightly basic condition precipitates the monoprotected compound with a free basic group.
Limited removal of protecting groups by other mechanisms, including but not limited to cleaving protecting groups that are cleaved by hydrogenation by using a limited amount of hydrogen gas in the presence of palladium catalysts. The resulting product is also an asymmetric partially deprotected diketopiperazine derivative. These derivatives can be isolated essentially as described above.
The monoprotected diketopiperazine is reacted to produce a diketopiperazine with one sidechain and protecting group. Removal of protecting groups and coupling with other side chains yields unsymmetrically substituted diketopiperazines with a mix of acidic, basic, and zwitterionic sidechains.
Other materials that exhibit this response to pH can be obtained by functionalizing the amide ring nitrogens of the diketopiperazine ring.
C. Transport Enhancers
In a preferred embodiment, the active agent is complexed with a transport enhancer which is degradable and capable of forming hydrogen bonds with the target biological membrane in order to facilitate transport of the agent across the membrane. The transport enhancer also is capable of forming hydrogen bonds with the active agent, if charged, in order to mask the charge and facilitate transport of the agent across the membrane. A preferred transport enhancer is diketopiperazine.
The transport enhancer preferably is biodegradable and may provide linear, pulsed or bulk release of the active agent. The transport enhancer may be a natural or synthetic polymer and may be modified through substitutions or additions of chemical groups, including alkyly, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
A preferred transport enhancer is fumaryl diketopiperazine. Other diketopiperazines which may be useful as a transport enhancer are described above.
Like most proteins and peptides, insulin is a charged molecule, which impedes its ability to cross charged biological membranes. It has been found that when insulin hydrogen bonds to fumaryl diketopiperazine, the charge of the peptide is masked, thereby facilitating or enhancing the passage of insulin across the membranes, such as mucosal membranes, and into the blood.
II. Methods
A. Encapsulation
In one embodiment, active agent is encapsulated within microparticles by dissolving a diketopiperazine with acidic side chains in bicarbonate or other basic solution, adding the active agent in solution or suspension, and then precipitating the microparticle by adding acid, such as 1 M citric acid.
In another embodiment, active agent is encapsulated within microparticles by dissolving a diketopiperazine with basic side chains in an acidic solution, such as 1 M citric acid, adding the active agent in solution or suspension, and then precipitating the microparticle by adding bicarbonate or another basic solution.
In still another embodiment, active agent is encapsulated within microparticles by dissolving a diketopiperazine with both acidic and basic side chains in an acidic or basic solution, adding the active agent in solution or suspension to be encapsulated, then precipitating the microparticle by neutralizing the solution.
The microparticles can be stored in the dried state and suspended for administration to a patient. In the first embodiment, the reconstituted microparticles maintain their stability in an acidic medium and dissociate as the medium approaches physiological pH in the range of between 6 and 14. In the second embodiment, suspended microparticles maintain their stability in a basic medium and dissociate at a pH of between 0 and 6. In the third embodiment, the reconstituted microparticles maintain their stability in an acidic or basic medium and dissociate as the medium approaches physiological pH in the range of pH between 6 and 8.
The impurities typically are removed when the microparticles are precipitated. However, impurities also can be removed by washing the particles to dissolve the impurities. A preferred wash solution is water or an aqueous buffer. Solvents other than water also can be used to wash the microspheres or precipitate the diketopiperazines, in order to remove impurities that are not water soluble. Any solvent in which neither the cargo nor the fumaryl diketopiperazine is soluble are suitable. Examples include acetic acid, ethanol, and toluene.
In an alternative embodiment, microparticles of diketopiperazine are prepared and provided in a suspension, typically an aqueous suspension, to which a solution of the active agent then is added. The suspension is then lyophilized or freeze dried to yield diketopiperazine microparticles having a coating of active agent. In a preferred embodiment, the active agent is insulin in a hexameric form. Zinc ions can then be removed by washing the microparticles with an appropriate solvent.
As used herein, the term "entrapped" with reference to an active agent in/with a diketopiperazine includes coating of the active agent onto microparticles of the diketopiperazine.
The diketopiperazine microparticles have been found to have a higher affinity for insulin than does zinc. Insulin has been found to be stabilized within an ordered lattice array of fumaryl diketopiperazine. In this state, in the sufficient absence of zinc ions, the insulin is predominately dimeric and monomeric, as opposed to it hexameric state. The insulin therefore more readily dissociates to its monomeric state, which is the state in which insulin exerts its biological activity.
Other complexing agents may be substituted for the diketopiperazine. Other representative complexing agents include serum albumin and other proteins, alginic acid, antibodies, cyclodextrins, phospholipids, and lecithin. For example, insulin contaminated with zinc can be complexed with bovine serum albumin. The complex can be dialyzed in tubing with a molecular weight cut-off below 1,000 Daltons to separate and remove the zinc. Once sufficient amounts of zinc have been dialyzed away, as evidenced by its presence in the dialysate, the dispersion is transferred to dialysis tubing with a molecular weight cut-off below 10,000 Daltons. Only monomeric insulin will pass through the tubing into the dialysate, leaving any remaining hexameric zinc complexed insulin behind. The purified insulin can be captured from the dialysate.
These materials may not, however, provide sufficient stabilization of unstable or labile drugs.
B. Administration
The compositions of active agent described herein can be administered to patients in need of the active agent. The compositions preferably are administered in the form of microparticles, which can be in a dry powder form for pulmonary administration or suspended in an appropriate pharmaceutical carrier, such as saline.
The microparticles preferably are stored in dry or lyophilized form until immediately before administration. The microparticles then can be administered directly as a dry powder, such as by inhalation using, for example, dry powder inhalers known in the art. Alternatively, the microparticles can be suspended in a sufficient volume of pharmaceutical carrier, for example, as an aqueous solution for administration as an aerosol.
The microparticles also can be administered via oral, subcutaneous, and intraveneous routes.
The compositions can be administered to any targeted biological membrane, preferably a mucosal membrane of a patient. In a preferred embodiment, the patient is a human suffering from Type II diabetes. In a preferred embodiment, the composition delivers insulin in biologically active form to the patient, which provides a spike of serum insulin concentration which simulates the normal response to eating.
In a preferred embodiment, hexameric insulin is entrapped in fumaryl diketopiperazine to form a solid precipitate of monomeric insulin in the fumaryl diketopiperazine, which then is washed with aqueous solution to remove the free zinc. This formulation demonstrates blood uptake following pulmonary administration at a rate 2.5 times the rate of insulin uptake following subcutaneous injection, with peak blood levels occurring at between 7.5 and 10 minutes after administration.
The range of loading of the drug to be delivered is typically between about 0.01% and 90%, depending on the form and size of the drug to be delivered and the target tissue. In a preferred embodiment using diketopiperazines, the preferred range is from 0.1% to 50% loading by weight of drug. The appropriate dosage can be determined, for example, by the amount of incorporated/encapsulated agent, the rate of its release from the microparticles, and, in a preferred embodiment, the patient's blood glucose level.
One preferred application is in the treatment of hyperinsulinemia. In a preferred embodiment, microparticles of the composition wherein the active agent is glucagon can be administered by continuous subcutaneous infusion. Glucagon is an extremely unstable peptide, but can be stabilized in particles of diketopiperazine, for example. The stabilized glucagon/diketopiperazine microparticles can be made by adding glucagon to a solution of diketopiperazine which hydrogen bonds to the glucagon and when the solution is acidified, such as by adding a food acid, both the diketopiperazine and the glucagon self-assemble to form uniform microspheres having a mean particle size of, for example, about 2 &mgr;m. In this process, approximately 95% of the glucagon is pulled out of solution and is evenly distributed within the diketopiperazine microparticle. These particles can readily be suspended and infused subcutaneously with a standard infusion pump. Then the glucagon/diketopiperazine particles are contacted with the near neutral pH environment of the subcutaneous fluid, where they dissolve, thereby releasing glucagon in its pharmacologically active state.
The compositions and methods described herein are further described by the following non-limiting examples.EXAMPLE 1 Removal of Zinc From U.S.P. Injectable Insulin
Insulin trapped in fumaryl diketopiperazine was analyzed to assess whether zinc was removed during the entrapment process. The insulin used as the starting material met U.S.P. standards for injectable insulin, and according to the certificate of analysis, the insulin contained a considerable quantity of zinc: 0.41%. This insulin was then entrapped in fumaryl diketopiperazine to form a solid fumaryl diketopiperazine/insulin mixture, as described above.
Following entrapment of the insulin in fumaryl diketopiperazine, the amount of zinc theoretically should be present in the same proportion as it existed in the neat insulin. Using the certificate of analysis value, it was calculated that one should expect to find 697 parts per million (ppm) of zinc per gram in the solid yield of fumaryl diketopiperazine/insulin. Surprisingly, the quantity of zinc present the solid fumaryl diketopiperazine/insulin was measured to be only 6 ppm. The "missing" zinc was presumably eliminated with the water used to wash the insulin/fumaryl diketopiperazine precipitate.EXAMPLE 2 Bioavailability of Insulin in Diketopiperazine Pulmonary Formulation
Subjects and Methods
The study was reviewed and approved by the ethical review committee of the Heinrich-Heine-University, Düsseldorf, and conducted according to local regulations, the Declaration of Helsinki and the rules of Good Clinical Practice.
The study was conducted with 5 healthy male volunteers. Inclusion criteria were good health, as judged by physical examination, age: 18 to 40 years, body mass index: 18 to 26 kg/m2, capability to reach peak inspiratory flow of ≧4 I/sec measured by a computer assisted spirometry and a FEV1 equal to or greater than 80% of predicted normal (FEV1=forced expiratory volume in one second). Exclusion criteria were Diabetes mellitus type 1 or 2, prevalence of human insulin antibodies, history of hypersensitivity to the study medication or to drugs with similar chemical structures, history or severe or multiple allergies, treatment with any other investigational drug in the last 3 months before study entry, progressive fatal disease, history of drug or alcohol abuse, current drug therapy with other drugs, history significant cardiovascular, respiratory, gastrointestinal, hepatic, renal, neurological, psychiatric and/or hematological disease, ongoing respiratory tract infection or subjects defined as being smokers with evidence or history of tobacco or nicotine use.
Conduct of the Study
On the morning of the study days, the subjects came to the hospital (fasting, except for water, from midnight onward) at 7:30 a.m. The subjects were restricted from excessive physical activities and an intake of alcohol for 24 hours before each treatment day. They were randomly assigned to one of the three treatment arms. The subjects received a constant intravenous regular human insulin infusion, which was kept at 0.15 mU min−1 kg−1 so that serum insulin concentrations were established at 10-15 &mgr;U/ml during a period of 2 hours before time point 0. This low-dose infusion was continued throughout the test to suppress endogenous insulin secretion. Blood glucose was kept constant at a level of 90 mg/dl throughout the glucose clamp by a glucose controlled infusion system (BIOSTATOR™). The glucose clamp algorithm was based on the actual measured blood glucose concentration and the grade of variability in the minutes before to calculate the glucose infusion rates for keeping the blood glucose concentration constant. The insulin application (5 U i.v. or 10 U s.c. injection or three deep breaths inhalation per capsule (2 capsules with 50 U each) applied with a commercial inhalation device (Boehringer Ingelheim)) had to be finished immediately before time point 0. The duration of the clamp experiment was 6 hours from time point 0. Glucose infusion rates, blood glucose, serum-insulin and C-peptide were measured.
Bioefficacy and Bioavailability
To determine bioefficacy, the areas under the curve of the glucose infusion rates were calculated for the first 3 hours (AUC0-180) after the administration and for the overall observation period of six hours after the administration (AUC0-360) and were correlated to the amount of insulin applied. To determine bioavailability, the areas under the curve of the insulin concentrations were calculated for the first 3 hours (AUC0-180) after the administration and for the overall observation period of six hours after the administration (AUC0-360) and correlated to the amount of insulin applied.
In this clamp study, inhalation of 100 U of TECHNOSPHERE™/Insulin was well tolerated and was demonstrated to have a substantial blood glucose lowering effect with a relative bioavailability of 25.8% for the first three hours as calculated from the achieved serum insulin concentrations. TECHNOSPHERES™ are microparticles (also referred to herein as microspheres) formed of diketopiperazine that of self-assembles into an ordered lattice array at particular pHs, typically a low pH. They typically are produced to have a mean diameter between about 1 and about 5 &mgr;m.
The pharmacokinetic results are illustrated in FIGS. 1 and 2 and in Table 1.
Efficacy Results
Inhalation of 100 U of TECHNOSPHERE™/Insulin (inhalation of 100 U) revealed a peak of insulin concentration after 13 min (intravenous (i.v.) (5U): 5 min, subcutaneous (s.c.) (10 U): 121 min) and a return of the insulin levels to baseline after 180 min (i.v.: 60 min, s.c. 360 min). Biological action as measured by glucose infusion rate peaked after 39 min (i.v. 14 min, s.c.: 163 min) and lasted for more than 360 min (i.v.: 240 min, s.c.: >360 min). Absolute bioavailability (comparison to i.v. application) was 14.6±5.1% for the first 3 hours and 15.5±5.6% for the first 6 hours. Relative bioavailability (comparison to s.c. application) was 25.8±11.7% for the first 3 hours and 16.4±7.9% for the first 6 hours.TABLE 1 Pharmacokinetic Parameters Intravenous Subcutaneous Administration Inhaled Administration Parameter Calculated on Glucose Infusion Rate T50%* 9 min 13 min 60 min Tmax 14 min 39 min 163 min T-50%** 82 min 240 min 240 min T to baseline 240 min >360 min >360 min Parameter Calculated on Insulin Levels T50%* 2 min 2.5 min 27 min Tmax 5 min 13 min 121 min T-50%** 6 min 35 min 250 min T to baseline 60 min 180 min 360 min *time from baseline to half-maximal values **time from baseline to half-maximal after passing Tmax
Safety Results
TECHNOSPHERE™/Insulin was shown to be safe in all patients. One patient was coughing during the inhalation without any further symptoms or signs of deterioration of the breathing system.
Inhalation of 100 U of TECHNOSPHERE™/Insulin was well tolerated and was demonstrated to have a substantial blood glucose lowering effect with a relative bioavailability of 25.8% for the first 3 hours as calculated from the achieved serum insulin concentrations.
In this study, the inhalation of TECHNOSPHERE™/Insulin (the formulation of example 1) was demonstrated in healthy human subjects to have a time-action profile with a rapid peak of insulin concentration (Tmax: 13 min) and rapid onset of action (Tmax: 39 min) and a sustained action over more than 6 hours. The total metabolic effect measured after inhalation of 100 U of TECHNOSPHERE™/Insulin was larger than after subcutaneous injection of 10 U of insulin. The relative bioefficacy of TECHNOSPHERE™/Insulin was calculated to be 19.0%, while the relative bioavailability was determined to be 25.8% in the first three hours.
The data also show that inhalation of TECHNOSPHERE™/Insulin resulted in a much more rapid onset of action than s.c. insulin injection that was close to the onset of action of i.v. insulin injection, while duration of action of TECHNOSPHERE™/Insulin was comparable to that of s.c. insulin injection.
The drug was well tolerated and no serious adverse events were reported during the entire trial.EXAMPLE 3 Removal of Impurity From Proprietary Peptide
A proprietary peptide containing an impurity was trapped in fumaryl diketopiperazine, forming a peptide/fumaryl diketopiperazine precipitate. The precipitate was washed with water to remove the impurity. The peptide is rather unstable and trapping it in fumaryl diketopiperazine markedly improves its stability; both as a dry powder and in aqueous suspension for injection.EXAMPLE 4 Stabilized Glucagon Formulations
Glucagon was formulated under sterile conditions, into a stabilized complex by precipitation in acidic solution with fumaryl diketopiperazine (3,6-bis[N-fumaryl-N-(n-butyl)amino]-2,5-diketopiperazine). The complex was washed and lyophilized, yielding a sterile dry powder formulation of diketopiperazine/glucagon (hereinafter referred to as "TG") containing from 1.2 to 8.2% glucagon by weight, depending upon the formulation parameters desired (allowing physicians to increase dose yet keep the volume constant). The TG powder was suspended in an appropriate media suitable for subcutaneous delivery in a MiniMed 507C infusion pump.
Stability Protocol
Glucagon and TG were suspended in infusion media and incubated at 40° C. in a water bath for varying amounts of time up to 150 hours.
Glucagon HPLC Analysis
An adaptation of USP method for glucagon analysis was employed. A Waters Symmetry Shield RP8 column (5 &mgr;m, 3.9×150 mm) and guard RP8 column (5 &mgr;m, 3.9×20 mm) were used at a flow rate of 1 mL/min. and a detection wavelength of 214 nm. The gradient method consisted of mobile phase A: 9.8 g NaH2PO4 (0.0816 M) and 170 mg L-cysteine (1.4 mM) per liter HPLC grade water, adjusted pH to 2.6 with phosphoric acid; and B: acetonitrile. Glucagon solutions were diluted as needed with water and injected. TG samples were prepared by adding {fraction (1/10)}th volume 1 M Tris pH 10.0 to sample to solubilize the fumaryl diketopiperazine.
Rat Study Protocol
Sprague Dawley rates 200-250 g were fasted overnight and given subcutaneous injection of glucagon or TG (0.75 mg/kg) in an appropriate media that had been held at 25° C. for 0, 24, or 48 hours. Blood samples were taken at −10, −5, 0, 5, 10, 15, 20, 30, 45, and 60 minutes post dose and analyzed for blood glucose (HemCue B-glucose analyzer, Hemocue AB, Angelholm Sweden). Mean baseline was determined (pre-dose measurements) and was subtracted from the subsequent data and plotted vs. time. This was done to assure that the TG formulation, which appeared to not degrade significantly, showed appropriate pharmacological activity.
Following 40° C. incubation, HPLC analysis showed an increase in breakdown products in the glucagon preparation. By contrast, TG has only one minor degradation peak (RT=6) which correlated with the slightly less active oxidative form of glucagon. Glucagon without diketopiperazine (i.e. without TECHNOSPHERES™) had many degradation peaks, some of which contributed to an enhanced effect and others that reduced the potency of glucagon.
The TG sterile lyophilized powder was shipped frozen to a hospital, where it was re-suspended in sterile media. The material re-suspended well and each vial was continuously infused over a 72 hour period.
Standard preparations of glucagon are not suitable for regulation of blood glucose by continuous subcutaneous infusion. Administration of such preparations containing variable amounts of the deamidated and hydrolysed forms resulted in highly variable blood glucose levels. Suspensions of TECHNOSPHERES™/glucagon, which is stabilized, does not aggregate and contains clinically irrelevant amounts of breakdown products. As such TG can be and has been used as a therapy for hyperinsulinemia, providing consistent, elevated glucose levels when administered subcutaneously over time.
Modifications and variations of the present invention will be obvious to those of skill in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.
1. A method for delivering insulin to a patient in need thereof, comprising
- administering to the patient an effective amount of the insulin in a composition which comprises microparticles of a diketopiperazine in which monomeric or dimeric insulin is complexed to the diketopiperazine.
2. The method of claim 1 wherein the diketopiperazine is fumaryl diketopiperazine.
3. The method of claim 2 wherein the composition is in a dry powder form administered to the lungs via inhalation.
4. The method of claim 1 wherein the patient is a Type II diabetic.
5. The method of claim 4 wherein the composition is administered concurrently with, or less than about 20 minutes prior to, the patient eating a meal.
6. The method of claim 1 wherein the composition is provided in one or more unit doses of insulin, each dose equivalent to about 6 IU of insulin.
|October 4, 1994
|Feldstein et al.
|April 2, 1996
|Steiner et al.
|August 20, 1996
|Anderson, Jr. et al.
|March 30, 1999
|Gonda et al.
|November 2, 1999
|June 6, 2000
|Steiner et al.
|August 6, 2002
|Steiner et al.
|September 3, 2002
|Steiner et al.
|0 220 958
|May 1987
|November 1996
|October 1999
- Cerasi, et al. "Decreased sensitivity of the pancreatic beta cells to glucose in prediabetic and diabetic subjects. A glucose dose-response study," Diabetes 21(4): 224-34 (1972).
- Gupta, et al., "Comtemporary approaches in aerosolized drug delivery to the lung," Journal of Controlled Release 17:129-148 (1991).
- Katchalski, et al., "Synthesis of lysine anhydride," J. Amer. Chem. Soc. 68:879-80 (1946).
- Kopple, et al., "A convenient synthesis of 2,5-piperazinediones," J. Org. Chem. 33(2): 862-64 (1968).
- Leahy, "Beta-cell dysfunction in type II diabetes mellitus," Curr. Opin. Endocrinol. Diabetes 2(4): 300-306 (1995).
- Lian, et al., "A self-complementary, self-assembling microsphere system: application for intravenous delivery of the antiepileptic and neuroprotectant compound felbamate," J Pharm Sci 89:867-875 (2000).
- Pfeiffer, "Insulin secretion in diabetes mellitus," Am. J. Med. 70(3): 579-88 (1981).
- Polonsky, et al., "Abnormal patterns of insulin secretion in non-insulin-dependent diabetes mellitus," N. England J. Med. 318(19): 1231-39 (1988).
Type: Grant
Filed: Aug 20, 2002
Date of Patent: Nov 25, 2003
Patent Publication Number: 20030013641
Assignee: MannKind Corporation (Danbury, CT)
Inventors: Solomon S. Steiner (Mount Kisco, NY), Rodney J. Woods (New Hampton, NY), Joseph W. Sulner (Paramus, NJ)
Primary Examiner: Thurman K. Page
Assistant Examiner: Liliana Di Nola-Baron
Attorney, Agent or Law Firm: Holland & Knight LLP
Application Number: 10/224,761
International Classification: A61K/914;


text: Just today I noticed something new covering my iPhone camera lens. It looks like a shutter or aperture blade over the optics, but knowing that the iPhone has neither, I'm really confused. The camera is able to take pictures still, but I can't imagine this would help. I have not dropped the phone to my recent knowledge and I do not believe this "blade" was covering the optics yesterday.

subset: commoncrawl


text:
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text:
Mama Kimchi from Mama is a Korean style noodle. Mama kimchi is a new flavor of Kimchi with a little spicy. The noodle is thick and tender. If you love spicy thing, this mama Kimchi is good to explore. Ingredients: Wheat flour, garlic, oil, chili, dried vegetable. Product of Thailand.
Copyright © 2004 - 2019 GroceryThai.com


text: First they came for the lead in your paint. Then they came for the lead in your gasoline. Then birdshot. Now, following the European Union's ban in 2005, lead wheel weights are to be phased out in California, thanks to a lawsuit by the Oakland-based Center For Environmental Health. It's like these do-gooders think lead is bad for you or something! Seems that 500,000 pounds of wheel weights fall off each year, with much of the lead ending up in drinking water supplies, so it'll be steel or zinc weights for those of us in the Golden State. [Los Angeles Times]


text:
About IDChief: We have been making fake ids for years as articles in google news shows. We were never shut down as some like to conclude, but decided to go on hiatus due to the media, and government attention. Do note you will Card How Id Professional Mobile With - Youtube To Android Design not find one complaint from any customer that we did not fulfill every pending order. Some news articles liked to parrot absurd gov scare tactics to stop customers who might read the article from ordering. Saying customers who have ordered had their credit compromised. Do note again not one complaint online from anyone that we ever did this. Common sense one would conclude this is impossible for us to do. Ordering from us you only provide basic details as when ordering anything online with the exception of your physical description which is defiantly not marketable. 99% of customers request IDChief make the license number for their ids. The 1% that provide one we have no way to know, if real, or not. Even with a valid name, address, and license number this is of no value. To even begin to assume an identity you need the social security number and mothers maiden name something we have no need for to make any id for a customer and never asked for.Cart Get 2018 How Food To License A
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Card How Id Professional Mobile With - Youtube To Android Design About our fake ids: We sell exact replicas of real ids and nothing less. Please do not email asking will your fake id do X or have X. Answer is yes. We correctly format and print 1D and 2D barcodes. We do use our reader/writers to encode the magnetic strip. From microprint, tactile print, laser engrave, and more that a real state card printer is capable of, we too are capable, and do replicate flawlessly. Exact card stocks used for printing the real id. PVC: Just a few cards we offer are printed on PVC as is the real cards. PVC is being phased out for all id making. Teslin: Synthetic paper material that is flexible and tear resistant. This is how the "bend test" originated by bouncers and the like as one way to ensure card authenticity. PolycarbonateBuy Scannable Id Make Fake Ids Alabama Premium - We: Does not come in a solid card stock you load for card printing as one might think like PVC or teslin. Polycarbonate is loaded in punched sheets and each sheet is used for a different part of our printing process same as a real made id. When you see OVD (optically variable device) windows, image shifting designs, as we show you on our site a sheet is used solely for embedding. States that require raised text based on our cloned template tactile print is the last step. Printer heats the carbon in the PC card and raises it to the surface. After a set time, again based on template design, the carbon rises beyond the surface to create tactile text/signatures in black. Adding together during printing each layer has tiny microscopic hairs that fuse together under heat and pressure creating a finished card. When you read all of this above we thank you. We think it is important you know that we know what we are doing. This is our passion and profession ID-Chief makes the best premium fake id.
Card How Id Professional Mobile With - Youtube To Android Design Your Image:This is of the utmost importance and we recommend two ways. One, is get a passport picture taken and scan to upload on the order form. You can ask as some places will email or put on USB for you. Many chain stores that provide passport pictures such as the UPS store, Fedex store, Staples, and Wallgreens. Most independent postal box stores and other pharmacy chains do passport pictures also.
The second way is take it yourself which is what most customers do. Not hard it is just keeping in mind you are replicating an image taken at a DMV. This means office type light, stand about 6 feet away from the camera, no smiling, (Real ID Act Rule, so facial recognition will work to spot you on any government video camera and keep tabs on you in the land of the free) hair as flat as you can make it, stand straight, and look directly into the camera.
Card How Id Professional Mobile With - Youtube To Android Design
We receive images that can only be described as 'what are they thinking'. To post them would be a violation of our customer's privacy, but how we wish you could see just what we are talking about. Please save us both much time of emailing back, and forth, and do your best the first time. Please do not use images on a current id of any kind. They either have security images over the face or are too small to edit in our background which we do for every image. Please do not edit the image yourself as we just said no matter the image we edit to place in our template background.
Do you make X id? If you do not see it available on our site we do not and we do not do one off fake ids. One offs are not worth the cost to you for us to dedicate a designer to make a replica template to print. When the id you want a one off has security laminate this too has to be cloned and printed by our source in laminate rolls. You are looking at thousands for time and cost for a one off.
Due to the Real ID Act all US states have to comply with new rules for what is acceptable to get a real id. For us the issue is the states must have certain security features on the id card to be complaint. Like everything with any government they wait until they no longer can to be complaint. While some states are in compliance, still many states are not. Normally a state comes out with a new style maybe once every 4 or more years. Now, some state is rolling out a new style pretty much every month. We work with our contacts and have a bounty also, if you want to make good, fast money, send us the new style and confirmation images. We like to have at least one fake to start our template to clone it. Confirmation images help to ensure the state has not made tweaks to the style and are staying with initial roll out style or making changes to it. Email us for more info on any country we make fakes for and do not have the state or providence for sale. We make the template from scratch and replicate the laminate depending the card material (polycarbonate does not use overlay rolls). We will eventually have all US states, but you can always consider using an out of state fake, if we do not yet offer the state you live in. Many go with an out of state id for different reasons. One being they move to another state for college and want a fake from their home state. While you are suppose to obtain a driver license from the state you move to within several months many never do. Permanent residency is back in your home state, and school is only temporary, while going back home during breaks making an out of state fake id very common.


text:
Willard van Orman Quine
American philosopher and logician
Willard Van Orman Quine (25 June 1908 – 25 December 2000) was an influential 20th Century American philosopher and logician.
From a Logical Point of View: Nine Logico-Philosophical Essays (1953)Edit
Willard van Orman Quine. From a Logical Point of View: Nine Logico-Philosophical Essays (1953). Harper and Row, New York
- How are we to adjudicate among rival ontologies? Certainly the answer is not provided by the semantical formula "To be is to be the value of a variable"; this formula serves rather, conversely, in testing the conformity of a given remark or doctrine to a prior ontological standard.
- "On What There Is"
- Nonbeing must in some sense be, otherwise what is it that there is not? This tangled doctrine might be nicknamed Plato's beard; historically it has proved tough, frequently dulling the edge of Occam's razor.
- "On What There Is"
- Wyman's overpopulated universe is in many ways unlovely. It offends the aesthetic sense of us who have a taste for desert landscapes.
- "On What There Is", p. 4. a humorous comment on the idea "unactualized possible".
- The word 'definition' has come to have a dangerously reassuring sound, owing no doubt to its frequent occurrence in logical and mathematical writings.
- "Two dogmas of Empiricism", p. 26
- Our argument is not flatly circular, but something like it. It has the form, figuratively speaking, of a closed curve in space.
- "Two Dogmas of Empiricism", p. 26
- Tactically, conceptualism is no doubt the strongest position of the three; for the tired nominalist can lapse into conceptualism and still allay his puritanic conscience with the reflection that he has not quite taken to eating lotus with the Platonists.
- A fancifully fancyless medium of unvarnished news.
- A mocking title for the 'protocol language' imagined by some of the logical positivists, in "Word and Object (1960), section 1
- Creatures inveterately wrong in their inductions have a pathetic but praiseworthy tendency to die before reproducing their kind.
- "Natural Kinds", in Ontological Relativity and Other Essays (1969), p. 126; originally written for a festschrift for Carl Gustav Hempel, this appears in a context explaining why induction tends to work in practice, despite theoretical objections. The hyphen in "praise-worthy" is ambiguous, since it falls on a line break in the source.
- Logic chases truth up the tree of grammar.
- Philosophy of Logic (1970)
- Set theory in sheep's clothing.
- Referring to Second-order logic, in Philosophy of Logic (1970)
- "Yields falsehood when preceded by its quotation" yields falsehood when preceded by its quotation.
- Quine's paradox, in "The Ways of Paradox" in "The Ways of Paradox and other Essays" (1976)
- Necessity resides in the way we talk about things, not in the things we talk about.
- Ways of Paradox and Other Essays (1976), p. 174
The Web of Belief (1970)Edit
- (2nd ed., 1978). With J. S. Ullian
- At root what is needed for scientific inquiry is just receptivity to data, skill in reasoning, and yearning for truth. Admittedly, ingenuity can help too.
- Implication is thus the very texture of our web of belief, and logic is the theory that traces it.
1980s and laterEdit
- It is within science itself, and not in some prior philosophy, that reality is to be identified and described.
- Theories and Things, Cambridge, MA: Harvard University Press. 1981
- We cannot stem linguistic change, but we can drag our feet. If each of us were to defy Alexander Pope and be the last to lay the old aside, it might not be a better world, but it would be a lovelier language.
- Quiddities: An Intermittently Philosophical Dictionary (1987), p. 231
- Possibly, but my concern is that there not be more things in my philosophy than are in heaven and earth.
- Response to being quoted William Shakespeare's statement from Hamlet: "There are more things in heaven and earth… than are dreamt of in your philosophy." As quoted in When God is Gone Everything Is Holy: The Making Of A Religious Naturalist (2008) by Chet Raymo
Quotes about QuineEdit
- I regard Quine as one of our most stimulating philosophers.
- Friedrich Hayek, quoted in David Gordon, "Friedrich Hayek as a Teacher" (2009)


text:
When Land Rover redesigned the mid-level LR4 SUV for 2010, many of my colleagues proclaimed that it was a leaps-and-bounds improvement over the model it replaces, the LR3.
I was not among them. Not because the LR4 isn't a very good vehicle, because it is -- but the LR3 was a solid, functional SUV, too, and though its styling may have been a little dowdy inside and out compared with the LR4, the LR3 got the job done. Years ago, one carried me over a 16,420-foot mountain road in Argentina -- the highest drivable mountain pass in the Western Hemisphere -- without missing a beat, so I've always thought favorably of the LR3.
Here's the problem: The LR3 and the LR4 remind me of a gourmet chef who works at the Pancake Parlor. Sure, the pancakes are great, but you know the cook is capable of so much more.
The vast majority of Land Rovers and their more deluxe siblings, Range Rovers, lead a pampered life in the hands of owners who have no idea what their vehicles are capable of. Case in point: The 2011 LR4's specifications list a "maximum wading depth" of 27.56 inches. That means it can "wade" through standing water that is 27.56 inches deep -- a level just under the headlights, almost to the top of the tires and nearly halfway up the tops of the doors -- without suffering mechanical failure or interior leakage. Try that with your Cadillac Escalade or Lincoln Navigator. (Or, better yet, please don't.)
Yes, Rover products are typically upscale, but much of the price is due to overbuilding them. The huge, thick rubber molding around the doors, for instance, is one reason you can drive an LR4 into more than 2 feet of water, and molding like that adds to the price, but probably few buyers have even noticed it.
The three LR4 models -- the base, the HSE and the HSE Lux -- are all four-wheel-drive and have the same engine and transmission. The differences between the $47,650 LR4, the $51,950 HSE and the $56,800 HSE Lux are the level of premium features.
On the road, the LR4 has a very smooth, surprisingly quiet ride. Though it's heavy at 5,617 pounds and fairly tall, it does not feel tipsy on winding roads, something that could not always be said for older Rover SUVs. There is a full roster of safety features, too, including all-terrain electronic stability control -- some other stability control systems on SUVs don't really work off-road -- plus six airbags.
Off the road, well, this is a Land Rover. Standard equipment includes a "terrain response system," with settings for snow, mud, sand, slow rock-crawling and general driving conditions. The test vehicle's air suspension raises and lowers the LR4 to the proper height per conditions.
The LR4's face-lift does indeed make it look more like a member of the Range Rover family than the sensible-shoes LR3 ever did, and this engine and transmission bolster the LR4 package to where it may create a problem for the company: Just as the Porsche Cayman is such a nice car that it's difficult to justify the extra expense for the top-of-the-line Porsche 911, I'm not sure why I'd bypass the LR4 for the top-of-the-line Range Rover, which starts at $79,685.


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Glendale Community CollegeHome Menu
District Policies
Weapons on Campus
Weapons are not allowed on campus at any time. Weapons include but are not limited to firearms, tasers, pellet guns, BB guns, batons, knives, dirks, razors, or nunchulks. Persons bringing weapons to campus will be arrested and prosecuted.
Administrative Regulation 3530 Weapons on Campus
Sexual and Other Assaults on Campus Policy
Administrative Regulation 3540 Sexual and Other Assaults on Campus
Registration of Sex Offenders
State law requires that all sex offenders attending the college register with the campus police department within 5 business days of enrollment or employment with the college, in addition to registering with the police department of the city they reside in. Information on registered sex offenders attending the campus may be obtained by viewing the California Department of Justice Online Megan's Law Website at www.meganslaw.ca.gov, or by calling the California Department of Justice Sex Offender Tracking Program at (916) 227-4974.
Timely Warnings
The GCC Police Department, in conjunction with the Office of the Superintendent/President will provide safety warnings to all parties involved, including employees, students, and members of the community whenever there is a credible threat to their safety. This will be done in accordance to State and Federal laws regarding privacy, the California Public Records Act and the Family Educational Rights and Privacy Act. For information regarding timely warnings, please contact the GCC Police at (818) 551-5205.
Administrative Regulation 3515 Reporting of Crime
Employee Drug Free Workplace
Administrative Regulation 7331 Drug Free Workplace
Smoking on Campus Information
It is the policy of the Glendale Community College District to not allow smoking on campus. This policy applies to all faculty, staff, students and the general public. View the complete policy.
Skateboarding/Rollerblading/Bicycles and Motorcycles on Campus
No person shall ride a skateboard, roller-skates, rollerblades or non motorized scooter on District property. No person shall operate a bicycle in excess of 5 mph while on District property. Bicycles and motorized bicycles shall not be operated on pedestrian walkways or in buildings. Bicycles and motorized bicycles must park only in designated areas. Motorcycles are not permitted to drive in any area that motor vehicles are prohibited from entering except designated motorcycle parking areas.
See Board Policy 6750 and Administrative Regulation 6750.
Animals on Campus Policy
Glendale Community College District is committed to providing its employees, students, and visitors with a healthy environment in which to work and study.
No animals are allowed on campus except those animals that are individually trained to provide services to individuals with disabilities.
See Board Policy 3535 and Administrative Regulation 3535.
Complete list of District Policies and Administrative Regulations


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Germans are now able to release pent up anger by calling up and verbally attacking a telephone operator—thanks to two German entrepreneurs who recently launched the "Schimpf-los" (swear away) hotline.
Motivated by their own stressful lives, the hotline's creators wanted to help others avoid arguments with loved ones and co-workers.
"If you're stressed out at work, you go home and your partner gets an earful even though it's not her fault," co-founder Ralf Schulte told Reuters.
The operators are available seven days a week and are trained to provoke callers if they stammer or their insults are mediocre. They egg on angry customers on with lines like, "That's the third time I've heard that today - is that all you've got?"
The hotline's 1.49 euros a minute is a "bargain," Schulte said.
"We don't judge people who are angry. It happens. It's natural. With us you can blow off steam no strings attached," the 41-year-old co-founder said.


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CHICAGO – Anze Kopitar scored, then Jarrett Stoll got a short-handed goal. There was another score in the final second of the second period.
Once the Kings got going, the Chicago Blackhawks just couldn't stop them.
Brown converted his own rebound with 1:27 remaining and the Kings scored all their goals in the final two periods of a 5-4 victory over the Blackhawks on Monday night.
"It was just a team effort," Kopitar said. "Going into the game we knew we had to get 20 guys on board with a team effort was the intention we had. We just stuck with it."
It was Los Angeles' highest-scoring game since a 5-2 victory over San Jose on March 16 and came on the heels of consecutive shutout losses to Dallas and Vancouver. The scoreless stretch reached 150 minutes, 35 seconds before Kopitar picked up his 10 th goal of the season at 4:21 in the second.
"We got good games out of our third and fourth lines, and Brownie scored a big goal at the end," Kings coach Darryl Sutter said. "They're probably not happy with the way they played, and if we'd have lost, we wouldn't have been happy with the way we played."
Brown's first attempt went off the back of Blackhawks defenseman Johnny Oduya, but it bounced right back to the captain, who sent it past Corey Crawford for his 12 th of the season. Kopitar set up the score by winning a faceoff in Chicago's end.
"We didn't respond to the challenge that was there tonight and I was disappointed with how we played and what we gave them," Blackhawks coach Joel Quenneville said.
Michael Frolik had two goals and an assist for Chicago, which has lost two in a row. Patrick Kane and Jonathan Toews also scored, and Duncan Keith finished with three assists.
The Blackhawks were without right wing Marian Hossa for the second straight game due to an upper-body injury. Hossa isn't expected to play Tuesday night against Calgary, but could return this weekend.
Toews gave Chicago the lead when he beat Jonathan Quick with a backhander early in the third period, but Tyler Toffoli responded with a tying snap shot with 11:54 remaining.
Dwight King also scored for Los Angeles, which will be honored at the White House on Tuesday for winning the Stanley Cup last season.
"We kept battling," Toffoli said. "Pucks started going in. We'd lost two at home, so we wanted to get the road trip going well. Now it's important to carry it into the next game."
Kopitar picked up a loose puck, skated in and sent a beautiful shot over a sliding Oduya and into the upper right corner of the goal to tie it at 1 in the second. It was the first goal for Los Angeles since Stoll scored a power-play goal in the third period of a 3-2 victory over Phoenix last Tuesday.
After Stoll and Frolik exchanged scores in the middle of the period, Kane gave the Blackhawks the lead when he waited until the perfect moment to slide the puck past a prone Quick on the left side of the net for his team-best 17 th of the season.
Los Angeles needed just 40 seconds to respond, getting the tying goal at 19:59 of the period. Drew Doughty made a strong push that was denied by Crawford, but the rebound went off King's skate and in for his fourth on the year.
And just like that, the Kings had equaled their output from the previous three games in just one period.
"For them to tie it up I think gave them the momentum," Kane said. "But we came back and took the lead and obviously didn't play very well with the lead. Tough loss for sure."
The Blackhawks used a quick burst of precision passing to jump in front 5:55 into the game. Toews won a faceoff in the Kings' end back to Keith, who quickly sent it forward to Brandon Saad. The rookie forward then passed it to the middle to Frolik, who whipped it past Quick for his first goal since Chicago's 5-2 victory at Los Angeles in the season opener on Jan. 19.
Chicago had a chance to add to the lead, but was unable to score during a four-minute power play in the first. Los Angeles hasn't allowed a power-play goal since the second period of a 4-3 loss to San Jose on March 14, killing off the past 17 chances it has faced.
"We worked hard," Brown said. "We didn't get discouraged. We battled against different things each period. The four-minute kill was huge in the first. It gave us a chance to battle back."
NOTES: Blackhawks LW Brandon Bollig and Kings C Jordan Nolan dropped their gloves and ditched their helmets for a fight in the second period. Bollig patted Nolan on the back of the head after it was broken up by the officials, then skated over to the penalty box and pounded the glass as the door closed. … The Kings have never been shut out in three consecutive games. … LW Daniel Carcillo and D Sheldon Brookbank also were scratched for the Blackhawks. … RWs Brad Richardson and Dustin Penner, along with D Davis Drewiske, were the scratches for Los Angeles.


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"Employment in heavy construction is projected to increase about as fast as the industry average. Growth is expected in highway, bridge, and street construction, as well as in repairs to prevent further deterioration of the Nation's highways and bridges.
Employment in special trades contracting, the largest segment of the industry, should grow at about the same rate as the entire construction industry. Demand for special trades subcontractors in building and heavy construction is rising, and, at the same time, more workers will be needed to repair and remodel existing homes."1
Three sites have information on heavy and highway construction. We highlight the sites and the content we think you'll find useful as you explore your career options in this area of construction.
For a quick overview of what each site below offers, click the link.
1 (Source: Bureau of Labor Statistics, U.S. Department of Labor, Career Guide to Industries, 2002-03 Edition, Construction, on the Internet at http://www.bls.gov/oco/cg/cgs003.htm)


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(ST. LOUIS) October 16, 2012 – Ascension Health has entered into negotiations with HCA Midwest Health System to sell St. Joseph and St. Mary's Medical Centers and other Carondelet Health subsidiaries in Kansas City, Mo. The three Carondelet Health long-term care facilities, Carondelet Manor, Villa St. Joseph, St. Mary's Manor, currently operated by Carondelet Health and Benedictine Health System, and the two hospital Foundations, would remain operated by Ascension Health.
"The decision to enter into these negotiations was made following a period of review and discernment by both the Carondelet Health Board and Ascension Health. This action supports our focus on providing a Vital Presence in the communities we serve, meaning we serve where we are needed and how we are needed to be there," said Robert J. Henkel, FACHE, President and Chief Executive Officer, Ascension Health. "Ascension Health will continue to serve the community by helping to meet the growing need for senior services."
"Being a part of a system with a large local presence would strengthen Carondelet's position in this competitive market which is critical to ensure the system's long-term success amidst the myriad of changes that are occurring as part of health care reform," said Fleury Yelvington, President and Chief Executive Officer of Carondelet Health.
If the negotiations lead to a sale, HCA Midwest has expressed a commitment to working with the local Diocese to preserve the hospitals' Catholic heritage.


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Not to brag, but we know our way around nail polish. From solid shades and classic French tips to jazzy colorblocking and full-on masterpieces, our digits have been painted to the
(half) moon and back. But, just when we thought we'd seen and tried everything, we laid eyes on Sally Hansen's I Heart Nail Art Studs Kit. Welcome to the world of 3-D embellishments, friends.
Not to be confused with a regular nail-art set, this kit includes everything you need for tips with texture, depth, and a serious cool factor. Watch the video above to see how our model gets a perfectly embellished mani with nothing more than a few studs and a bottle of Camelflage* polish — no 3-D glasses needed.
*Available in June.
Like this post? There's more. Get tons of beauty tips, tutorials, and news on the Refinery29 Beauty Facebook page!


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A panel exhibit on the "Ellis Island of the West," where one million immigrated from 80 countries between 1910 and 1940. Sponsored by the Angel Island Immigration Station Foundation.
Related Program:
Carved in Silence: Film screening of the dramatic story of Angel Island, the "Ellis Island of the West." After the Chinese Exclusion Act (1882), potential immigrants suffered detainment and vigorous interrogation for up to two years on this small island within sight of San Francisco. This documentary features scenes recreated in the actual barracks and interviews with former detainees. Film producer and director Felicia Lowe will give a short talk and answer question.
Wednesday, June 9
Latino/Hispanic Meeting Room, Main Library, Lower Level


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2000 Toyota Tacoma Vent Visors & Window Deflectors
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Great British trainer Sir Henry Cecil dies at 70
LONDON (AP) — Henry Cecil, who trained unbeaten superstar Frankel and was one of British horse racing's greatest trainers in a career spanning nearly half a century, died Tuesday following a long battle with cancer. He was 70.
Cecil's death was announced on the website of Warren Place Stables, where the Scotsman worked as a trainer for 44 years.
Knighted by Queen Elizabeth II in 2011, Cecil was champion trainer in Britain 10 times. He capped his career by training one of racing's all-time great horses, Frankel, who was retired last year after winning all 14 of his races.
"His unique talents as one of Britain's greatest racehorse trainers, epitomized by his successes with Frankel, have played a major part in growing the sport's profile around the world, for which we will be forever in his debt," said Rod Street, chief executive of the British Champions Series.
The popular Cecil, who hailed from an aristocratic background and started his racing career as an assistant to his stepfather, was diagnosed with stomach cancer in 2006. He looked frail in his last few public appearances, speaking in a whisper.
After capturing his first classic winner at the 2,000 Guineas with Bolkonski in 1975, he trained four Derby winners and the same number of winners in the St. Leger.
In 1985, Cecil's Oh So Sharp became the first filly since 1955 to win English racing's three classics — the 1,000 Guineas, Oaks and St. Leger.
American jockey Steve Cauthen rode Oh So Sharp to England's Triple Crown in 1985 at the start of their successful six-year partnership with Cecil.
"He was a super intelligent guy and really knew how to place his horses," Cauthen said. "He tried to have fun. The atmosphere during most of the time I was up at Warren Place was just fantastic.
"It was all due to him. It was a team effort but everyone looked to him. He was the one making decisions about where to run the horses."
A minute's silence was held before each of the four meetings in Britain on Tuesday. More tributes are expected next week at Royal Ascot where Cecil claimed a record 75 victories.
"His record as a trainer was one of almost unparalleled achievement, but more than that he generated a level of affection from both racing's participants and followers that few others in sport can ever have matched," British Horseracing Authority chairman Paul Roy said.
Cecil was born into a racing family and started off at 21 as assistant to his stepfather, the royal trainer Cecil Boyd-Rochfort.
When Boyd-Rochford retired in 1968, Cecil took over his stables and the first of 10 champion trainer titles came in 1976.
But there were low points later on. Sheikh Mohammed removed all of his horses from Cecil's Warren Place yard in 1995, and an acrimonious split with jockey Kieren Fallon came four years later.
By 2000, Cecil's career faltered and there was speculation he could retire.
But Cecil bounced back, claiming 25 British winners in 2006. The following year, Light Shift won the Oaks for Cecil's first classic win since 1999.
A series of other Group One triumphs followed, with Frankel providing a fitting finale to Cecil's stellar training career by winning all 14 races before being declared among the best racehorses of all time.
"He went through a rough patch, but he came back," Cauthen said. "To have a horse like Frankel and finish off his career when he wasn't in great health was incredible. He did a perfect job in making Frankel the horse he was."
The British Horseracing Authority has granted a temporary license to Cecil's wife, Jane.
He is survived by two children from his first marriage, Katie and Noel, and son Jake from his second marriage.