US20070275069A9 - Hydrophobic biomolecular structure - Google Patents
Hydrophobic biomolecular structure Download PDFInfo
- Publication number
- US20070275069A9 US20070275069A9 US11/179,673 US17967305A US2007275069A9 US 20070275069 A9 US20070275069 A9 US 20070275069A9 US 17967305 A US17967305 A US 17967305A US 2007275069 A9 US2007275069 A9 US 2007275069A9
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- rhgh
- biostructure
- polymer
- hydrophobe
- polar
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH], i.e. somatotropin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
Definitions
- the present invention relates to a new hydrophobe biomolecular structure, a method for the preparation of the structure and use of the new structure for the manufacture of a medicament.
- the molecule When a cell should be intro-transfected with DNA and/or a plasmid the molecule should not be bigger than 10 nm and have hydrophobic properties. It also must be stable enough so that the plasmid/DNA can be handled.
- the administration of a peptide to a patient is normally performed by injection, as biological uptake of a peptide by other routes of administration is difficult.
- the molecule to be introduced should not larger than 10 nm and have hydrophobic properties.
- Hyaluronic acid is a naturally occurring glycosaminoglycan consisting of a linear polymer of repeating units of glucuronic acid and N-acetyl-glucosamine.
- the molecular weight can vary over a wide range depending on the source.
- Hy is present in several tissues of animals, and in some organs, such as rooster combs, in concentrations high enough for commercial scale extraction. Such tissue contains Hy of a wide range of molecular weights and during a complex series of extraction, purification and sterilisation-steps, high molecular weight chains are more or less degraded resulting in a final product having a considerably narrower molecular weight range.
- Hy is non-toxic and is decomposed in the body and thus suitable for pharmacological use.
- HEALON® Korean Pharmacia AB, Uppsala, Sweden
- This product is produced as outlined in U.S. Pat. No. 4,141,973 and is an ultra-pure product.
- Hy is known in slow release formulations and in WO 9005522 HA is mentioned as a slow release carrier together with a binding protein for e.g. GH or IGF.
- Low molecular weight hyaluronic-acid LMWHA is known as carrier for pharmaceutical active agents and for pharmaceutical activity by itself.
- EP 522 491 discloses a freeze-dried composition comprising hyaluronic acid and a polypeptide, which is administered by injection after reconstitution of the composition.
- Hyaluronic acid molecular mass 300-5000 kDalton is claimed to increase viral vectors uptake by adjuvant effect page 33.
- Hy binds to the cell receptor and to an adeno-virus construct increasing the contact time which facilitate and increase the efficiency of transfection page 55.
- the goal of the compaction of biomolcules is to improve the oral bioavailability of peptides and thereby avoid the need of parental administration.
- the used procedure will also improve the transfection efficiency of genes into mammalian cells for a stable expression and thereby avoid the need of toxic plasmid-DNA complex resulting in a short living expression and in side effects.
- Peptides are compacted from 2.4 nm to ⁇ 0.3 nm, improving its ability to penetrate across biological membranes e.g. the gut wall and the stratum corneum.
- the plasmid-DNA construct is compacted from 87 nm to ⁇ 10 nm, improving the DNA-structure for free passage through the nuclear pore.
- These compacted structures can be administrated by new routes and thereby avoid the need of parental administration.
- these structures are peptides-such as growth factors, metabolic regulators such as insulin and the like, plasmid-DNA and related bio-active molecules such as naked DNA, RNA and poly-electrolyte structures such us heparin and heparin derivatives.
- the routes include oral-, pulmonary-, nasal-, topical administration and intra cellular trafficking. These routes will make drug application more convenient especially for children. The drug will also be easier to handle which will result in an improved compliance. The cost of production for most of these drugs will be less since there are no requirements for sterile production of drugs administered the suggested way.
- Bioadhesive gels such as bioadhesive gels, liposomes or micro-beads.
- Another approach is to replace peptides and related biological molecules with organic compounds designed for oral delivery and with a molar mass less than 500 Dalton. These structures are screened for activation of soluble receptors in tracks with around 96 probes. In about 75 000 structures can easily be evaluated in a short time. Up til now however no active structure has been found although the screening started for around 10 years ago.
- one embodiment of the present invention is a hydrophobe biomolecular structure containing a polymer and a polar biostructure.
- the structure is derived from collapsing the structure, when the structure is made to pass over the structure's point of collapse. The result will be a compacted hydrophobic structure with buried polar groups and a minimum size, and the polymer is surrounding the biostructure.
- Another embodiment of the present invention is a method for the production of the hydrophobic biomolecular structure.
- the method includes the steps of:
- the acid in step a) is hydrochloric acid, sulphuric acid, phosphoric acid or acetic acid and the pH is less than 3, preferable in the range of 1.0 to 2.5.
- the electrolyte of step b) contains cations such as NH 4 + , K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Fe 3+ and anions such as sulfates, chloride, acetates.
- the hydrophobic biomolecular structure is used as a medicament.
- Hy is normally in the form of loops, but to our surprise, we have found that it can become straight in the presence of protons, H+.
- Hy is here meant Hyaluronan with a molecular weight of 150 kDa in a range of 80-360 kDa.
- a stable complex/polymer is formed by Hy surrounding the plasmid, the peptides respectively when Hy is changing its structure back to a curling like structure when pH is changed to pH 6. It is then possible to dilute the solutions to the desired strength.
- transfection of plasmid/DNA into cells has been performed by either using a signal substance or by passive diffusion.
- a compaction compression of plasmid, which are about 60 to 500 nm, results in a much smaller molecules.
- Transfection with small molecules has-earlier been performed with the use of lipid-amine-complexes or sodium chloride. However, these amines result in a toxic complex for the cell and are thus not suitable. When NaCl alone is used, the complex is not stable.
- the compaction according to our invention occurs when NaCl or Na 2 SO 4 and HCl are mixed with the plasmid.
- the water, which was included in the plasmid, is evacuated and hydrogen bridges are eliminated.
- the molecules become more negatively charged and more hydrophobic.
- This complex is stable for at least 3 month in aqueous solution and is non-toxic.
- Recombinant growth hormone rhGH is here used as an example of a peptide of great medical interest.
- rhGH has a size of about 2.4 nm and cannot be given orally because of its size, its hydrophobic properties and its ease to be bio-graded. This unable rhGH like most other peptides to pass bio-membranes. When rhGH is in a solution at its isoelectric point the molecule is not charged and is hydrophobic. If straightened Hy is added, complexes are formed in which the rhGH molecule is “surrounded” by Hy and the particles are less than 1 nm. These particles appear stable and could be administered orally.
- HI-HPLC assays of none compact (n-compact.) and compact rhGH suggest activity of compact rhGH to be within acceptable limits and of the same magnitude as for of none compact rhGH. No agglomerates or particles are found upon dilution.
- Parental administration in H-x rats of Compact rhGH resulted in a dose response of growth gain and of Tibia elongation.
- None compact rhGH also resulted in a dose response of growth gain and of Tibia elongation in H-x rats. The growth gain and the Tibia elongation were of similar magnitude for compact and none compact rhGH.
- rhGH has been compacted.
- One rhGh unit has been compact from radii of 2.4 nm to 0.22 nm.
- FIGS. 1 to 6 are showing the correlation function and relaxation time distributions of hyaluronic acid.
- FIG. 7 is showing the hydrodynamic radii Rn in nm (0-300) as a function of the concentration of hyaluronic acid in ⁇ g/ml.
- FIG. 8 is illustrating the solubility of rhGH.
- FIG. 9 is illustrating the proton-binding capacity of rhGH as a function of pH.
- FIG. 10 is illustrating total weight gained inc of administrated dose.
- Hy The particle size of Hy was studied upon pH-changes. Dynamic light scattering, 500 mV at Fysicum, Uppsala University, followed three concentrations of Hy 90, 30 and 10 ⁇ g/ml. Malvern, ZetaMaster S, version PCS: v 1.26 was also used to determine pH and ⁇ -potential
- FIGS. 1-6 The data from dynamic light scattering are presented in FIGS. 1-6 .
- the figures are showing the correlation function and relaxation time distributions for the following concentrations of Hy:
- the resulting relaxation time distributions are essentially single-modal under all conditions.
- the peak width decreases systematically with increasing concentration due to a greater signal to noise ratio.
- FIG. 7 is showing the hydrodynamic radii (Rn) in nm (0-300) as a function of concentration of Hy in ⁇ g/ml.
- the calculated hydrodynamic radii ( FIG. 7 ) fall on the same line for the undiluted samples and those diluted with water. Those samples diluted with acid have considerably larger particle size. The true particle sizes in contrast to the apparent values which are influenced by interactions at finite concentrations are 65 nm (water solutions) and 105 (acid diluted). It is noteworthy that the scattered intensities are the same for the undiluted and water diluted samples, but about the double in value for the acid-diluted. For example, 11 kHz for the water-diluted and 26 kHz for the acid-diluted solutions. This is consistent with an increase in particle dimensions in acid-diluted solutions of Hy. This is indicative for that the Hy structure upon strong acid addition is stretching out from a curling cylinder to straight line.
- Hy with a molecular weight of 150 k Dalton becomes positively charged (3.1-16.9 mV) when pH changes from pH 6.5 to 1.5.
- Particle size measurements by dynamic light scattering 500 mV indicate that the Hy structure is stretching out from a curling cylinder to straight line upon addition of a strong acid. pH is changed from 6.5 to 1.5 and the hydrodynamic radii is almost doubled or changed from 65 to 105 nm. TABLE 1 ⁇ - potential, pH and hydrodynamic radii of Hy 150k Dalton upon pH-changes.
- Example 2 illustrates the mechanisms of Hy to obtain a durable compact plasmid. -DNA-structure.
- the gene expression of the plasmid is encoded for Chloramphenicol Acetyl Transferace, CAT.
- Solution 1 Plasmid pRc/CMV-CAT, double strained closed-ring structure ⁇ 6400 base pair, molecular weight 4 250 000 Dalton 200 ⁇ L was used in the concentration of 130 ⁇ g/ml.
- Solution 2 10.4 mg Hy molecular weight 150 000 Dalton was dissolved in 100 ml water in a concentration of 104 ⁇ g/ml.
- Solution 5 1 M HCl
- the aimed final solution: 5 00 ⁇ l was formulated to contain Conc. 500 ⁇ l in ⁇ g/ml ( ⁇ g/ ⁇ l) pRc/CMV-CAT, 200 ⁇ L 0.13 ⁇ g/ ⁇ l 26 ⁇ g 52 (0.052) Hy 150 Da 50 ⁇ L 0.1 ⁇ g/ ⁇ l 5 ⁇ g 10 (0.010) ⁇ 250 ⁇ L 250 ⁇ L is diluted to 500 ⁇ L by pH shift pH 7.4/2.4 and precipitated by NaCl 2 M. Dialysing Procedure
- the tube is softened in distilled water for 1 h.
- the bag is dialysed for 30 h.
- Hy present in strong acid-solutions should be avoided due to a breakdown of the Hy structure and to a loss of the acetyl-group.
- the time to form a complex between Hy and different plasmid in strong acid solution should therefore be limited to around 30 min.
- Hy solution pH 1.75, is used in the studies to form a complex with the plasmid. Samples are dialyzed within 30 min against 0.15 M NaCl to rise the pH.
- Hy-plasmid-complex is formulated to 510 ⁇ L contain in ⁇ g in ⁇ g/ml in nM pRc/CMV-CAT 26 50.98 12.0 Hy 150 000 Dalton 11.1 21.76 145 NaCl* 2.1.4 ⁇ 10 9 4.2 ⁇ 10 9 150 ⁇ 10 6 *The concentration of NaCl is high and will be lowered in the coming studies Physical-Chemical Evaluation
- the methods involve acidification, forming a complex, salting and dialyse treatment, at a molar ratio of plasmid/Hy 150 000 Dalton 0.83-0.85:1.
- Dynamic light scattering identifies the diameter of the complex pRc/CMV-CAT-Hy (7.5 nm).
- Hy-structure Hy pH 8.8 ⁇ -potential 51 to ⁇ 69 mV Hy pH 1.75 1.6 ⁇ 2.3 mV
- Preparation is done with a newly prepared plasmid (conc. 0.67 ⁇ g/ml) with the following changes; a new molar ratio plasmid/Hy 0.1128 (molar ratio plasmid/Hy 0.085), batch size 1020 ⁇ l (5201), pH 1.65 and 1.8(pH 1.75 and 1.08).
- the complexion is based on a positive charging of the Hyaluronan (Hy) structure (HY Dest. W. ⁇ , ⁇ 61.5 ⁇ 8.5 mV/Hy ⁇ , pH 1.65 ⁇ 2.4 ⁇ 2.2 mV, Hy Dest. W. ⁇ , ⁇ 61.5 ⁇ 8.5 mV/Hy ⁇ , pH 1.80 ⁇ 4.0 mV ⁇ 1.9 mV)
- the complex is formed by a pH-shift (pH ⁇ 2/ ⁇ 6,) and an ionic concentration change (NaCl 2 M/0.075M).
- the Plasmid-Hy complex is determined after 3 months of storage at 5-8° C.
- Solution 1 Plasmid pRc/CMV-CAT molecular weight 4 250 kDa, concentration 0.67 ⁇ g/ ⁇ L.
- the original plasmid concentrate is diluted in dist. w. to a concentration of 0.194 ⁇ g/ ⁇ L and frozen at ⁇ 22° C.
- the tube is softened in distilled water for 1 h.
- the bag is dialysed for 30 h.
- Containers for the final solutions, pipettes, pipette tubes, beakers, holders for tubes and other materials were sterilised according to Autoclave program.
- the preparation steps were performed under aseptic conditions in a Laminar Air Flow (LAF-) cabinet, LAF Holten HB 2472 S
- Plasmid-Complex formed by pH shift 1.65(1.63-1.67) ⁇ 6.0, by pH shift 1.80 (1.78-1.79) ⁇ 6.0 and the naked plasmid at pH 6.0
- Time used to dispense the solutions 45 min Total time used step 3-5, 131 min.
- Solubility of peptides was examined as a function of pH.
- the peptides were concentrated by ultra centrifugation at pH 8 and dialysed towards different pH.
- the dialysate in the dialyse-tube was kept in equilibrium with a precipitate of the peptide at different pH for 24 hours at 20° C.
- the precipitate was removed and the solution was filtered through a 0,22 ⁇ m membrane filter.
- the concentration was determined by UV absorption at 276 nm.
- Different buffer systems were used for solubility determination at different pH. These data are used for dissolving and compacting peptides at different pH.
- Recombinant growth hormone 33 mg/ml was used for solubility determination.
- a minimum of solubility was found at pH 5.0 around 1 mg/ml. This occurs at the iso-electric point (pI).
- pI iso-electric point
- the solubility for rhGH rose sharply.
- pH 7.5 rhGH could be concentrated in a Tris-HCl buffer to 160 mg/ml In a sodium-acetate buffer pH 4.0 a concentration of 62.6 mg/ml was observed.
- pI iso-electric point
- This example shows how to improve the hydrophobicity of a peptide structure.
- the hydrophobicity is gained in an acid solution with pH on the acid-side of its pI.
- Two different forms of rhGH were observed in a study to determine buffer capacity.
- One a deprotonised form was obtained in an acid solution.
- the other a protonised form was obtained in an alkaline solution.
- the deprotonised form of rhGH was found to have a stronger proton binding capacity compared to protonised-rhGH.
- a more nonpolar character of the protonised rhGH compared to deprotonize rhGH the former burying the polar groups explains this.
- Recombinant Human Growth Hormone (rhGH molar mass 22124) used in the study was concentrated on DEAE-FF-gel (Pharmacia Biotech AB) and desalted on Sephadex G 25 gel (Pharmacia Biotech AB) to contain 33.3 mg/ml in distilled water. Hydrophobic Interaction Chromatography (HIC) was used to determine purity of rhGH 99.2%. The buffer capacity of rhGH was determined by potentiometric acid-base titration. By adding acid and base to the peptide a protonised and a deprotonised form of rhGH is obtained. These forms are then titrated back with base and acid.
- HIC Hydrophobic Interaction Chromatography
- Acidification of peptides is here used to get a hydrophobic state of the peptide and to improve its oral bioavailability.
- This example illustrates the compaction of rhGH.
- Hy By changing pH to a strong acid-solution (pH 1.5) Hy becomes charged stretching out from a curling cylinder to straight line. At this pH rhGH is easily soluble. The size of the rhGH particles is moderated by the speed of the pH-change and the ionic concentration change from 2 M to 0.15 M NaCl.
- the pH-interval used is 7-1.5-5.0(pI) at a constant ratio of rhGH: Hy.
- Hy stabilise the dispersion in changing its structure back to curling like structure at pH 6-7.
- the preparations were assayed with HI-HPLC.
- Compact rhGH was determined after storage at 5-8° C. for 30 days by light scattering, Z-master. The diameter of the measured particles is given in means of six measurements.
- Hy 20.78 mg Hy with a molar mass of 150 KDa was dissolved in 900 ⁇ l distilled water and was allowed to react for more than 1 hour.
- the pH 9.2 of the Hy-solution was adjusted to pH 1.51 with 50 ⁇ l water and 50 ⁇ l 1 M HCl.
- the above-prepared Compact rhGH and None Compact rhGH are used for analytical assay (in situ determined biological activity and for particle sizing.
- concentrations used for particle sizing of Compact rhGH was 7.6 mg/ml and 9.3 mg/ml and for None Compact rhGH 15 mg/ml.
- the measurements, means of six measurements were done at 25° C.
- HI-HPLC of Compact and of None Compact rhGH HI-HPLC assay is commonly used to determine biological activity of rhGH TABLE 16 None Compact Preparation Compact rhGH rhGH Experiment # 4 1 Bag 1 4 1 Bag 2 4 (Bag 1 + 2) Quantitative Assay, mg/ml 7.6 9.3 15.2 Monomer in % 97.3 93.8 98.3 LMWG, % 0.8 0.7 0.7 Clipped Forms in % 1.9 5.5 0.9 Retention Time, min Approved Approved Approved Approved pH in dialyze-tube 7.4 7.4 7.6 Appearance in dialyze-tube cake particles clear
- the ionic concentration was altered from 2 M to 0.15 M NaCl* in Example 6 to in Example 7 1 M [NH 4 ] 2 SO 4 dialysed against 0.15 M NaCl**, and to 1 M Na 2 SO 4 dialysed against 0.15 M NaCl.
- the following anions; phosphates chlorine acetate and cat ions; NH 4 , potassium, magnesium were also evaluated. Only Na 2 SO 4 was considered for further studies.
- the preparations were assayed by HI-HPLC-technique.
- the particle size of compact and none compact rhGH was determined With DynaPro-801 after storage at 5-BOC for 21 days. * “2 M/0.15 M NaCl”
- the solutions ionic properties are changed from 2 M to 0.15 M by dialyse. ** 1 M [NH 4 ] 2 SO 4 was found to give a heavy precipitation and therefore omitted.
- Hy 14.9 mg Hy with a molar mass of 150 KDa was dissolved in 1200 ⁇ l 1 M Na 2 SO 4 and was allowed to react for more than 1 hour.
- the pH of the Hy-solution pH 7.6 was adjusted to pH 1.51 with 100 ⁇ l water and 330 ⁇ l HCl 1 M to a clear solution. HCl was added slowly when passing pH 3.8 a precipitate was observed. 22 mg of lyophilised rhGH was dissolved in the Hy-solution.
- the molar ratio rhGH: Hy is 10:1. In portions of 10 ⁇ l 1M HCl was added to obtain a clear solution. pH at start is 2.77. 40 ⁇ l of 1 M HCl was added to change pH in the solution from 2.77 to 1.52.
- the solution was not totally clear. A loss of the solution was obtained when transferred to the dialyse bag. The final volume was 1240 ⁇ l.
- the solutions were divided in two parts and transferred in two bags prepared as a dialyse tube of MWCO 6-8000, Spectra Pore R. softened in a tris buffer 10 mM pH 7.8.
- the final volume of the solution was adjusted to 1240 ⁇ L.
- the solution was divided in two parts and transferred in two bags (Bag1, Bag2) softened in a tris buffer 10 mM pH 7.8.
- the above-prepared Compact rhGH and None Compact rhGH are used for analytical assay (in situ determined biological activity), in vivo biological activity and for particle sizing.
- concentrations used for particle sizing of Compact rhGH were 5,3 mg and 1.5 mg and for None Compact rhGH 5-10 mg.
- the sizing is given in means of ten measurements and was done at 18, 25 and 30° C.
- Injection solutions of compact and of none compact rhGH were prepared by dilution with Diluent 1.25% Albumin solution to desired strength, low dose 0.04 IU/ml and high dose 0.16 IU/ml. Diluent 1.25% Albumin solution was used as placebo.
- FIG. 10 is illustrating total weight gained in corp of administered dose (treatment of rhGH in i.u./kg body weight for different doses).
- Equation The estimated molecular weight is calculated in DynaPro-801 from R h and the sample temperature using standard curve of MW vs. R h for globular proteins.
- the particle size of Compact and None Compact rhGH was determined at 18, 25 and 30° C. after storage at 5-8° C. for 21 days.
- Compact rhGH was studied at two different concentrations 1.5 mg/ml, and 5.3 mg/ml at 25 and 30° C.
- None Compact rhGH was studied for one concentration, 14.2 mg/ml, at 18, 25 and 30° C. These were the concentrations obtained from the dialyze bags.
- a bimodal function is applied for Compact rhGH and the results present a two-size distribution with its own amplitude, size and MW.
- the distribution is mono-modal and is fully resolved.
- TABLE 21 Particle size of Compact and of None compact rhGH determined with DynaPro-801. Given radii represents mean of 10 measurements Compact rhGH Compact rhGH None Compact rhGH 1.5 mg/ml 5.3 mg/ml 14.2 mg/ml Temp. ° C. 18 25 25 30 18 25 30
- the particle size of Compact rhGH was determined at a concentration of 1.5 mg/ml at 18 and 25° C.; 18° C.
- 100% of the mass contains of a polymer with radii of 5.3 nm and a molar mass of 165 kDa
- 100% of the mass contains of a polymer with radii of 4.9 nm and a molar mass of 142 kDa
- the polymer contains of 6.4 rhGH units (142/22.124) with radii of 0.77 nm (4.9/6.4)
- the particle size of Compact rhGH was determined at a concentration of 5.3 mg/ml at 25 and 30° C. 25° C.
- the polymer contains of 32.4 rhGH units (716/22.124) with radii of 0.29 nm (9.4/32.4) 30° C.
- the polymer contains 50.8 rhGH units (1125/22.124) with a radii of 0.22 nm (11.4/50.8).
- the radii of Compact rhGH are smaller than None Compact rhGH. That is, at a concentration of 5.3 mg/ml one compacted unit of rhGH is estimated to have hydrodynamic radii of 0.22-0.29 nm and at 1.5 mg/ml 0.70-0.77 nm. At a concentration of 14.5 mg/ml None Compact rhGH is estimated to have a hydrodynamic radii of 2.4 nm. The number of units of compacted rhGH contained in a polymer is found to depend on the concentration of the solution. Upon dilution no agglomerate of the polymers were found.
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Abstract
The present invention relates to a new hydrophobe biomolecular structure, which is compacted due to the passing of the structure over its point of collapse, a method for the preparation of the structure and use of the new structure for the manufacture of a medicament.
Description
- The present invention relates to a new hydrophobe biomolecular structure, a method for the preparation of the structure and use of the new structure for the manufacture of a medicament.
- When a cell should be intro-transfected with DNA and/or a plasmid the molecule should not be bigger than 10 nm and have hydrophobic properties. It also must be stable enough so that the plasmid/DNA can be handled.
- The administration of a peptide to a patient is normally performed by injection, as biological uptake of a peptide by other routes of administration is difficult. The molecule to be introduced should not larger than 10 nm and have hydrophobic properties.
- Hyaluronic acid (Hy) is a naturally occurring glycosaminoglycan consisting of a linear polymer of repeating units of glucuronic acid and N-acetyl-glucosamine. The molecular weight can vary over a wide range depending on the source. Hy is present in several tissues of animals, and in some organs, such as rooster combs, in concentrations high enough for commercial scale extraction. Such tissue contains Hy of a wide range of molecular weights and during a complex series of extraction, purification and sterilisation-steps, high molecular weight chains are more or less degraded resulting in a final product having a considerably narrower molecular weight range.
- Hy is non-toxic and is decomposed in the body and thus suitable for pharmacological use.
- A commercial available hyaluronic acid product is HEALON® (Kabi Pharmacia AB, Uppsala, Sweden) which has a average molecular weight of about 4 000 000 Dalton. This product is produced as outlined in U.S. Pat. No. 4,141,973 and is an ultra-pure product. There are many literature references relating to the use of viscoelastic products of HA in ophthalmologic application and the preparation of such products, including the preparation of chemically modified HA.
- Hy is known in slow release formulations and in WO 9005522 HA is mentioned as a slow release carrier together with a binding protein for e.g. GH or IGF.
- Low molecular weight hyaluronic-acid LMWHA is known as carrier for pharmaceutical active agents and for pharmaceutical activity by itself.
- EP 522 491 discloses a freeze-dried composition comprising hyaluronic acid and a polypeptide, which is administered by injection after reconstitution of the composition.
- A patent application, WO 97/15330, claiming Hyaluronic acid as DNA carrier for gene Therapy and VEGF antisense DNA to treat abnormal retinal vascularisation was published May 1st 1997.
- Hyaluronic acid molecular mass 300-5000 kDalton is claimed to increase viral vectors uptake by adjuvant effect page 33.
- The mechanisms is suggested to be of targeting nature, that is, Hy binds to the cell receptor and to an adeno-virus construct increasing the contact time which facilitate and increase the efficiency of transfection page 55.
- The goal of the compaction of biomolcules is to improve the oral bioavailability of peptides and thereby avoid the need of parental administration.
- The used procedure will also improve the transfection efficiency of genes into mammalian cells for a stable expression and thereby avoid the need of toxic plasmid-DNA complex resulting in a short living expression and in side effects.
- Full biological effect of compact peptide is demonstrated in biological assay and in vivo. The size and the durable hydrophobic properties obtained at
pH 6 of the peptide suggest an improved oral bioavailability. Plasmid-DNA complex has been compact from 87 nm to 7.5 nm suggesting an improved transfection efficiency of genes. - Peptides are compacted from 2.4 nm to <0.3 nm, improving its ability to penetrate across biological membranes e.g. the gut wall and the stratum corneum. The plasmid-DNA construct is compacted from 87 nm to <10 nm, improving the DNA-structure for free passage through the nuclear pore. These new hydrophobic structures are obtained by pH-shifts, by elimination of molecular charge and bindings, by evacuation of water and ions, and finally stabilised with Hyaluronan.
- These compacted structures can be administrated by new routes and thereby avoid the need of parental administration. Examples of these structures are peptides-such as growth factors, metabolic regulators such as insulin and the like, plasmid-DNA and related bio-active molecules such as naked DNA, RNA and poly-electrolyte structures such us heparin and heparin derivatives. The routes include oral-, pulmonary-, nasal-, topical administration and intra cellular trafficking. These routes will make drug application more convenient especially for children. The drug will also be easier to handle which will result in an improved compliance. The cost of production for most of these drugs will be less since there are no requirements for sterile production of drugs administered the suggested way.
- Pharmaceutical research is today devoted to improve the ability of the drug to penetrate biological membrane. One approach is to entrap bio-molecules in carrier system such as bioadhesive gels, liposomes or micro-beads.
- Another approach is to replace peptides and related biological molecules with organic compounds designed for oral delivery and with a molar mass less than 500 Dalton. These structures are screened for activation of soluble receptors in tracks with around 96 probes. In about 75 000 structures can easily be evaluated in a short time. Up til now however no active structure has been found although the screening started for around 10 years ago.
- Compacting of well-known peptides such as growth hormones and insulin as well as uncomplicated DNA structures will eliminate expansive and extended toxicological studies. Clinical studies of these compacted structures will also be less complicated than for those studies required for new chemical entities and for entrapped bio-structures in liposome, microspheres and related carrier systems.
- Thus, one embodiment of the present invention is a hydrophobe biomolecular structure containing a polymer and a polar biostructure. The structure is derived from collapsing the structure, when the structure is made to pass over the structure's point of collapse. The result will be a compacted hydrophobic structure with buried polar groups and a minimum size, and the polymer is surrounding the biostructure.
- In a prefered embodyment of the inventive hydrophobic biomolecular structure is:
- the polymer a glycosaminoglycan such as hyaluronic acid or a cationic polysaccharide such as chitosan; and
- the polar biostructure a nucleotide such as plasmid-DNA, DNA or RNA, a peptide such as insulin, growth hormone, recombinat growth hormone, heparin, heparin derivatives or enzyme, or a monoclonal.
- Another embodiment of the present invention is a method for the production of the hydrophobic biomolecular structure. The method includes the steps of:
- a) solubilisation of the polymer and solubilisation of the polar biostructure by addition of an acid;
- b) collapsing the polar biostructure by addition of an electrolyte by passing the polar biostructure over polar biostructure's point of collapse and changing the hydrodynamic randii to a minimum compact size; and
- c) dialysing the biomolecular structure, thereby obtaining a compacted hydrophobic structure with the polar groups buried and the polymer surrounding the biostructure.
- In a prefered embodiment of the inventive method the acid in step a) is hydrochloric acid, sulphuric acid, phosphoric acid or acetic acid and the pH is less than 3, preferable in the range of 1.0 to 2.5.
- In another prefered embodiment of the inventive method the electrolyte of step b) contains cations such as NH4 +, K+, Na+, Ca2+, Mg2+, Zn2+, Fe2+, Fe3+ and anions such as sulfates, chloride, acetates.
- In another embodiment of the present invention the hydrophobic biomolecular structure is used as a medicament.
- Preferred embodiments and other aspects of the present invention are defined in the independent and the dependent claims.
- Hy is normally in the form of loops, but to our surprise, we have found that it can become straight in the presence of protons, H+.
- Thus, when HCl is added to a solution of Hy, the molecules will become straight and will be positively charged.
- By Hy is here meant Hyaluronan with a molecular weight of 150 kDa in a range of 80-360 kDa.
- An optimal compaction (=size) and hydrophobic properties of plasmid (<10 nm) and peptides (<0.3 nm) are found by elimination of charge and molecular bindings by addition of HCl to pH<2 and of different ions. These ions are cationic ions: NH4 +, Keg Na+ and anionic ions: sulfates, chloride, carbonates, acetates.
- A stable complex/polymer is formed by Hy surrounding the plasmid, the peptides respectively when Hy is changing its structure back to a curling like structure when pH is changed to
pH 6. It is then possible to dilute the solutions to the desired strength. - I. Plasmids
- Historically transfection of plasmid/DNA into cells has been performed by either using a signal substance or by passive diffusion. A compaction=compression of plasmid, which are about 60 to 500 nm, results in a much smaller molecules. Transfection with small molecules has-earlier been performed with the use of lipid-amine-complexes or sodium chloride. However, these amines result in a toxic complex for the cell and are thus not suitable. When NaCl alone is used, the complex is not stable.
- The compaction according to our invention occurs when NaCl or Na2 SO4 and HCl are mixed with the plasmid. The water, which was included in the plasmid, is evacuated and hydrogen bridges are eliminated.
- The molecules become more negatively charged and more hydrophobic.
- When the straightened Hy and the compressed plasmid are mixed at low pH and dialysed to a pH of about 6.5 a stable complex is formed with a diameter of about 7.5 nm.
- This complex is stable for at least 3 month in aqueous solution and is non-toxic.
- II Peptides
- Recombinant growth hormone, rhGH is here used as an example of a peptide of great medical interest.
- rhGH has a size of about 2.4 nm and cannot be given orally because of its size, its hydrophobic properties and its ease to be bio-graded. This unable rhGH like most other peptides to pass bio-membranes. When rhGH is in a solution at its isoelectric point the molecule is not charged and is hydrophobic. If straightened Hy is added, complexes are formed in which the rhGH molecule is “surrounded” by Hy and the particles are less than 1 nm. These particles appear stable and could be administered orally.
- Method of Compaction
- By changing pH to a strong, acid solution (pH 1.5) peptides gets easily soluble and Hy becomes a charged structure stretching out from a curling cylinder to straight line. By adding electrolytes to the solution a minimum of charge occurs in the peptide. This results in a total collapse of the peptide structure as it passes its pI (isoelectric point). In regulating the ions (NaCl and Na2SO4) and the rhGH concentrations with the speed of the pH-change when passing the pI. the hydrodynamic radii of the particles in the dispersion of the peptide are changed from 2.4 to 0.22 nm. Hy stabilise the dispersion in changing its structure back to a curling like structure when pH is changed to
pH 6. It is then possible to dilute the dispersions to the concentration desired. The hydrophobic properties of the peptide structure is found to be optimal as pH is changed in a strong acid solution pH<2 and by dialyses transferred to aneutral solution pH 6. - HI-HPLC assays of none compact (n-compact.) and compact rhGH suggest activity of compact rhGH to be within acceptable limits and of the same magnitude as for of none compact rhGH. No agglomerates or particles are found upon dilution. Parental administration in H-x rats of Compact rhGH resulted in a dose response of growth gain and of Tibia elongation. None compact rhGH also resulted in a dose response of growth gain and of Tibia elongation in H-x rats. The growth gain and the Tibia elongation were of similar magnitude for compact and none compact rhGH.
- No growth gain or Tibia elongation was recorded for Placebo.
- The structure of rhGH has been compacted. One rhGh unit has been compact from radii of 2.4 nm to 0.22 nm.
- Full biological effect of compact rhGH is demonstrated in vivo. Hydrophobic properties of rhGH is obtained in acid solutions pH<2 and a durable hydrophobic structure is demonstrated when pH is changed to
pH 6. The size and the hydrophobic properties of the complex suggest an improved oral bioavailability of rhGH. - The present invention will be further described in more detail in the following figures, examples and tables.
- FIGS. 1 to 6 are showing the correlation function and relaxation time distributions of hyaluronic acid.
-
FIG. 7 is showing the hydrodynamic radii Rn in nm (0-300) as a function of the concentration of hyaluronic acid in μg/ml. -
FIG. 8 is illustrating the solubility of rhGH. -
FIG. 9 is illustrating the proton-binding capacity of rhGH as a function of pH. -
FIG. 10 is illustrating procent weight gained in procent of administrated dose. - A substantial buffer capacity in aqueous Hy-solution was observed when diluted acids were added. Strong acid-solutions should be avoided due to breakdown of the polymer Hy-structure and also to loss of the acetyl-group. The time for studies of Hy with different plasmid/peptides should therefore be limited to around 30 min.
- The particle size of Hy was studied upon pH-changes. Dynamic light scattering, 500 mV at Fysicum, Uppsala University, followed three concentrations of
Hy 90, 30 and 10 μg/ml. Malvern, ZetaMaster S, version PCS: v 1.26 was also used to determine pH and ξ-potential - The data from dynamic light scattering are presented in
FIGS. 1-6 . The figures are showing the correlation function and relaxation time distributions for the following concentrations of Hy: - 10 μg/ml (with dilutions with water and acid to 5 μg/ml)
- 30 μg/ml (with dilutions with water and acid to 15 μg/ml)
- 90 μg/ml (with dilutions with water and acid to 45 μg/ml)
- The resulting relaxation time distributions are essentially single-modal under all conditions. The peak width decreases systematically with increasing concentration due to a greater signal to noise ratio.
-
FIG. 7 is showing the hydrodynamic radii (Rn) in nm (0-300) as a function of concentration of Hy in μg/ml. The calculated hydrodynamic radii (FIG. 7 ) fall on the same line for the undiluted samples and those diluted with water. Those samples diluted with acid have considerably larger particle size. The true particle sizes in contrast to the apparent values which are influenced by interactions at finite concentrations are 65 nm (water solutions) and 105 (acid diluted). It is noteworthy that the scattered intensities are the same for the undiluted and water diluted samples, but about the double in value for the acid-diluted. For example, 11 kHz for the water-diluted and 26 kHz for the acid-diluted solutions. This is consistent with an increase in particle dimensions in acid-diluted solutions of Hy. This is indicative for that the Hy structure upon strong acid addition is stretching out from a curling cylinder to straight line. - pH, μ-potential and hydrodynamic radii are presented in Table 1. The μ-potential of the Hy-structure is changed from a negative charge (−59 mV) to positive charge (+3.1-+16.9 mV) as pH is changed from
pH 6 to pH 1-2. - Conclusion: Hy with a molecular weight of 150 k Dalton becomes positively charged (3.1-16.9 mV) when pH changes from pH 6.5 to 1.5. Particle size measurements by dynamic light scattering 500 mV indicate that the Hy structure is stretching out from a curling cylinder to straight line upon addition of a strong acid. pH is changed from 6.5 to 1.5 and the hydrodynamic radii is almost doubled or changed from 65 to 105 nm.
TABLE 1 ξ- potential, pH and hydrodynamic radii of Hy 150k Dalton upon pH-changes. Lightscattering HCl Distilled pH Zeta Master, version PCS: v 1.26 500 mV 1 M Water separate ξ-potential Hydrodynamic Hy in μg/ml in ml in ml measurement KCPs pH in mV radii in nm 10 — 1 5.4-7.8 2844 6.5 −59 65 10 1 1 1.4 2.4 3.1 — 5 1 1 1.6 2542 — 16.9 105 30 — 1 — — — — 65 15 1 1 2.3 — 2.5 — 105 90 — 1 — — — — 65 45 1 1 — — — — 105 - Example 2 illustrates the mechanisms of Hy to obtain a durable compact plasmid. -DNA-structure.
- The gene expression of the plasmid is encoded for Chloramphenicol Acetyl Transferace, CAT.
- Solution 1: Plasmid pRc/CMV-CAT, double strained closed-ring structure −6400 base pair,
molecular weight 4 250 000Dalton 200 μL was used in the concentration of 130 μg/ml. - Solution 2: 10.4 mg Hy
molecular weight 150 000 Dalton was dissolved in 100 ml water in a concentration of 104 μg/ml. - Solution 3: 2 M NaCl
- Solution 3.1: 0.15 M NaCl was used
- Solution 4: 1 M NaOH
- Solution 5: 1 M HCl
The aimed final solution: 5 00 μl was formulated to contain Conc. 500 μl in μg/ml (μg/μl) pRc/CMV-CAT, 200 μL 0.13 μg/μl 26 μg 52 (0.052) Hy 150Da 50 μL 0.1 μg/ μl 5 μg 10 (0.010) Σ 250 μL
250 μL is diluted to 500 μL by pH shift pH 7.4/2.4 and precipitated by NaCl 2 M.
Dialysing Procedure - Dialyse-tube, Spectra/Pore Membrane MWCO 6-9,000 Record No 132645
- 1. The tube is softened in distilled water for 1 h.
- 2. The tube is cut and a dialyse-claim is fitted at one edge of the tube
- 3. 500 μL is filled in the bag. Filling is done with a sterile micro-pipette
- 4. The other edge is fitted with a dialyse-claim
- 5. The bag is dialysed for 30 h.
- 6. Control the integrity of the membrane.
- 7. The dialysed solution is sucked up with a sterile micro-pipette
- 8. Control the volume in μL.
- Preparation Steps
- 1. Thaw at 24° C. 200 μL of the plasmid solution 1 (0.13 μg/μl) 26 μg
- 2. Prepare positive charged Hy.
- pH of
Solution 2 was checked; pH 8.33. - 3. The pH-value of the Hy solutions were studied to obtain a positive ζ,-potential of the Hy structure sees Table 2.
TABLE 2 Acidification of Hy Solution 2 1 M HCl Hy in HCl in μL in μL μg/ml in M pHPrim. pH5 min pH30 min pH30tim 2000 500 83.2 0.2 0.76 ND 0.68 0.81 2000 1000 69.3 0.33 5.19* 2.28 2.93 2.9 2000 1500 59.4 0.42 0.37 ND 0.36 0.47 2000 2000 52 0.5 0.32 ND 0.26 0.39
*pH-value deviates from expected values and was therefore controlled.
-
TABLE 3 Control of deviating pH Solution 2 1 M HCl Hy HCl in μL in μL in μg/ml in M pHPrim. 2000 1000 69.3 0.33 0.85 1000 50 99.0 0.047 1.18 1000 25 101.4 0.024 1.69 - In example 1 it was found that in weak acid solutions the ζ-potential changes from a negatively to a positively charged structure (ζ-potential. from −69.3 to +8.9-+16.9 mV). The results were not consistent that is when the acidity goes towards a lower pH<pH 1.75 the measured ξ-potential indicate an increased negative charged structure of Hy (−4.4, −5.1, −17.9) see table 4.
TABLE 4 Positive charging of the Hy-structure Zeta MasterS Version PCS: v1.26 n = 6 Solution 2 in1 M HCl Hy in HCl -potential μL in μl μg/ml in M pH KCPs in mV 2000 100 99 0.0476 1.3 2301 −8.5 4000 100 101 0.0244 1.75 2241 −4.4 2000 200 94.5 0.0909 1.08 2088 −5.1 2000 1000 69.3 0.33 0.53 1900 −17.5 - Hy present in strong acid-solutions should be avoided due to a breakdown of the Hy structure and to a loss of the acetyl-group. The time to form a complex between Hy and different plasmid in strong acid solution should therefore be limited to around 30 min.
- Solution to form a complex.
- 3. Hy solution, pH 1.75, is used in the studies to form a complex with the plasmid. Samples are dialyzed within 30 min against 0.15 M NaCl to rise the pH.
- In this study 101 μg/ml Hy, pH 1.75, was chosen for preparing the compaction, although the measured ζ,-potential was negative. In solution 94.5 μg/ml Hy, pH 1.08 some measured values were positive but the values of 101 μg/ml Hy, pH 1.75, were more even contributed.
- 4. Mix 101 μg/ml Hy, pH 1.75, (0.11×101.4=11.1 μg Hy) or 110 μl Hy 11.1 μg Hy with
-
Solution 1, Plasmid solution (pRc/CMV-CAT, 0.13 μg/μl) or 200 μL pRc/CMV-CAT, 26.0 μg plasmid - add within 30 min
-
Solution 3, 2M NaCl 50 μl - add
- Distilled.
water 150 μl - Final solution Σ 510 μl
- 4. Final solution 510 μl is incubated for 1 hour at 25° C.
- 5. Dialyzed against Solution 3.1, 0.15 M NaCl. The dialyze procedure and the membrane was checked 9 p.m. and 4 a.m. every day.
-
Day 1 6 h. -
Day 2 24 h -
Day 3 24 h -
Day 4 24 h -
Day 5 13 h - Σ 91 h
Hy-plasmid-complex is formulated to 510 μL contain in μg in μg/ml in nM pRc/CMV-CAT 26 50.98 12.0 Hy 150 000 Dalton11.1 21.76 145 NaCl* 2.1.4 × 109 4.2 × 109 150 × 106
*The concentration of NaCl is high and will be lowered in the coming studies
Physical-Chemical Evaluation -
Solution 1, plasmid pRc/CMV in 0.15 M NaCl (1+1) 130/2 μg/ml 30.57/2 ρM). -
Solution 2,Hya 150 000 Dalton 104 μg/ml (69.3 ρM) in distilled water. - The final solution the complex—(pRc/CMV-CAT+Hy) 50.98+21.76 μg/ml in 0.15 m NaCl.
- These were examined by Dynamic Light Scattering, Malvern Instruments England Zeta Master Version PCS: v1.26. The examined volume was≅500 μL.
TABLE 5 Diameter by Volume (Diameter in nm.) of the Hy-plasmid-complex compared with plasmid pRc/CMV and Hy 150 000 Dalton.Concentration Diameter by Solution used in μg/ml nM volume in nm Hy 150 000 Dalton 104 69.3 16.41) in water pRc/CMV-CAT in 65 15.25 87.1 0.15 M NaCl (1 + 1) Xn=6 Complex-(pRc/CMV- 50.98 + 21.76 12.0 + 145 7.5 CAT-Hy) in 0.15 M NaCl Xn=6
1)16.4 too low intensity of the laser for measurement
Conclusion the plasmid pRc/CMV-CAT has been compact from 87.1 nm to 7.5 nm.
- The methods involve acidification, forming a complex, salting and dialyse treatment, at a molar ratio of plasmid/
Hy 150 000 Dalton 0.83-0.85:1. - Dynamic light scattering identifies the diameter of the complex pRc/CMV-CAT-Hy (7.5 nm).
- Positive charging of the Hy-structure (Hy pH 8.8 ζ-potential 51 to −69 mV Hy pH 1.75 1.6±2.3 mV),
- PH-shift (pH<2 pH 6) and an ionic concentration change of NaCl 2 M to 0.075 M forms the complex.
- It is indicated that the compact state of the complex is persistent after
storage 3 months at 5-8 C. - Objective Preparation of a plasmid—Hyaluronan complex for transfection studies in cells and physicochemical characterisation after three (3) months of storage.
- Preparation is done with a newly prepared plasmid (conc. 0.67 μg/ml) with the following changes; a new molar ratio plasmid/Hy 0.1128 (molar ratio plasmid/Hy 0.085), batch size 1020 μl (5201), pH 1.65 and 1.8(pH 1.75 and 1.08).
- Results The complexion is based on a positive charging of the Hyaluronan (Hy) structure (HYDest. W. ζ, −61.5±8.5 mV/Hy ζ,pH 1.65 −2.4±2.2 mV, HyDest. W. ζ, −61.5±8.5 mV/Hy ζ,pH 1.80 −4.0 mV±1.9 mV) The complex is formed by a pH-shift (pH≦2/≅6,) and an ionic concentration change (NaCl 2 M/0.075M). The Plasmid-Hy complex is determined after 3 months of storage at 5-8° C. and characterised by a plasmid compression of the plasmid from 356 (98.9-507) nm at pH-shift 1.65≧6.0 to 84.41 nm at pH-shift 1.80≧6.0 to 69.0 nm identified by dynamic light scattering (see Table 6)
TABLE 6 Solutions prepared for transfection studies and particle size determination of these Plasmid-complex formed by pH shift Plasmid-complex Hy 104 μg/ml 1.65 ≧ 6.0 formed by pH shift in Dist. w. Hy pH 1.65 −2.4 ± 2.2 mV 1.8 ≧ 6.0 Naked plasmid −61.5 ± 8.5 mV Complex/ mV Hy pH 1.80 −4.0 mV ± 1.9 mV at pH 6.0 mV Plasmid/ 1020 μL in in 1020 μL in in 1020 μL in in in in Hy in μg μg/ml nM in μg μg/ml nM in μg μg/ml nM μg/ml nM pRc/CMV- 72 70.58 16.6 72 70.58 16.6 — — — — CAT + + + + + + + Hy 22.52 22.08 147.2 22.66 22.2 148.1 — — — 105 700 pRc/CMV- — — — — — — 72 70.6 16.6 — — CAT NaCl 2.23 4.38 75 2.23 4.38 75 2.23 4.38 75 — — 103 103 106 103 103 106 103 103 106 Particle size 84.41 69.0 98.9-507 98.9 ± 43.1 Diameter by volume in nm - Conclusions an optimal molar ratio for the plasmid compression was not found in. After
storage 3 months at 5-8° C. the molar ratio plasmid/Hy 0.1128 resulted in compression from 98.9-507 nm to 69.0 at pH 1.8 and to 84.41 nm at pH 1.65. For a molar ratio plasmid/Hy 0.085 the plasmid was compressed from 87 nm to 7.5 nm at pH 1.75. It is also indicated that the new plasmid was impure that is a two size distribution with a peak at 98.9 and one peak at 507 nm. - Experimental
- Material
- Solution 1: Plasmid pRc/CMV-CAT
molecular weight 4 250 kDa, concentration 0.67 μg/μL. The original plasmid concentrate is diluted in dist. w. to a concentration of 0.194 μg/μL and frozen at −22° C. -
Solution 2; 10.5 mg Hy mol. wt. 150 kDa was dissolved in 100.0 ml Dist. water.Concentration 105 μg/ml -
Solution 3; 2 M NaCl - Solution 3.1; 0.15 M NaCl
- Solution 3.2; 0.075 M NaCl
-
Solution 4; 1 M NaOH -
Solution 5; 1 M HCl - All chemicals were of analytical grade P.A. Solutions Titrisol Merck
- Dialyze-tube, Spectra/Poor Membrane MWCO 6-9,000 Record No 132645
- Operating and checking the dialyse-tube.
- 1. The tube is softened in distilled water for 1 h.
- 2. The tube is cut and a dialyse-claim is fitted at one edge of the tube
- 3. 500 μL is filled in the bag. Filling is done with a sterile Micropipette
- 4. The other edge is fitted with a dialyse-claim
- 5. The bag is dialysed for 30 h.
- 6. Control the integrity of the membrane.
- 7. The dialysed solution is sucked up with sterile Micro-pipette tubes
- 8. The volume is controlled and given in μl
- Preparation Steps
- Containers for the final solutions, pipettes, pipette tubes, beakers, holders for tubes and other materials were sterilised according to Autoclave program.
- The preparation steps were performed under aseptic conditions in a Laminar Air Flow (LAF-) cabinet, LAF Holten HB 2472 S
- Three different plasmid solutions were prepared Plasmid-Complex formed by pH shift 1.65(1.63-1.67)≧6.0, by pH shift 1.80 (1.78-1.79)≧6.0 and the naked plasmid at pH 6.0
- 1. Thaw at 24° C., 9 frozen (−24° C.) tubes containing≅139 μL (0.194 μg/μl) of the plasmid, “
Solution 1”. 372.μl or 72.μg (372 μL×0.194 μg/μL=0.72 μg) was used (52 μg will be needed since 26 μg was used to prepare 510 μg and resulted in a good compression. - 2. Prepare the pH-adjusted Hy-solutions. Start time=0 (Total time for
preparation steps - Hy-solutions pH 1.65 Hy-solutions pH 1.8
- 4000 μL Hy-
solution 105 μg/ml 4000 μl Hy-solution, 105 μg/ml - 100 μL 1M HCL (approximate amount 75 μL 1M HCl Pipette out of order)
- The solutions were mixed for 20 min magnetic stirring, low speed. The pH and the ξ-potential were checked. For control of the t-potential the solutions were placed in a refrigerator at 5-8° C. and the ζ-potential was measured 5 days later Concentration of Hy; 102.4 μg/ml Concentration of Hy; 103 μg/ml
- pH; 1.63-1.67 at 24° C. pH; 1.78, 1.79 at 24° C.
- Hy ζ,pH 1.65; −2.4±2.2 mV Hy ζ, pH 1.8; −4.0±1.9 mV Time used 30 min
- 3. Mix the plasmid with the Hy-solution by magnetic stirring low speed. Measure the time spend for the preparation Time used 30 min, Σ2,3 60 min
- 4. Add 2 M NaCl and Dist. w. and incubate the solutions above at 24° C. for 30 min Low magnetic stirring Time used 26 minΣ2,3,4 86 min
TABLE 7 Preparation of the plasmid-complexes and the naked plasmid, step Plasmid-complex formed at pH 1.65 Plasmid-complex formed at pH 1.8 Naked plasmid at pH 6.0 Hy pH 1.65 −2.4 ± 2.2 mV Hy pH 1.8 −4.0 ± 1.9 mV pH 6.0 N.D. Hy-solution pH 1.65 220 μL Hy-solution pH 1.8 220 μL Plasmid 0.194 ug/ul 372 μL Plasmid 0.194 ug/ul 372 μL Plasmid 0.194 ug/ul 372 μL 2 M NaCl 100 μL 2 M NaCl 100 μL 2 M NaCl 100 μL Dist .w. 328 μL Dist .w. 328 μL Dist .w. 548 μL Total amount 1020 μL Total amount 1020 μL Total amount 1020 μL - 5. Stabilise the complexes by a pH-shift (pH≦2/≅6,) and an ionic concentration change (NaCl, 2M/0.075M). The total time for
preparation steps - Aseptic transfer and close 1020 μL Plasmid-complex formed at pH 1.65, 1020 μl Plasmid-complex formed at pH 1.8 and 1020 μL Naked plasmid at
pH 6 into the dialyse tubes according to handling direction. - Place the closed tubes into a dialyse-bath containing 1000 ml 0.075 M NaCl. Start the magnetic stirring. Record the dialyse-time.
- Time used to dispense the solutions 45 min Total time used step 3-5, 131 min.
- 6 Dialyse the tubes (48 h, change of 0.075 M NaCl, twice)
- Dialyse Procedure
- 6.1 The closed tubes were dialysed at 24° C. in 0.075
M NaCl 1000 ml for 44 h, magnetic stirring. - 6.2 The solution in the dialyse-bath was renewed 0.075
M NaCl 1000 ml and the closed tubes were placed into a dialyse-bath for 4 h at 24° C. magnetic stirringTABLE 8 Solutions (Plasmid-complex and plasmid) for transfection studies are formulated to contain (pRc/CMV- CAT + Hy) in 75 mM NaCl (pRc/CMV- CAT + Hy) in 75 mM NaCl pRc/CMV- CAT Complex formed by pH shift 1.65 ≧ 6.0 Complex formed by pH shift 1.8 ≧ 6.0 in 75 mM NaCl Hy pH 1.65 −2.4 ± 2.2 mV Hy pH 1.8 −4.0 ± 1.9 mV pH 6.0 1020 μL 1020 μL 1020 μl in μg in μg/ml in nM in μg in μg/ml in nM in μg in μg/ml in nM 72 + 70.58 + 16.6 + 72 + 70.58 + 16.6 + 72 70.6 16.6 22.52 22.08 147.2 22.66 22.2 148.1 — — — - 7 Aseptic dispense the solutions into sterilized tubes with sterile Micro-pipette tubes in portions of 100 μL to fill tubes in an amount of 500 μL* 2
- 8 Measure the volume obtained, determine the pH-values with pH-paper-indicator and label the tubes with date for ready-packed and signature
TABLE 9 Volume obtained, pH and label Volume obtained in Volume obtained in μl μl pH of plasmid- pH of plasmid-complex complex formed at formed at pH shift Volume obtained in μl pH shift 1.65 ≧ 6.0 1.8 ≧ 6.0 pH naked plasmid Label Label Label 500/150 Σ 650 500/250 Σ 750 500/300 Σ 800 6.0 6.0 6.0 pRc/CMV-CAT-Hy pRc/CMV-CAT-Hy pRc/CMV-CAT Complex .pH 1.65/6.0 Complex .pH .pH 6.0 1.8/6.0 - 9. Store the plasmid-solutions at 5-8° C. in a dark place.
- Physical-Chemical Evaluation
- Positive charging of Hy Determination of pH, ζ,-potential and particle size for Hy 150 k D
TABLE 10 Determination of pH, -potential and particle size for Hy 150 000Dalton Hy 150 000 ZetaMasterS Dalton Conc. Version PCS: v1.26 - Particle Diameter in Additives pH mean of potential size ZAve by volume μg/ml in nM in M 6 measurements KCPs in mV KCPs in nm in nm 105 700 Dist. 7.0-7.5 1694 −61.5 ± 8.5 0.5 116.5 ± 51 98.9 ± 43.1 water 103 687 HCl 0.0184 1.80 1890 −4.0 ± 1.9 0.4 112.5 ± 89.9 128.5 ± 31.1 102.4 683 HCl 0.0244 1.65 2108 −2.4 ± 2.2 N.D. N.D. N.D. -
TABLE 11 Diameter by volume of the Hy-plasmid-complex compared with plasmid pRc/CMV-CAT and Hy 150KDa storage 3months 8° C.Diameter Preparation Concentration by volume examined in μg/ml nM Xn−6 in nm Hy 150 KDa in 105 700 98.9 ± 43.1 Dist w pH 7.0-7.5 Hy 150 KDa in103 687 128.5 ± 31.1 Dist w pH 1.80 pRc/CMV-CAT in 97 22.82 98.9/507 0.038 M NaCl pH ≅ 6 Complex-(pRc/CMV- 70.58 + 102.4 16.6 + 683 84.41** CAT + Hy) in 0.075 M NaCl pH 1.65 ≧ 6 Complex-(pRc/CMV- 70.58 + 103 16.6 + 687 69.0** CAT + Hy) in 0.075 M NaCl pH 1.8 ≧ 6
*Values are deviating; indicating two different particle sizes see Table 12
**Prime values (Storage, 3months 8° C.) see Table 8
-
TABLE 12 Determination of pH and the particle size for plasmid pRc/CMV-CAT ZetaMasterS Version PCS: v1.26 Plasmid Conc. mean of 6 measurements 0.0375 M NaCl Particle size diameter in ZAve by volume μg/ml in nM pH KCPs in nm in nm 97 22.8 6 1.3 ± 0.1 258.5 ± 111.8 N.D. 97 22.8 6 0.8 ± 0.2 100 ± 33 93.9-507 -
TABLE 13 Determination of the particle size of the plasmid-complex formed by pH shift 1.65 ≧ 6.0 and 1.8 ≧ 6.0 in 0.075 M NaCl after storage 3 month at 8° C.ZetaMasterS Version PCS: v1.26 Plasmid Conc. mean of 6 measurements 0.075 M NaCl Particle size diameter in in pH ZAve by volume μg/ml nM shift KCPs in nm in nm 70.58 16.6 1.65 ≧ 6.0 1.4 ± 0.2 60.6 ± 9.3 84.41 70.58 16.6 1.8 ≧ 6.0 1.4 ± 0.1 55.8 ± 18.1 69.0 - This example illustrates the influence pH-changes have on the compaction and the solubility of peptides. It also shows on methods to determine solubility.
- Solubility of peptides was examined as a function of pH. The peptides were concentrated by ultra centrifugation at
pH 8 and dialysed towards different pH. For solubility evaluation the dialysate in the dialyse-tube was kept in equilibrium with a precipitate of the peptide at different pH for 24 hours at 20° C. The precipitate was removed and the solution was filtered through a 0,22 μm membrane filter. The concentration was determined by UV absorption at 276 nm. Different buffer systems were used for solubility determination at different pH. These data are used for dissolving and compacting peptides at different pH. - Recombinant growth hormone 33 mg/ml was used for solubility determination. A minimum of solubility was found at pH 5.0 around 1 mg/ml. This occurs at the iso-electric point (pI). On both sides of this pH the solubility for rhGH rose sharply. At pH 7.5 rhGH could be concentrated in a Tris-HCl buffer to 160 mg/ml In a sodium-acetate buffer pH 4.0 a concentration of 62.6 mg/ml was observed. On the acid-side of pI equilibrium of clear solutions and precipitates were found indicating that rhGhH only consist of one genuine form. This in comparison to the base-side where colloid dispersions co-exists with clear solutions of rhGH. Different and diverting clarity of rhGH solutions on the base-side indicate polymorph forms of rhGH. Solubility of rhGH is illustrated in
FIG. 8 and Table 14TABLE 14 dialysis of rhGH in buffers of different pH. Concentration Conditions in of rhGH pH, measured in dialysis sack after filtration Buffer (25 mM) dialysis sample after equilibrium (mg/mL) Na-acetat pH 4.5 4.0 Precipitate 62.6 Na-acetat pH 4.7 4.7 Precipitate 3.1 Na-acetat pH 5.0 5.0 Precipitate 1.3 Na-acetat pH 5.3 5.3 Precipitater 2.2 Bis-Tris pH 6.0 6.0 Precipitate/gel 18.9 Bis-Tris pH 6.5 6.5 Slightly 129.3 opalescent - Conclusion Equilibrium of clear solutions and precipitates were found on the acid-side of pI, indicating that rhGH only exists in one genuine form at this pH. A minimum of solubility, 0.1 mg/ml was found at
pH 5. On the base-side of pI colloid dispersions co-exists with clear solutions indicating polymorph forms of rhGH. These observations were used to get a homogenous precipitate of rhGH when condensing rhGH. This procedure involves the dissolution in an acid environment of a high concentration of rhGH, the dialysing of the solution throughout pI and the stabilising of the precipitate by a polymer. The stabilised suspension is then transferred in the polymeric-form to a pH not exceeding pH 6-6.5. - This example shows how to improve the hydrophobicity of a peptide structure. The hydrophobicity is gained in an acid solution with pH on the acid-side of its pI. Two different forms of rhGH were observed in a study to determine buffer capacity. One a deprotonised form was obtained in an acid solution. The other a protonised form was obtained in an alkaline solution. The deprotonised form of rhGH was found to have a stronger proton binding capacity compared to protonised-rhGH. A more nonpolar character of the protonised rhGH compared to deprotonize rhGH the former burying the polar groups explains this.
- Recombinant Human Growth Hormone (rhGH molar mass 22124) used in the study was concentrated on DEAE-FF-gel (Pharmacia Biotech AB) and desalted on
Sephadex G 25 gel (Pharmacia Biotech AB) to contain 33.3 mg/ml in distilled water. Hydrophobic Interaction Chromatography (HIC) was used to determine purity of rhGH 99.2%. The buffer capacity of rhGH was determined by potentiometric acid-base titration. By adding acid and base to the peptide a protonised and a deprotonised form of rhGH is obtained. These forms are then titrated back with base and acid. - It was found that the proton binding capacity is stronger for the deprotonised form of rhGH compared to the protonised rhGH. A more nonpolar character explains a lesser uptake of protons by the protonised rhGH burying its polar groups. This property a more nonpolar character of a peptide resulting in a more hydrophobic character is used to improve the oral bioavailability. This is done by bringing the peptides in an acid state at pH−1 and keeping it where for about one hour and transferring it back to pH 6.5. Graphic presentation of the protonbinding of deprotonised and protonised rhGH is illustrated in
FIG. 9 .FIG. 9 is illustrating the proton binding capacity of rhGH as a function of pH, suggesting that protonised rhGH (at the acid side) is more hydrophobic when protonised rhGH (at the base side). In changing the pH in the interval 4-10 a precipitation and cloudiness is seen in the interval 4.6-5.0. Above pH 9.0 a slight cloudiness is seen indicating that clear solution of rhGH are obtained in a pH interval 6.5-8.5. This property a clear solution is used in judging time for dialysing a Compact rhGH, see Example 6 and 7. - Conclusion: Acidification of peptides is here used to get a hydrophobic state of the peptide and to improve its oral bioavailability.
- This example illustrates the compaction of rhGH. By changing pH to a strong acid-solution (pH 1.5) Hy becomes charged stretching out from a curling cylinder to straight line. At this pH rhGH is easily soluble. The size of the rhGH particles is moderated by the speed of the pH-change and the ionic concentration change from 2 M to 0.15 M NaCl. The pH-interval used is 7-1.5-5.0(pI) at a constant ratio of rhGH: Hy. Hy stabilise the dispersion in changing its structure back to curling like structure at pH 6-7.
- The preparations were assayed with HI-HPLC. The particle size of the Compact and the None. Compact rhGH was determined after storage at 5-8° C. for 30 days by light scattering, Z-master. The diameter of the measured particles is given in means of six measurements.
- Preparation:Compact rhGH
- 20.78 mg Hy with a molar mass of 150 KDa was dissolved in 900 μl distilled water and was allowed to react for more than 1 hour.
- The pH 9.2 of the Hy-solution was adjusted to pH 1.51 with 50 μl water and 50 μl 1 M HCl.
- 30.65 mg of lyophilised rhGH was dissolved in the Hy-solution. The molar ratio rhGH: Hy is 10:1. In portions of 10 μl 1 M HCl was added to obtain a clear solution and to change pH in the solution from 1.7 to 1.48 at that pH the solution was clear. The final volume was 1000 μl. The solutions were divided in two parts and transferred in two bags prepared as a dialyse tube of MWCO 6-8000, Spectra Pore R. softened in a
tris buffer 10 mM pH 7.8 for 24 hours. - None Compact rhGH
- 30.7 mg rhGH was dissolved in 990 μl distilled water pH 7.4 the final volume of the solution pH 7.0 was adjusted to 1000 μL. The solution was divided in two parts and transferred in two bags (Bag1, Bag2) softened in a
tris buffer 10 mM pH 7.8 for 24 hours. - Dialyse Procedure
- The tubes were dialysed in tris—Buffer 10-mM pH 7.9. The solution, volume 600-ml, was exchanged 3 times. Dialyse Procedure MWCO 6-8000, Spectra Pore R
TABLE 15 Compact rhGH None Compact rhGH Experiment # 41(Bag 1) 41(Bag 2) 4, (Bag 1) 4, (Bag 2) Dialyse pH in Ocular/ Ocular/ Ocular/ Ocular/ time in Dialyse volume volume volume volume hour Solution pH in μl pH in μl pH in μl pH in μl 0 7.9 1.5 Clear/ 1.5 Clear/ 1.5 Clear 1.5 Clear 500 500 500 500 1 7.4 N.D. precipi- N.D precipitate N.D. Clear N.D. Clear tate 19 7.4 N.D. Cake in N.D. Particles N.D. Clear N.D. Clear Solution in Solution 42 7.8 7.4 Cake in 7.4 Particles in 7.6 Clear/ 7.6 Clear/ Solution solution 200 200 300 750 - The above-prepared Compact rhGH and None Compact rhGH are used for analytical assay (in situ determined biological activity and for particle sizing. The concentrations used for particle sizing of Compact rhGH was 7.6 mg/ml and 9.3 mg/ml and for
None Compact rhGH 15 mg/ml. The measurements, means of six measurements were done at 25° C. - Result
- Analytical Assay, HI-HPLC of Compact and of None Compact rhGH HI-HPLC assay is commonly used to determine biological activity of rhGH
TABLE 16 None Compact Preparation Compact rhGH rhGH Experiment # 41 Bag 141 Bag 24 ( Bag 1 + 2)Quantitative Assay, mg/ml 7.6 9.3 15.2 Monomer in % 97.3 93.8 98.3 LMWG, % 0.8 0.7 0.7 Clipped Forms in % 1.9 5.5 0.9 Retention Time, min Approved Approved Approved pH in dialyze-tube 7.4 7.4 7.6 Appearance in dialyze-tube cake particles clear - Conclusion: The values obtained for compact rhGH, 41 Bag1 and 41
Bag 2, Table 1, are within limits for an approved biological activity. Values are also of the same magnitude as for the untreated peptide, none compact rhGH. This suggests that the procedure to compact rhGH does not change the biological activity of rhGH as determine with HI-HPLC-method. - Particle sizing of Compact and of None Compact rhGH was performed by light scattering, Z-master.
TABLE 17 Particle size of None Compact Particle size of Compact rhGH diluted 1:10 rhGH diluted 1:10 Diameter by volume Preparation Diameter by volume in nm in nm Experiment # 41 Bag 141 Bag 24 Bag 1 + 2Colloidal solution 23.1 23.3 75 Cake in equilibrium N/A 114 N/A with a solute - Conclusion: The values obtained of the diameter by volume for None Compact rhGH and Compact rhGH indicate that for Compact rhGH the diameter is changed from 75 nm to 23 nm and that a cake or particles are obtained. This suggests that the procedure to compact rhGH lessen the particle size of rhGH
- This example illustrates the biological activity of Compact and None Compact rhGH in rats with ectomised hypophysis (Hx-rats).
- For optimising the compaction of the rhGH structure the ionic concentration was altered from 2 M to 0.15 M NaCl* in Example 6 to in Example 7 1 M [NH4]2SO4 dialysed against 0.15 M NaCl**, and to 1 M Na2SO4 dialysed against 0.15 M NaCl. The following anions; phosphates chlorine acetate and cat ions; NH4, potassium, magnesium were also evaluated. Only Na2SO4 was considered for further studies. The preparations were assayed by HI-HPLC-technique. The particle size of compact and none compact rhGH was determined With DynaPro-801 after storage at 5-BOC for 21 days.
* “2 M/0.15 M NaCl” The solutions ionic properties are changed from 2 M to 0.15 M by dialyse.
** 1 M [NH4]2SO4 was found to give a heavy precipitation and therefore omitted.
- Low dose, 0.04 IU/ml, and high dose, 0.16 IU/ml, of compact and none compact rhGH were compared in Hx-rats to confirm biological activity and to examine an expected dose-response. A group of the Hx-rats were also treated with a placebo solution***.
*** Bovine albumin 12.5 ml (200 mg/ml) was diluted with isotonic NaCl to 1000 ml. This solution, Diluent 1.25% Albumin, was used as placebo and to dilute the solutions of Compact rhGH and None Compact rhGH in doses for animal trials.
- Preparation Compact rhGH
- 14.9 mg Hy with a molar mass of 150 KDa was dissolved in 1200 μl 1 M Na2SO4 and was allowed to react for more than 1 hour. The pH of the Hy-solution pH 7.6 was adjusted to pH 1.51 with 100 μl water and 330 μl HCl 1 M to a clear solution. HCl was added slowly when passing pH 3.8 a precipitate was observed. 22 mg of lyophilised rhGH was dissolved in the Hy-solution. The molar ratio rhGH: Hy is 10:1. In portions of 10 μl 1M HCl was added to obtain a clear solution. pH at start is 2.77. 40 μl of 1 M HCl was added to change pH in the solution from 2.77 to 1.52.
- The solution was not totally clear. A loss of the solution was obtained when transferred to the dialyse bag. The final volume was 1240 μl. The solutions were divided in two parts and transferred in two bags prepared as a dialyse tube of MWCO 6-8000, Spectra Pore R. softened in a
tris buffer 10 mM pH 7.8. - None Compact rhGH
- 22 mg rhGH was dissolved in 990 μl distilled water pH 7.4.
- The final volume of the solution was adjusted to 1240 μL. The solution was divided in two parts and transferred in two bags (Bag1, Bag2) softened in a
tris buffer 10 mM pH 7.8. - Dialyse Procedure
- The tubes were dialysed in tris—Buffer 10-mM pH 7.9. The solution, volume 600-ml, was exchanged 3 times.
TABLE 18 Dialyse Procedure MWCO 6-8000, Spectra Pore R Compact rhGH None Compact rhGH EXPERIMENT # 51(Bag 1) 51(Bag 2) 5(Bag 1) 5(Bag 2) Ocular/ Ocular/ Ocular/ Ocular/ Dialyse time pH in Dialyse volume volume volume volume in hour Solution pH in μl pH in μl pH in μl pH in μl 0 7.9 1.5 Clear/ 1.5 Clear/ 1.5 Clear 1.5 Clear 620 620 620 620 19 7.4 N.D. precipi- N.D precipi- N.D. Clear N.D. Clear tate tate 24 7.5 N.D. Cloudy N.D. precipi- N.D. Clear N.D. Clear tate 42 7.6 7.3 almost 7.3 almost 7.6 Clear/ 7.4 Clear/ clear clear 550 450 420 1100 - The above-prepared Compact rhGH and None Compact rhGH are used for analytical assay (in situ determined biological activity), in vivo biological activity and for particle sizing. The concentrations used for particle sizing of Compact rhGH were 5,3 mg and 1.5 mg and for None Compact rhGH 5-10 mg. The sizing is given in means of ten measurements and was done at 18, 25 and 30° C.
- Result
- Analytical Assay, HI-HPLC of Compact and of None Compact rhGH HI-HPLC assay is commonly used to determine biological activity of rhGH
TABLE 19 Compact rhGH None Compact rhGH Experiment # 41 41 51 4 5 Preparation (Bag 1) (Bag 2) ( Bag 1 + 2)( Bag 1 + 2)( Bag 1 + 2)Quantitative Assay, mg/ml 7.6 9.3 5.3 15.2 14.2 Monomer, % 97.3 93.8 100 98.3 98 LMWG, % 0.8 0.7 <0.3 0.7 0.6 Clipped forms, % 1.9 5.5 <0.2 0.9 0.2 Retention Time, min Approved Approved Approved Approved Approved pH in dialyse-tube 7.4 7.4 7.3 7.6 7.4 Appearance in dialyse-tube cake particles almost clear clear clear Volume of clear solution μl 300 750 1520 400 1000 - Conclusion: The values obtained for compact rhGH, 41(Bag 1) 41 (Bag 2), and 51 (Bag1+2), Table 2, are within limits for an approved biological activity. Values are also of the same magnitude as for the untreated peptide, none compact rhGH (4 and 5 Bag1+2) this suggests that the procedure to compact rhGH does not change the biological activity of rhGH as determine with HI-HPLC-method.
- Biological activity of Compact rhGH and None Compact rhGH were evaluated after parental injections in Hypophysis-Ectomised (H-x) rats.
- The dose response of 0.04 IU/ml and 0.16 IU/ml of Compact rhGH and of None Compact rhGH were evaluated in groups of 10 H-x rats. A group was treated with a placebo solution*.
* Bovine albumin 12.5 ml (200 mg/ml) was diluted with isotonic NaCl to 1000 ml. This solution, Diluent 1.25% Albumin, was used as placebo and to dilute the solutions of Compact rhGH and None Compact rhGH to doses for animal trials.
- Solutions from the dialyse-tubes Compact rhGH, 5.3 mg/ml and none compact rhGH 14.2 mg/ml, were aseptically diluted with distilled water and assayed; Compact rhGH, 0.23 mg/ml or 0.69 IU/ml and None Compact rhGH to 0.48 mg/ml or 1.45 IU/ml.
- Injection solutions of compact and of none compact rhGH were prepared by dilution with Diluent 1.25% Albumin solution to desired strength, low dose 0.04 IU/ml and high dose 0.16 IU/ml. Diluent 1.25% Albumin solution was used as placebo.
-
FIG. 10 is illustrating procent weight gained in procent of administered dose (treatment of rhGH in i.u./kg body weight for different doses). Dose response of Compact and None Compact rhGH in % weight gain is demonstrated inFIG. 10 of and Table 20 after subcutaneous injection in H-x rats.TABLE 20 Weight Gain in % given as mean of ten H-x rats. (X n=10 in %) Preparation Compact rhGH None compact rhGH Placebo Dose in IU/kg Body- 0.32 1.22 0.32 1.24 0 weight % Weight Gain 8 10 8 12 −2 - A dose response in % weight gain is demonstrated for Compact and for None Compact rhGH in
FIG. 1 and Table 3. - That is for:
Compact rhGH 8% weight gain for 0.32 IU/kg and 10% for 1.22 IU/kg -
None Compact rhGH 8% weight gain for 0.32 IU/kg and 12% for 1.24 IU/kg - For Placebo no weight gain is obtained.
- Conclusion: The dose response obtained for Compact rhGH in H-x rats suggests that full biological effect be obtained after the compacting of the rhGH structure.
- Particle sizing of Compact and of None Compact rhGH was performed with DynaPro-801. The hydrodynamic radius (Rh) is derived from the Transnational Diffusion Coefficient using Stokes-Einstein—
- Equation. The estimated molecular weight is calculated in DynaPro-801 from Rh and the sample temperature using standard curve of MW vs. Rh for globular proteins.
- The particle size of Compact and None Compact rhGH was determined at 18, 25 and 30° C. after storage at 5-8° C. for 21 days. Compact rhGH was studied at two different concentrations 1.5 mg/ml, and 5.3 mg/ml at 25 and 30° C. None Compact rhGH was studied for one concentration, 14.2 mg/ml, at 18, 25 and 30° C. These were the concentrations obtained from the dialyze bags.
- A bimodal function is applied for Compact rhGH and the results present a two-size distribution with its own amplitude, size and MW. For None Compact rhGH the distribution is mono-modal and is fully resolved.
TABLE 21 Particle size of Compact and of None compact rhGH determined with DynaPro-801. Given radii represents mean of 10 measurements Compact rhGH Compact rhGH None Compact rhGH 1.5 mg/ml 5.3 mg/ml 14.2 mg/ml Temp. ° C. 18 25 25 30 18 25 30 Type of bimodal bimodal bimodal bimodal Mono Mono Mono function modal modal modal % mass 100 0 100 0 98 2 98 2 100 100 100 Est. MW in 165 41281 142 32115 716 63666 1125 83646 24 24 23 KDa Particle size 5.3 51.5 4.9 46.2 9.4 61.6 11.4 68.9 2.4 2.4 2.4 Radii in nm
Comments to Table 20 - The particle size of Compact rhGH was determined at a concentration of 1.5 mg/ml at 18 and 25° C.; 18° C.
- 100% of the mass contains of a polymer with radii of 5.3 nm and a molar mass of 165 kDa
- The polymer contains of 7.5 rhGH units (molar mass of polymer/molar mass of rhGH; or 165 000/22.124=7.5) with radii of 0-0.70 nm (radii of polymer/number of rhGH units contained in the polymer; or 5.3/7.5=0.7) 25° C.
- 100% of the mass contains of a polymer with radii of 4.9 nm and a molar mass of 142 kDa
- The polymer contains of 6.4 rhGH units (142/22.124) with radii of 0.77 nm (4.9/6.4)
- The particle size of Compact rhGH was determined at a concentration of 5.3 mg/ml at 25 and 30° C. 25° C.
- 98% of the mass contains a polymer with radii of 9.4 nm and a molar mass of 716 kDa
- The polymer contains of 32.4 rhGH units (716/22.124) with radii of 0.29 nm (9.4/32.4) 30° C.
- 98% of the mass contains a polymer with radii of 11.4 nm and a molar mass of 1125 kDa.
- The polymer contains 50.8 rhGH units (1125/22.124) with a radii of 0.22 nm (11.4/50.8).
- MW rhGH=22124 Da
TABLE 21 Properties of compacted rhGH Compact rhGH structure Assayed concentration in mg/ml 1.5 5.3 Temp. at size measurements° C. 18 25 25 30 Molar mass of the polymers in 165 142 716 1125 kDa Hydrodynamic radii of the 5.3 4.9 9.4 11.4 polymers in nm Number of compacted units rhGH 7 6 32 50 in the polymers Estimated hydrodynamic radii of 0.70 0.77 0.22 0.29 one compacted rhGH-unit in the polymer in nm - Conclusion: The radii of Compact rhGH are smaller than None Compact rhGH. That is, at a concentration of 5.3 mg/ml one compacted unit of rhGH is estimated to have hydrodynamic radii of 0.22-0.29 nm and at 1.5 mg/ml 0.70-0.77 nm. At a concentration of 14.5 mg/ml None Compact rhGH is estimated to have a hydrodynamic radii of 2.4 nm. The number of units of compacted rhGH contained in a polymer is found to depend on the concentration of the solution. Upon dilution no agglomerate of the polymers were found.
- Conclusion of example 5: By changing pH to a strong acid solution (pH 1.5) rhGH gets easily soluble and Hy becomes a charged structure stretching out from a curling cylinder to straight line. By adding electrolytes to the solution a minimum of charge occurs in the peptide. This results in a total collapse of the peptide structure as it passes its pI (isoelectricpoint). In regulating the ions (NaCl and Na2SO4) and the rhGH concentrations with the speed of the pH-change when passing the pI. the hydrodynamic radii of the particles in the dispersion of the peptide are changed from 2.4 to 0.22 nm. Hy stabilize the dispersion in changing its structure back to a curling like structure when pH is changed to
pH 6. It is then possible to dilute the dispersions to the concentration desired. The hydrophobic properties of the rhGH structure is found to be optimal as pH is changed in a strong acid solution pH<2 and by dialyses transferred toneutral solution pH 6 as demonstrated in Example 5.
Claims (16)
1. A method for treating diabetes, growth deficiency diseases, deficiencies related to free radicals, infections, inflammatory conditions, decomposition of gene structures, inherited diseases, muscular dystrophy, atrophy or haemostatic diseases in a patient in need of treatment thereof, comprising administering to said patient an effective amount of a hydrophobe biomolecular structure comprising a polymer and a polar biostructure, wherein the polymer is a hyaluronic acid, which is less than 400 kDa, wherein said hydrophobe biomolecular structure is a compacted hydrophobic structure with buried polar groups and a minimum size, wherein the polymer surrounds the biostructure, and wherein the hydrodynamic radii of the hydrophobe biomolecular structure is compacted to 1/10 of the structures original size.
2. The method according to claim 1 , wherein said patient has an infection, an inflammatory condition, a decomposition of a gene structure, muscular dystrophy, atrophy or an haemostatic disease.
3. The method according to claim 1 , wherein said patient has diabetes.
4. The method according to claim 1 , wherein said patient has a growth deficiency disease.
5. The method according to claim 1 , wherein said patient suffers from a haemostatic disease.
6. The method according to claim 1 , wherein said haemostatic disease is a stroke or infarctus.
7. The method according to claim 6 , wherein said effective amount of the hydrophobe biomolecular structure is administered parenterally.
8. The method according to claim 1 , wherein said patient suffers from diabetes and the polar biostructure is insulin.
9. The method according to claim 1 , wherein said polar biostructure is selected from the group consisting of a growth hormone and recombinant growth hormone.
10. The method according to claim 1 , wherein said polar biostructure is DNA or RNA.
11. The method according to Claim 1 , wherein said polar biostructure is heparin or heparin derivative.
12. The method according to claim 1 , wherein said polar biostructure is an enzyme.
13. The method according to claim 1 , wherein said polar biostructure is a monoclonal antibody
14. A method for improving the oral bioavailability of a peptide, comprising administering a protein with a hydrophobe biomolecular structure comprising said protein, a polymer and a polar biostructure, wherein the polymer is a hyaluronic acid, which is less than 400 kDa, wherein said hydrophobe biomolecular structure is a compacted hydrophobic structure with buried polar groups and a minimum size, wherein the polymer surrounds the biostructure, and wherein the hydrodynamic radii of the hydrophobe biomolecular structure is compacted to 1/10 of the structures original size.
15. A method for administering an enzyme to a patient, comprising administering to said patient an effective amount of said enzyme with a hydrophobe biomolecular structure comprising a polymer and a polar biostructure, wherein the polymer is a hyaluronic acid, which is less than 400 kDa, wherein said hydrophobe biomolecular structure is a compacted hydrophobic structure with buried polar groups and a minimum size, wherein the polymer surrounds the biostructure, and wherein the hydrodynamic radii of the hydrophobe biomolecular structure is compacted to 1/10 of the structures original size.
16. A method for the sustained release of a molecule, comprising administering said molecule with a hydrophobe biomolecular structure comprising a polymer and a polar biostructure, wherein the polymer is a hyaluronic acid, which is less than 400 kDa, wherein said hydrophobe biomolecular structure is a compacted hydrophobic structure.
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US11/179,673 Expired - Fee Related US7338931B2 (en) | 1999-11-15 | 2005-07-13 | Hydrophobic biomolecular structure |
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US09/455,472 Expired - Fee Related US6448093B1 (en) | 1999-11-15 | 1999-12-06 | Hydrophobe biomolecular structure |
US10/186,732 Expired - Fee Related US6926910B2 (en) | 1999-11-15 | 2002-07-02 | Hydrophobe biomolecular structure |
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EP (1) | EP1231926B1 (en) |
AT (1) | ATE287720T1 (en) |
AU (1) | AU1748101A (en) |
CA (1) | CA2391647C (en) |
DE (1) | DE60017783T2 (en) |
ES (1) | ES2236004T3 (en) |
SE (1) | SE9904121D0 (en) |
WO (1) | WO2001036656A2 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
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DE60036915T2 (en) | 1999-01-13 | 2008-08-07 | Alchemia Oncology Pty Ltd., Hawthorn | USE OF HYALURONAN FOR THE MANUFACTURE OF A MEDICAMENT FOR INCREASING THE EFFICACY OF CYTOTOXIC DRUGS |
SE9904121D0 (en) * | 1999-11-15 | 1999-11-15 | Gustaf Jederstroem | Hydrophobic biomolecular structure |
AUPQ879500A0 (en) | 2000-07-14 | 2000-08-10 | Meditech Research Limited | Hyaluronan as cytotoxic agent, drug presensitizer and chemo-sensitizer in the treatment of disease |
US9066919B2 (en) | 2000-07-14 | 2015-06-30 | Alchemia Oncology Pty Limited | Hyaluronan as a chemo-sensitizer in the treatment of cancer |
WO2003070256A1 (en) * | 2002-02-15 | 2003-08-28 | Research Development Foundation | Hyaluronic acid mediated adenoviral transduction |
US20060293263A1 (en) * | 2003-05-16 | 2006-12-28 | Bbk Bio Corporation | Preparation for preventing contact of pathogenic matter with living organism |
CA2616607C (en) | 2005-07-27 | 2015-06-02 | Alchemia Oncology Pty Limited | Therapeutic protocols using hyaluronan |
CN101287475B (en) * | 2005-09-07 | 2012-11-14 | 阿尔卡米亚肿瘤学股份有限公司 | Therapeutic compositions comprising hyaluronan and therapeutic antibodies as well as methods of treatment |
US20100135979A1 (en) * | 2007-05-07 | 2010-06-03 | Jederstrom Pharmaceuticals Ab | Stabilized suspension |
FR2925333B1 (en) * | 2007-12-19 | 2012-04-13 | Farid Bennis | PHARMACEUTICAL COMPOSITIONS COMPRISING AT LEAST ONE PROTEIN ACTIVE INGREDIENT PROTECTS DIGESTIVE ENZYMES |
RU2014120626A (en) * | 2014-05-22 | 2015-11-27 | Владимир Андреевич Сабецкий | INSULIN-CONTAINING MEDICINE OF LONG-TERM ACTION |
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DE3482812D1 (en) * | 1983-10-11 | 1990-08-30 | Fidia Spa | FRACTIONS OF HYALURONIC ACID WITH PHARMACEUTICAL ACTIVITY, METHOD FOR THE PRODUCTION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME. |
JPH02213A (en) * | 1987-10-19 | 1990-01-05 | Taiho Yakuhin Kogyo Kk | Long-acting physiologically active peptide preparation |
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EP0859636A4 (en) | 1995-10-23 | 2002-05-02 | Hyal Pharmaceutical Australia | Hyaluronic acid as dna carrier for gene therapy and vegf antisense dna to treat abnormal retinal vascularization |
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SE9904121D0 (en) * | 1999-11-15 | 1999-11-15 | Gustaf Jederstroem | Hydrophobic biomolecular structure |
-
1999
- 1999-11-15 SE SE9904121A patent/SE9904121D0/en unknown
- 1999-12-06 US US09/455,472 patent/US6448093B1/en not_active Expired - Fee Related
-
2000
- 2000-11-15 EP EP00980185A patent/EP1231926B1/en not_active Expired - Lifetime
- 2000-11-15 CA CA002391647A patent/CA2391647C/en not_active Expired - Fee Related
- 2000-11-15 AT AT00980185T patent/ATE287720T1/en active
- 2000-11-15 DE DE60017783T patent/DE60017783T2/en not_active Expired - Lifetime
- 2000-11-15 AU AU17481/01A patent/AU1748101A/en not_active Abandoned
- 2000-11-15 WO PCT/SE2000/002245 patent/WO2001036656A2/en active IP Right Grant
- 2000-11-15 ES ES00980185T patent/ES2236004T3/en not_active Expired - Lifetime
-
2002
- 2002-07-02 US US10/186,732 patent/US6926910B2/en not_active Expired - Fee Related
-
2005
- 2005-07-13 US US11/179,673 patent/US7338931B2/en not_active Expired - Fee Related
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US4141973A (en) * | 1975-10-17 | 1979-02-27 | Biotrics, Inc. | Ultrapure hyaluronic acid and the use thereof |
US4141973B1 (en) * | 1975-10-17 | 1989-08-08 | ||
US5637566A (en) * | 1988-08-24 | 1997-06-10 | Southern Cross Biotech Pty. Ltd. | Method of improving carcass quality by administering growth hormone |
US5614212A (en) * | 1992-04-08 | 1997-03-25 | International Medical Associates, Inc. | Method of transdermally administering high molecular weight drugs with a polymer skin enhancer |
US5654006A (en) * | 1993-02-12 | 1997-08-05 | Mayo Foundation For Medical Education And Research | Condensed-phase microparticle composition and method |
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US5844107A (en) * | 1994-03-23 | 1998-12-01 | Case Western Reserve University | Compacted nucleic acids and their delivery to cells |
Also Published As
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US20050255162A1 (en) | 2005-11-17 |
WO2001036656A3 (en) | 2001-11-15 |
CA2391647C (en) | 2008-03-11 |
WO2001036656A2 (en) | 2001-05-25 |
DE60017783D1 (en) | 2005-03-03 |
US6448093B1 (en) | 2002-09-10 |
AU1748101A (en) | 2001-05-30 |
ES2236004T3 (en) | 2005-07-16 |
EP1231926B1 (en) | 2005-01-26 |
EP1231926A2 (en) | 2002-08-21 |
US6926910B2 (en) | 2005-08-09 |
DE60017783T2 (en) | 2006-01-05 |
SE9904121D0 (en) | 1999-11-15 |
CA2391647A1 (en) | 2001-05-25 |
US20030096266A1 (en) | 2003-05-22 |
WO2001036656B1 (en) | 2002-02-28 |
US7338931B2 (en) | 2008-03-04 |
ATE287720T1 (en) | 2005-02-15 |
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