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  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/005—Sugars; Derivatives thereof; Nucleosides; Nucleotides; Nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
  • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/001—Acyclic or carbocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B63/00—Purification; Separation; Stabilisation; Use of additives
  • C07B63/04—Use of additives
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/534—Production of labelled immunochemicals with radioactive label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

• the present disclosure relates to the field of oncology and more particularly to the radiolabeling of a cancer detection agent.
• Sentinel node biopsy is rapidly gaining acceptance as a common practice for melanoma and breast cancer diagnosis (Vera, D. R. et al. (2001 ) J. Nucl. Med. 42, 951-959).
• This technique has not been standardized; it typically involves the use of a 99m Tc-colloid and a blue dye.
• the radioisotope "Technetium, that is used in the colloid imaging agent and in the current invention, has several desirable properties: its ready availability, relatively low cost, excellent imaging quality, and its short half- life of 6 hours.
• This radiotracer is employed preoperatively to ascertain the location of the sentinel node and, then, it is used intraoperatively to pinpoint the dissection of the sentinel node(s).
• the blue dye which is cleared rapidly through the lymph channels and nodes, is used to visually confirm the selection of the radioactive node as the sentinel node. Because this biopsy procedure varies with individual practitioners, it is difficult to train practitioners with a consistent skill set and consequently, these biopsies result in a wide range of reported false-negative rates ⁇ i.e., 0 to 12% - see Vera, D. R. ibid.).
• the present disclosure provides a composition containing a dextran conjugated with a bifunctional chelating agent, such as, DTPA, with ease of use as an "instant" kit involving a single lyophilized vial and a liquid diluent vial, having high radiochemical purity upon radiolabeling.
• a bifunctional chelating agent such as, DTPA
• the present disclosure also provides long- term storage stability, as well as sufficient reconstituted stability to facilitate its pharmaceutical or clinical use for ease of manipulation and administration as a diagnostic imaging agent.
• the present disclosure Upon addition of Sodium 99m Tc-pertechnetate, the present disclosure displays high radiochemical purity (i.e., >90% 99m Tc-DTPA-dextran purity) for the bifunctional ligand, DTPA, which are conjugated to a number of amino-terminated leashes on to a dextran molecule via an amide bond with one of its five carboxylic arms. While free DTPA undoubtedly coordinates all five of its deprotonated carboxylic groups to bind to heavy metal ions, such as, for example, 111 lndium, as a potential octadentate ligand (also contains three nitrogen atoms - see H. R. Maecke, et al. (1989) J. Nucl. Med. 30, 1235-1239), the heptadentate DTPA binds with decreased thermodynamic stability, which makes it more susceptible to competition for binding 99 Tc ions, possibly resulting in decreased radiochemical purity.
• DTPA bifunctional ligand
• the high radiochemical purity of 99m Tc-DTPA-dextran was achieved by decreasing the pH to between about 2 and 4, screening for non-competing constituents and identifying the ideal transchelator, Glycine (which also serves as a pH buffer), and utilizing the following facts: (1 ) the distribution of competing ligands for 99m Tc is determined by association rate constants, and (2) the dissociation rate constants for 99rn Tc from its DTPA-dextran complex is very slow and pH-dependent.
• the high efficiency of radiolabeling DTPA-dextran is enhanced by the transient binding to Glycine under highly acidic conditions, Glycine transferring the radioisotope to DTPA-dextran that more avidly binds it and the retention of the Technetium-99m (due to its slow dissociation rate constant) after the pH of this "instant" kit is shifted to mildly acidic conditions by its diluent.
• the present disclosure further provides a phosphate buffered saline diluent, enabling patient comfort by shifting pH from harsh acidic conditions (i.e., pH between about 3 and 4), which would cause pain on injection, to moderately acidic conditions (i.e., pH > -5), which would be well tolerated (M.
• the present disclosure further provides a reducing agent, such as, for example, L-ascorbic acid, which further stabilizes a radiolabeled DTPA-dextran preparation containing excess stannous or stannic ions, preventing the formation of Sn-colloids or other radiochemical impurities, such as, Sn 4+ .
• a reducing agent such as, for example, L-ascorbic acid
• the present disclosure yet prevents the oxidative degradation of the drug substance and its constituents and the autoradiolysis of the radiolabeled drug product by containing L-ascorbic acid in the formulation.
• the present disclosure further provides a stable and esthetically pleasing environment for the DTPA-dextran in an amorphous disaccharide lyophilization cake, allowing for quick reconstitution with Sodium 99m Tc-pertechnetate and addition with a buffered saline diluent to produce a clear, non-particulate liquid for ease of use.
• the present disclosure also provides an inert gaseous headspace by backfilling the lyophilized vials with pharmaceutical-grade nitrogen gas, further stabilizing the stannous ions to provide an excess capacity over the storage lifetime of this invention for reducing Sodium 99m Tc-pertechnetate (or, 99m Tc0 4 ⁇ ).
• the present method is an improved method for generating high radiochemical purity 99m Tc(lll) (and possibly, 99m Tc(IV)) complexes of DTPA-dextran with a single, lyophilized vial that is further reconstituted with pH-buffered Diluent to shift final solution pH, resulting in a solution that is stable for at least 6 hours and that facilitates patient comfort (Russell, CD. (1980) J. Nucl. Med. 21, 354-360; Russell, CD. and Suiter, A.G. (1982) Int. J. Appl. Radiat. Isot. 33, 903-906).
• the formulation of the lyophilized cold kit for DTPA-dextran is an "instant" kit, stabilizing the stannous chloride necessary to reduce Sodium 99m Tc-pertechnetate in a solid white lyophilized cake under a nitrogen environment, which has long-term storage stability.
• This kit generates high radiochemical purity by the Sn 2+ reduction of 99m Tc- pertechnetate under highly acidic conditions, while maintaining the 99m Technetium- DTPA-dextran complex in greater than 90% radiochemical yield following dilution with a phosphate-buffered saline solution to shift the reconstituted solution pH toward neutrality.
• Fig. 1 shows a typical Size Exclusion Chrmotagraphy (SEC) elution profile for reconstituted 99m Technetium-labeled Lymphoseek® (registered trademark of
• Neoprobe Corporation Dublin, OH U.S. Patent No. 6,409,990
• ligand drug product
• Fig. 2 displays a typical elution profile for 99m Technetium-labeled DTPA
• Fig. 3 shows that three excipients (Citrate, Mannitol and L-Cysteine) of the initial pilot formulation display significant 99m Tc-labeled peaks;
• Fig. 4 displays a comparison of the initial drug product formulation with liquid drug substance formulation pilots;
• Fig. 5 is a stacked SEC radiochemical elution profile for liquid DTPA- mannosyl-dextran drug substance placebo formulation pilots containing a sodium phosphate pH buffer and different combinations of transchelator (Citrate), reducing agents (Ascorbic Aicd) and bulking agents (Polyethylene Glycol (PEG) 8000), as measured by the SEC radiochemical purity method;
• Fig. 6 is a stacked SEC radiochemical elution profiles for the corresponding liquid DTPA-mannosyl-dextran drug substance placebo formulation pilots containing a sodium phosphate pH buffer;
• Figs. 7A and 7B are the stacked SEC radiochemical elution profiles for liquid
• Fig. 8 are stacked SEC radiochemical elution profiles for liquid DTPA- mannosyl-dextran drug substance and placebo formulation pilots containing 20 mM sodium acetate buffer at pH 4 and 6;
• Figs. 9A and 9B are screening studies employing a reducing sugar with a primary amine and a zwitterionic amino acid, i.e., Sodium Glucosamine (GIcNH) and Glycine (GIy);
• Figs. 1OA and 10B are studies relating to the range of the excipients Glycine and Sodium Ascorbate at two final concentrations: for Glyi and GIy 2 , it is 0.5 and 2.0 mg Glycine/mL, respectively; and for AA-i and AA 2 , it is 1.5 and 0.38 mg/mL Sodium Ascorbate, respectively;
• Fig. 1 1 are stacked SEC radiochemical elution profiles for the liquid drug substance formulation pilots with 20 mM sodium acetate buffer ranging from pH 5 to 4 with Glycine, Sodium Ascorbate and ⁇ , ⁇ -Trehalose;
• Fig. 12 are stacked SEC radiochemical elution profiles for the DMD drug substance formulations (containing 25 ⁇ M DTPA-mannosyl-dextran (0.5 mg/mL), pH buffer, 0.5 mg/mL Glycine, 0.5 mg/mL Sodium Ascorbate, 2% (w/v) ⁇ , ⁇ -Trehalose, 38.5 mm Sodium Chloride and 75 ⁇ g/mL SnCL 2 -2H 2 O) with the addition of 12.5 mCi 99m Tc-pertechnetate.
• the pH buffer is Acetate, pH 4; Phosphate, pH 3; and Phosphate, pH 2 (from top panel to bottom); and Fig.
• the key to development of a commercial "instant" kit for sentinel node diagnosis is the rational design of an imaging agent that will possess the properties required for optimal sentinel node detection. These properties are a small molecular diameter and high receptor affinity, yielding a radiopharmaceutical agent with a rapid injection site clearance rate and low distal lymph node accumulation (Vera, D. R. ibid.).
• the drug substance that is employed uses a dextran platform to deliver the radiolabel.
• the dextran backbone is a pharmaceutical-grade, average molecular-weight polymer of about 9,500 that is very hydrophilic, lacking in charge, and flexible. All these physical properties reduce migration across membrane walls, which facilitate rapid injection site clearance.
• the dextran polymer is conjugated to amine-terminated tethers that are coupled to DTPA groups, giving the molecule high receptor affinity to complex "Technetium.
• the high signal density of 99m Tc-DTPA-mannosyl-dextran enables better detection of the sentinel node(s) due to a higher signal-to-background ratio.
• DTPA-mannosyl- dextran binds avidly to mannose-terminated glycoprotein receptors in vitro (Vera, D. R. ibid.). In rabbit biodistribution studies, it was shown that 99m Tc-DTPA-mannosyl- dextran diffuses into lymph channels, flows to the sentinel node, and binds to mannose-binding glycoprotein receptors in macrophages and dendritic cells present in the sentinel node (Hoh, CK.
• 99m Tc-DPTA-mannosyl-dextran is a superior targeted 99m Tc-labeled diagnostic agent for sentinel node detection (Hoh, C. K., ibid.). While the pre-clinical and Physician Phase I trials of 99m Tc-DTPA-mannosyl-dextran successfully employed a radiolabeling procedure that used multiple fluid transfers and multiple vials, this dosing format would have been undesirable for commercial usage.
• the composition accomplishes this delicate balancing act by utilizing a newly identified transchelator, Glycine, under highly acidic conditions.
• a transchelator is a weak chelator that transiently binds reduced "Technetium, facilitating the transfer of this radioisotope to a stronger chelator, or ligand.
• the ligand for reduced "Technetium is derivatized diethylenetriaminepentaacetic Acid (DTPA), a heptadentate bifunctional ligand that coupled to the dextran amine- terminated tether by one of its five carboxylic groups. This ligand is well known to the practitioners of the art.
• DTPA is a bifunctional chelator conjugated to peptides and proteins, usually as an anhydride form covalently attached through its carbon backbone.
• transchelators such as, for example, Citrate, Tartrate, Phosphate, Phosphonate, Glucoheptonate and even, Ascorbic Acid.
• these transchelators are largely employed in mildly acidic to neutral pH formulations and can interfere with radiolabeling the active ingredient with high efficiency.
• the optimal pH for using Ascorbic Acid as a transchelator is from pH 4.5 to 6.2 (Liang et al. (1987) Nucl. Med. Biol. 14, 555-562). This stems from the pK a of its carboxylic group, pH 4.10 (CRC: Handbook of Chemistry and Physics, 75 th Edition, David R. Lide, Ph.D. (CRC Press, London)).
• the pK a of the carboxylic group of Glycine is 2.34. Its carboxylic group remains functional under highly acidic conditions ⁇ e.g., partially deprotonated at pH 2 and is fully deprotonated at pH 4). At the preferred embodiment in this disclosure, Ascorbic Acid is fully protonated. Thus, the composition reduces the potential interference of ascorbic acid, utilizing the beneficial properties of this antioxidant, while employing Glycine as an optimal transchelator.
• the preferred embodiment is to have the composition range from pH about 3 to about 4 to enable high radiochemical efficiency, while shifting pH to greater than about pH 5 on dilution of the reconstituted "instant" kit with phosphate-buffered saline Diluent, which would be well tolerated by patients.
• all ingredients are desired to be USP-grade (United States Pharmacopeia).
• q.s.” has its standard pharmaceutical meaning of "as much as is sufficient”.
• Fig. 1 shows a typical eiution profile for reconstituted 99m Technetium-labeled Lymphoseek Ligand Drug Product ( 99m Tc-DTPA-mannosyl-dextran), Lot NMK001 , measured by a radioactivity (NaI, set at 1000 cps/Volt) detector using Size Exclusion Chromatography (SEC).
• the conditions for this SEC radiochemical purity method are as follows: a TSKgel column, Tosoh Bioscience, G3000PW XL (7.8 x 30 cm, 6 ⁇ m, with a column temperature of 25 ⁇ 5 0 C) is employed with an isocratic mobile phase of 50 mM phosphate buffer, pH 7.2, and 300 mM sodium chloride.
• the lyophilized vial is reconstituted with O. ⁇ cc of 10 milliCuries of 99m Tc-pertechnetate, mixed, and allowed to radiolabel for at least 10 minutes at ambient room temperature prior to partially neutralizing the sample in 0.2 cc Phosphate-buffered saline.
• a refrigerated drug product sample 15 ⁇ L, is injected and run at 0.6mL/minute for a run time of 40 minutes; the retention time of the 99m Tc-DTPA-mannosyl-dextran ( 99 Tc-D M D) peak is about 12 to 12.5 minutes, stretching between 9 and 15 minutes with a tailing shoulder of 99m Tc-labeled excipients eluting at a radioactive peak of about 15 to 15.5 minutes.
• the elution profile is very similar to that the potency method using the same column and mobile phase, employing a Refractive Index detector (due to the absence of a UV/VIS absorbance).
• the broad elution peak for 99m Tc-DTPA- mannosyl-dextran is a result of the heterogeneity of the dextran polymer, which is further acerbated by the heterogeneity of the coupling of mannosyl and DTPA groups to amino-terminated leases on dextran (Vera, D. R. et al. (2001 ) J. Nucl. Med. 42, 951-959).
• the goal of the DTPA-mannosyl-dextran formulation was to achieve greater than 95% radiochemical purity in the bulk liquid drug substance formulation and greater than 90% radiochemical purity in the reconstituted lyophilized drug product.
• Fig. 2 displays a typical elution profile for 99m Technetium-labeled DTPA Standard radiolabeled with 10 milliCuries 99m Tc-pertechnetate using lyophilized Lymphoseek Ligand Drug Product Placebo (i.e., 4.5 mM L-Glycine, pH 3, 2.5 mM Sodium L(+)-Ascorbic Acid, 2% (w/v) ⁇ , ⁇ -Trehalose and 75 ⁇ g/mL Stannous Chloride Dihydrate), measured by a Radioactivity (NaI) Detector using SEC radiochemical purity method, described above.
• the 99m Tc-DTPA peak retention time is about 15 minutes, eluting between 14 and 16 minutes, which are the approximate retention times for almost all of the 99m Tc-labeled low-molecular-weight excipients (data not shown).
• the topmost stacked radiochemical elution profile shows the initial lyophilized formulation pilot (5 ⁇ M (0.1 mg/mL) DTPA-mannosyl-dextran, 20 mM Sodium Citrate, pH 5.6, 5.7 mM Sodium L-Cysteine, 2% (w/v) D-Mannitol and 75 ⁇ g/mL Stannous Chloride, Dihydrate) reconstituted with 10 milliCuries 99m Tc- pertechnetate and run via the SEC radiochemical purity method. (The initial lyophilized drug product formulation pilot just preceded the development of the SEC radiochemical purity method.) This elution profile clearly shows that the 99m Tc-DMD peak has less than about 25% radiochemical purity.
• Fig. 3 shows that three excipients of the initial pilot formulation display significant 99m Tc-labeled peaks. Proceeding from the topmost stacked radiochemical elution profile in a downward manner, the second elution profile shows a substantial 99m Tc-Citrate peak at RT-14.5 min, which may account for a significant amount of the 99m Tc-labeled interference at RT-14.5 min in the topmost pattern.
• Citrate is a known transchelator of DTPA at a pH range of 5 to 6 (Hnatowich, D. J., Chapter 8, pg. 175, Cancer Imaging with Radiolabeled Antibodies (Goldenberg, D.
• the fifth elution profile at 1 mg/mL L-Cysteine clearly shows that Cysteine binds 99m Tc and interferes with the transchelation of Citrate, eluting at retention times ranging from 21 to 23 minutes.
• the sixth radiochemical elution profile involves the addition of 1 mg/mL sodium L(+)-Ascorbic Acid Dihydrate to a Citrate formulation; Ascorbic Acid does not appear to interfere with 99m Tc-Citrate.
• Fig. 4 displays a comparison of the initial drug product formulation with liquid drug substance formulation pilots.
• the topmost stacked radiochemical elution profile is the initial drug product formulation and the second profile is that of Sodium Citrate in saline with SnCL 2 added to reduce 12.5 mCi 99m Tc-pertechnetate.
• the DTPA-mannosyl-dextran drug substance is partially radiolabeled with a significant 99m Tc-Citrate eluting at about 14.5 minutes.
• the use of Sodium Citrate is not a suitable pH bufferUranschelator choice.
• FIG. 5 is a stacked radiochemical elution profile for liquid DTPA-mannosyl- dextran drug substance placebo formulation pilots containing a Sodium Phosphate pH buffer and different combinations of transchelator, reducing agents and bulking agents, as measured by the SEC radiochemical purity method.
• the topmost stacked radiochemical elution profile shows a small 99m Tc-labeled interference peak with the 20 mM Sodium Phosphate buffer at pH 4 and 1.5 mg/mL Sodium Ascorbate.
• the second through the sixth elution profiles shows 20 mM Sodium Phosphate, pH 4, 75 ⁇ g/mL SnCL 2 -2H 2 O and 12.5 mCi 99m Tc-pertechnetate with the following respective potential excipients: 1 mg/mL Sodium Citrate; 1% PEG 8000; 1 mg/mL Sodium Citrate and 1.5 mg/mL Sodium Ascorbate; 1.5 mg/mL Sodium Ascorbate and 1 % PEG 8000; and 1.5 mg/mL Sodium Ascorbate, 1 mg/mL Sodium Citrate and 1 % PEG 8000.
• the third profile from the top displays background levels of 99m Tc-DMD, indicating that phosphate and PEG 8000 do not serve as satisfactory transchelators.
• Citrate is less efficient in radiolabeling drug substance and still interferes in these formulations.
• PEG 8000 apparently interferes with the drug substance yield with its hydroxyl groups and is unsuitable as a bulking agent. Since Sodium Phosphate is not an ideal pH buffer for lyophilization, alternative generally recognized as safe (GRAS) pH buffers were screened.
• Figs. 7A and 7B the stacked radiochemical elution profile for liquid DTPA- mannosyl-dextran drug substance and placebo formulation pilots containing 20 mM Sodium Acetate buffer at pH 4 are interspersed.
• the topmost radiochemical elution profile is the drug substance formulation with the potential transchelator, Sodium Tartrate at 1.5 mg/mL, displaying an enhanced radiolabeling of the drug substance with a significant interfering peak, 99m Tc-Tartrate (see fourth elution profile for corresponding placebo formulation).
• the second and third elution profiles in Fig. 7A show little difference in the presence of Sodium Ascorbate and PEG 8000.
• Fig. 7A show little difference in the presence of Sodium Ascorbate and PEG 8000.
• the radiochemical elution profiles demonstrate that the drug substance formulations for the Sodium Ascorbate and PEG 8000 combinations with Tartrate have less efficiency in radiolabeling the drug substance.
• the DTPA Standard has a small tailing edge shoulder with Sodium Acetate and Sodium Ascorbate at pH 4. Hence, the selection of Sodium Tartrate as potential transchelator in an Acetate pH buffer is unsatisfactory.
• the stacked radiochemical elution profile for liquid DTPA-mannosyl- dextran drug substance and placebo formulation pilots containing 20 mM Sodium Acetate buffer at pH 4 and 6 are also interspersed.
• the topmost and second radiochemical elution profiles show at pH 6, the presence of 1.5 mg/mL Sodium Ascorbate enhances the radiochemical purity of the drug substance, but the third and fourth profiles indicate that Ascorbate may contribute to a significant and a smaller interference peak of 99m Tc-Ascorbate at RT-13.5 and -15 minutes, respectively.
• the fifth elution profile demonstrates that the radiochemical purity is pH-sensitive, primarily radiolabeling the drug substance at pH 4 in the presence of Sodium Ascorbate.
• the fifth profile may contain some interfering material co-eluting with the "" 1 Tc-DMD peak, as observed in the slight shoulder of the trailing edge of the drug substance peak as well as the small 99m Tc-labeled peak at RT-16 minutes.
• the "" 1 Tc-DMD peak as observed in the slight shoulder of the trailing edge of the drug substance peak as well as the small 99m Tc-labeled peak at RT-16 minutes.
• Glycine and Sodium Ascorbate appeared compatible with enhanced radiochemical purity of the drug substance, the range of these excipients was investigated. Glycine and Sodium Ascorbate were evaluated at two final concentrations: for Glyi and GIy 2 , it is 0.5 and 2.0 mg/mL, respectively; and for AA 1 and AA 2 , it is 1.5 and 0.38 mg/mL, respectively.
• the mean average of two radiolabeling studies for GIy 1 and GIy 2 are 90.7 and 88.7% 99m Tc- DMD, respectively, as measured by the SEC radiochemical purity method.
• the mean average of two radiolabeling studies for the AAi and AA 2 drug substance formulations are 80.3 and 90.3% 99m Tc-DMD purity, respectively (see Figs. 10A and 10B).
• the topmost elution profile displays the Acetate formulation at pH 4, which has 96.9% 99m Tc-DMD purity, as measured by the SEC radiochemical purity method.
• Glycine Hydrochloride As an acidic pH buffer and a transchelator, because the pH 3 and pH 2 drug substance formulations exhibit 97.6 and 97.1 % 99m Tc-DMD purity, respectively, and meeting the desired goal of the drug substance formulation (see Fig. 13).
• the Acetate formulation at pH 4 failed to meet the goal for drug substance formulation (93.6% versus > 95% 99 TC-DMD purity), but this may due to day-to-day variability in the preparation of the formulation, incomplete degassing of the solutions, the inadequate mixing of the Stannous Chloride Dihydrate, etc.
• the Glycine Hydrochloride buffer may be utilized in pH 4 formulations in addition to the Acetate buffer.
• the Class I glass vials were filled with sterile-filtered aliquots of this pH study, 1.05 ml_, into 3 ml_ vials. Stoppers were placed in the necks of these vials, and the vials were placed on the VirTis Lyophilizer shelves for lyophilization. After the lyophilization cycle was completed, the vials were backfilled with nitrogen gas and stoppered. Subsequently, the stoppered vials were crimped with aluminum seals. On visual inspection, the lyophilized cakes for the Acetate, pH 4, and the Glycine, pH 3, drug product formulation vials retained their amorphous structure and appeared to have dried to low residual moisture.
• the preferred embodiment of this invention is the pH 3 drug product formulation (i.e., 12.5 to 25 ⁇ M DTPA-mannosyl-dextran (0.25 to 0.5 mg/mL), 0.5 mg/mL Glycine, pH 3, 0.5 mg/mL Sodium Ascorbate, 2% (w/v) ⁇ , ⁇ -Trehalose and 75 ⁇ g/mL SnCL 2 -2H 2 O).
• Sodium 99m Tc-pertechnetate is eluted from a Molybdenum-99 generator with 0.9% Sodium Chloride, or isotonic saline.
• the Lymphoseek Ligand Drug Product is formulated to meet the recommendations of the Infusion Nursing Society to be less than 500 mOsm ⁇ L following reconstitution with 1 mL of Sodium 99m Tc-pertechnetate.
• a suitable Diluent was identified for use with human parenterals, Buffered Saline for Injection from Greer Laboratories.
• this Diluent is: 0.107% Sodium Phosphate, Heptahydrate, 0.036% Potassium Phosphate (desirably USP-NF, United States Pharmacopeia-National Formulary), 0.5% Sodium Chloride and 0.4% Phenol. It is recommended that the lyophilized Lymphoseek Ligand Drug Product vial is reconstituted with 0.7 cc of 10 to 50 mCi of Sodium 99m Tc-pertechnetate for at least 10 minutes at ambient room temperature, mixed intermittently and then, diluted with 0.3 cc of Buffered Saline for Injection.
• the Lymphoseek® Ligand Drug Product has at least twelve hours of reconstituted stability, but it is recommended that the reconstituted drug product be administered within six hours (data not shown). Hence, the neutralized 99m Tc-labeled Lymphoseek Ligand Drug Product should be well tolerated by patients upon intradermal injection.

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