PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF NEUROLOGICAL DISEASES
20230141988 · 2023-05-11
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K9/0056
HUMAN NECESSITIES
A61K31/473
HUMAN NECESSITIES
A61K9/006
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to pharmaceutical compositions for administration to mammals that include (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient that provides pharmacokinetic profiles useful for the treatment of neurodegenerative diseases.
Claims
1. A method of treating a neurodegenerative disease, the method comprising: administering a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, wherein the composition provides a pharmacokinetic profile comprising a t.sub.1/2 at about 0.25 to about 4 hours after oral administration to a mammal.
2. (canceled)
3. (canceled)
4. A method of treating a neurodegenerative disease, the method comprising: administering a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, wherein the composition provides a pharmacokinetic profile comprising a t.sub.1/2 at about 0.5 to about 7 hours after intramuscular, subcutaneous, sublingual, buccal, or intranasal administration.
5. (canceled)
6. (canceled)
7. A method of treating a neurodegenerative disease, the method comprising: administering a pharmaceutical composition comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, wherein the composition provides a t.sub.1/2 at about 0.05 to about 5 hours after intravenous administration.
8. (canceled)
9. (canceled)
10. The method of claim 1, wherein the composition provides a pharmacokinetic profile comprising a T.sub.max at about 0.25 to about 10 hours after oral administration.
11. The method of claim 4, wherein the composition provides a pharmacokinetic profile comprising a T.sub.max at about 0.05 to about 10 hours after intramuscular, subcutaneous, sublingual, buccal, or intranasal administration.
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. The method of claim 4, wherein the composition provides a pharmacokinetic profile comprising a C.sub.max at about 1 ng/mL to about 5000 ng/mL after intramuscular, subcutaneous, sublingual, buccal, or intranasal administration.
19. (canceled)
20. (canceled)
21. The method of claim 1, wherein the composition provides a pharmacokinetic profile comprising a C.sub.max at about 10 ng/mL to about 2000 ng/mL after oral administration.
22. (canceled)
23. The method of claim 1, wherein the composition provides a brain to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6 following administration.
24. The method of claim 1, wherein the composition provides a CSF to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6 following administration.
25. (canceled)
26. The method of claim 1, wherein the composition provides an oral bioavailability of about 2% to about 30% relative to intravenous administration.
27. (canceled)
28. The method of claim 4, wherein the composition provides an intramuscular or subcutaneous bioavailability of about 40% to about 99% relative to intravenous administration.
29. The method of claim 4, wherein the composition provides a sublingual or buccal bioavailability of about 2% to about 80% relative to intravenous administration.
30. The method of claim 4, wherein the composition provides an intranasal bioavailability of about 20% to about 80% relative to intravenous administration.
31. (canceled)
32. (canceled)
33. The method of claim 1, wherein the composition provides an oral bioavailability of about 2% to about 30% relative to intramuscular or subcutaneous administration.
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. The method of claim 4, wherein the composition provides a provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after intramuscular or subcutaneous administration.
41. (canceled)
42. The method of claim 4, wherein the composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after sublingual or buccal administration.
43. (canceled)
44. The method of claim 4, wherein the composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after intranasal administration.
45. (canceled)
46. The method of claim 1, wherein the composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after oral administration.
47. (canceled)
48. The method of claim 7, wherein the composition provides an AUC.sub.last/C.sub.0 ratio from about 0.0001 to about 20 hours after intravenous administration.
49. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION
[0031] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
[0032] It must also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
[0033] As used herein, the term “about” means deviation of up to 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 40%-60%.
[0034] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic directly to a subject, whereby the agent positively impacts the target. “Administering” a composition may be accomplished by, for example, injection, oral administration, topical administration, or by these methods in combination with other known techniques. Such combination techniques include heating, radiation, ultrasound and the use of delivery agents. When a compound is provided in combination with one or more other active agents (e.g. other anti-atherosclerotic agents such as the class of statins), “administration” and its variants are each understood to include concurrent and sequential provision of the compound or salt and other agents.
[0035] By “pharmaceutically acceptable” it is meant the carrier, diluent, adjuvant, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0036] “Composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to “pharmaceutical composition” is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound o the present invention and a pharmaceutically acceptable carrier.
[0037] As used herein, the term “agent,” “active agent,” “therapeutic agent,” or “therapeutic” means a compound or composition utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. Furthermore, the term “agent,” “active agent,” “therapeutic agent,” or “therapeutic” encompasses a combination of one or more of the compounds of the present invention.
[0038] A “therapeutically effective amount” or “effective amount” of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit, block, or reverse the activation, migration, proliferation, alteration of cellular function, and to preserve the normal function of cells. The activity contemplated by the methods described herein includes both medical therapeutic and/or prophylactic treatment, as appropriate, and the compositions of the invention may be used to provide improvement in any of the conditions described. It is also contemplated that the compositions described herein may be administered to healthy subjects or individuals not exhibiting symptoms but who may be at risk of developing a particular disorder. The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. However, it will be understood that the chosen dosage ranges are not intended to limit the scope of the invention in any way. A therapeutically effective amount of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.
[0039] The terms “treat,” “treated,” or “treating” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the state of the condition, disorder, or disease; delay in onset or slowing of the progression of the condition, disorder, or disease; amelioration of the condition, disorder, or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder, or disease. Treatment includes prolonging survival as compared to expected survival if not receiving treatment.
[0040] The term “AUC” refers to the area under the plasma concentration-time curve. “AUC.sub.last” refers to the area under the plasma concentration-time curve from zero to the last quantifiable concentration time. The term “AUC.sub.all” refers to the area under the plasma concentration-time curve from the time of dosing to the time of the last observation, regardless of whether the last concentration is measureable or not. The term “AUC.sub.inf” refers to the area under the plasma concentration-time curve from time zero extrapolated to infinity. The term “C.sub.max” refers to maximum concentration. The term “C.sub.0” refers to initial or back-extrapolated drug concentration at time zero following bolus intravenous injection, and the term “T.sub.max” refers to the time when C.sub.max is achieved. The term “t.sub.1/2” refers to the half-life, which is the period of time required for the concentration or amount of drug in the body to be reduced by one-half. The term “(6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol” or a pharmaceutically acceptable salt thereof may also be referred to as “Compound 1”.
[0041] The term “bioavailability” or “F %” refers to the rate and extent to which (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol is absorbed into a biological system from an administered drug product and becomes available at the site of biological action. The values for bioavailability may be represented in relative terms. For example, oral bioavailability may be represented in terms of percentages relative to intravenous administration or subcutaneous administration.
[0042] Administration and Compositions
[0043] The pharmaceutical compositions included within the scope of the present invention comprise a therapeutically effective amount Compound 1 and at least one pharmaceutically acceptable excipient. The term “excipient” refers to a pharmaceutically acceptable, inactive substance used as a carrier for the pharmaceutically active ingredient (Compound 1), and includes anti adherents, binders, coatings, disintegrants, fillers, diluents, solvents, flavors, bulkants, colours, glidants, dispersing agents, wetting agents, lubricants, preservatives, sorbents and sweeteners. The choice of excipient(s) will depend on factors such as the particular mode of administration and the nature of the dosage form. Solutions or suspensions used for injection or infusion can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, including autoinjectors, or multiple dose vials made of glass or plastic.
[0044] In the methods of various embodiments, pharmaceutical compositions including the active agent can be administered to a subject in an “effective amount” or “therapeutically effective amount,” which may be any amount that provides a beneficial effect to the subject.
[0045] A pharmaceutical formulation of the present invention may be in any pharmaceutical dosage form. The pharmaceutical formulation may be, for example, a tablet, capsule, nanoparticulate material, e.g., granulated particulate material or a powder, a lyophilized material for reconstitution, liquid solution, suspension, emulsion or other liquid form, injectable suspension, solution, emulsion, etc., suppository, or topical or transdermal preparation or patch. The pharmaceutical formulations generally contain about 1% to about 99% by weight of Compound 1 and 99% to 1% by weight of a suitable pharmaceutical excipient. In one embodiment, the dosage form is an oral dosage form. In another embodiment, the dosage form is a parenteral dosage form. In another embodiment, the dosage form is an enteral dosage form. In another embodiment, the dosage form is a topical dosage form. In one embodiment, the pharmaceutical dosage form is a unit dose. The term ‘unit dose’ refers to the amount of Compound 1 administered to a patient in a single dose.
[0046] A pharmaceutical formulation may be, for example, an oral dosage form for controlled release. By way of example only, controlled or modified release oral dosage forms can be prepared by using methods known in the art. For example, a suitable controlled release form of Compound I may be a matrix tablet or a capsule dosage composition. Suitable materials for matrix dosage forms include, for example, waxes (e.g. carnauba, bees wax, paraffin wax, ceresine, shellac wax, fatty acids, and fatty alcohols), oils, hardened oils or fats (e.g., hardened rapeseed oil, castor oil, beef tallow palm oil, and soya bean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and polyethylene glycol). Other suitable matrix tableting materials include microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, ethyl cellulose, with other carriers, and fillers. Tablets may also contain granulates, coated powders, or pellets. Tablets may also be multi-layered. Multi-layered tablets are especially preferred when the active ingredients have markedly different pharmacokinetic profiles. The finished tablet may also be coated or uncoated.
[0047] The coating composition typically contains an insoluble matrix polymer (approximately 15-85% by weight of the coating composition) and a water soluble material (e.g., approximately 15-85% by weight of the coating composition). Optionally an enteric polymer (approximately 1 to 99% by weight of the coating composition) may be used or included. Suitable water soluble materials include polymers such as polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, and monomeric materials such as sugars (e.g., lactose, sucrose, fructose, mannitol and the like), salts (e.g., sodium chloride, potassium chloride and the like), organic acids (e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), and mixtures thereof. Suitable enteric polymers include hydroxypropyl methyl cellulose, acetate succinate, hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, shellac, zein, and polymethacrylates containing carboxyl groups. The coating composition may be plasticised according to the properties of the coating blend such as the glass transition temperature of the main component or mixture of components or the solvent used for applying the coating compositions. Suitable plasticisers may be added from 0 to 50% by weight of the coating composition and include, for example, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, acetylated citrate esters, dibutylsebacate, and castor oil. If desired, the coating composition may include a filler. The amount of the filler may be 1% to approximately 99% by weight based on the total weight of the coating composition and may be an insoluble material such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or polacrilin potassium. The coating composition may be applied as a solution or latex in organic solvents or aqueous solvents or mixtures thereof. If solutions are applied, the solvent may be present in amounts from approximate by 25-99% by weight based on the total weight of dissolved solids. Suitable solvents are water, lower alcohol, lower chlorinated hydrocarbons, ketones, or mixtures thereof. If latexes are applied, the solvent is present in amounts from approximately 25-97% by weight based on the quantity of polymeric material in the latex. The solvent may be predominantly water.
[0048] In some embodiments, a pharmaceutical composition of the present invention is delivered to a subject via a parenteral route, an enteral route, or a topical route. Examples of parental routes the present invention include, without limitation, any one or more of the following: intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, intracoronary, intracorporus, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intraocular, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous (bolus or drip), intraventricular, intravesical, and/or subcutaneous.
[0049] Enteral routes of administration of the present invention include administration to the gastrointestinal tract via the mouth (oral), stomach (gastric), and rectum (rectal). Gastric administration typically involves the use of a tube through the nasal passage (NG tube) or a tube in the esophagus leading directly to the stomach (PEG tube). Rectal administration typically involves rectal suppositories. Oral administration includes sublingual and buccal administration.
[0050] Topical administration includes administration to a body surface, such as skin or mucous membranes, including intranasal and pulmonary administration. Transdermal forms include cream, foam, gel, lotion or ointment. Intranasal and pulmonary forms include liquids and powders, e.g., liquid spray.
[0051] The dose may vary depending upon the dosage form employed, sensitivity of the patient, and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
[0052] In one embodiment, the daily dose of Compound 1 administered to a patient is selected from: up to 200 mg, 175 mg, 150 mg, 125 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, 0.5 mg, or up to 0.1 mg.
[0053] In another embodiment, the daily dose is at least 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or at least 200 mg. In another embodiment, the daily dose is 0.05-1 mg, 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, or more than 200 mg.
[0054] In another embodiment, a single dose of Compound 1 administered to a patient is selected from about: 0.05 mg, 0.1 mg, 0.5, mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.
[0055] In another embodiment, a single dose of Compound 1 administered to a patient is selected from about: 0.05-1 mg, 1-2 mg, 2-4 mg, 1-5 mg, 5-7.5 mg, 7.5-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, or more than 200 mg.
[0056] In one embodiment, the single dose is administered by a route selected from any one of: oral, buccal, or sublingual administration. In another embodiment, said single dose is administered by injection, e.g., subcutaneous, intramuscular, or intravenous. In another embodiment, said single dose is administered by inhalation or intranasal administration.
[0057] As a non-limited example, the dose of Compound 1 administered by intramuscular or subcutaneous injection may be about 0.05 to 50 mg per day to be administered in divided doses. A single dose of Compound 1 administered by intramuscular or subcutaneous injection may be about 0.05-6 mg, preferably about 1-4 mg, 1-3 mg, or 2 mg. Intra muscular or subcutaneous infusion may be preferable in those patients requiring division of injections into more than 10 doses daily. The continuous intramuscular or subcutaneous infusion dose may be 1 mg/hour daily and is generally increased according to response up to 4 mg/hour.
[0058] The fine particle dose of Compound 1 administered by pulmonary administration, e.g., inhalation using a pressurized metered dose inhaler (pMDI), dry powder inhaler (DPI), soft-mist inhaler, nebulizer, or other device, may be in the range of about, 0.5-15 mg, preferably about 0.5-8 mg or 2-6 mg. The Nominal Dose (ND), i.e., the amount of drug metered in the receptacle (also known as the Metered Dose), of Compound 1 administered by pulmonary administration may be, for example, in the range of 0.5-15 mg, 3-10 mg, 10-15 mg, 10-12.5 mg, 12.5-15 mg, 15-17.7 mg, 17.5-20 mg, 20-25 mg, 20-22.5 mg, 22.5-25 mg, 25-30 mg, 25-27.5 mg, 27.5-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg.
[0059] Long-acting pharmaceutical compositions may be administered, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times daily (preferably: 1 times per day), every other day, every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
[0060] Pharmaceutical compositions comprising (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and pharmaceutically-acceptable salts thereof can be administered by means that produces contact of the active agent with the agent's site of action. They can be administered by conventional means available for use in conjunction with pharmaceuticals in a dosage range of 0.001 to 1000 mg/kg of mammal body weight per day in a single dose or in divided doses. One dosage range is 0.01 to 500 mg/kg body weight per day in a single dose or in divided doses. Administration can be delivered as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but typically are administered with a pharmaceutically acceptable excipient selected on the basis of the chosen route of administration and standard pharmaceutical practice.
[0061] Indications Suitable for Treatment
[0062] The pharmaceutical compositions of the present invention may be employed to treat or reduce the symptoms associated with protein misfolding or conditions that would benefit from a reduction of such activity. Because (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol modifies the protein misfolding leading to neurodegenerative diseases, preferred embodiments of the present invention include pharmaceutical compositions that have (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient that provide a pharmacokinetic profile conducive to the treatment of neurodegenerative diseases.
[0063] Conditions suitable for treatment according to this invention include neurodegenerative diseases include Alzheimer's disease, Parkinson's Disease, multiple sclerosis, Huntington's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, muscular dystrophies prion-related diseases, cerebellar ataxia, Friedrich's ataxia, SCA, Wilson's disease, RP, Gullian Barre syndrome, Adrenoleukodystrophy, Menke's syndrome, cerebral autosomal dominant arteriopathy with subcortical infarcts (CADASIL), Charcot Marie Tooth diseases, neurofibromatosis, von-Hippel Lindau, Fragile X, spastic paraplegia, tuberous sclerosis complex, Wardenburg syndrome, spinal motor atrophies, Tay-Sach's, Sandoff disease, familial spastic paraplegia, myelopathies, radiculopathies, encephalopathies associated with trauma, radiation, drugs and infection, and disorders of the sympathetic nervous system (e.g., Shy Drager (familial dysautonomia), diabetic neuropathy, drug-induced and alcoholic neuropathy).
[0064] Pharmacokinetic (PK) Profile
[0065] Various embodiments further include a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, and a pharmacokinetic profile after administration to a mammal. When used at the appropriate dosages the described pharmacokinetic profiles provide levels of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol for the treatment of neurological diseases which include, Alzheimer's disease, Parkinson's Disease, multiple sclerosis, Huntington's Disease, ALS, spinal muscular atrophy, muscular dystrophies prion-related diseases, cerebellar ataxia, Friedrich's ataxia, SCA, Wilson's disease, RP, Gullian Barre syndrome, Adrenoleukodystrophy, Menke's syndrome, cerebral autosomal dominant arteriopathy with subcortical infarcts (CADASIL), Charcot Marie Tooth diseases, neurofibromatosis, von-Hippel Lindau, Fragile X, spastic paraplegia, tuberous sclerosis complex, Wardenburg syndrome, spinal motor atrophies, Tay-Sachs, Sandoff disease, familial spastic paraplegia, myelopathies, radiculopathies, encephalopathies associated with trauma, radiation, drugs and infection, and disorders of the sympathetic nervous system (e.g., Shy Drager (familial dysautonomia), diabetic neuropathy, drug-induced and alcoholic neuropathy); and preferably ALS, Huntington's disease, Parkinson's disease, and Friedreich's ataxia. In particular, the pharmaceutical compositions of the present invention provide a pharmacokinetic profile that provides (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to the site of biological action. Preferably in the central nervous system (CNS) of the subject. More preferably in the cerebral spinal fluid (CSF) and the brain of the subject.
[0066] One embodiment of the present invention includes a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides one or more pharmacokinetic properties including a t.sub.1/2 at about 0.1 to about 72 hours, a T.sub.max at about 0 to about 24 hours, a C.sub.max at about 1 ng/mL to about 50,000 ng/mL, a brain to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6, a CSF to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6, a bioavailability of about 0.1% to about 99% relative to intravenous administration, and/or an AUC.sub.last/C.sub.max or AUC.sub.last/C.sub.0 ratio from about 0.0001 to about 100 hours.
[0067] One embodiment of the present invention includes a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a pharmacokinetic profile that includes a t.sub.1/2 at about 0.1 to about 72 hours after administration to mammal. In another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.3 to about 48 hours after intramuscular or subcutaneous administration. In another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.3 to about 25 hours after intramuscular or subcutaneous administration. In another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.3 to about 10 hours after intramuscular or subcutaneous administration. Another embodiment of the pharmaceutical composition exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.5 to about 7 hours after administration. In certain embodiments the pharmaceutical composition of exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.5 to about 7 hours after intramuscular or subcutaneous administration. Yet in other embodiments, the pharmaceutical composition of exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.5 to about 7 hours after sublingual or buccal administration. In another embodiment, the pharmaceutical composition of exhibits a pharmacokinetic profile that includes a t.sub.1/2 at about 0.5 to about 7 hours after intranasal administration. Another embodiment includes the pharmaceutical composition provides a t.sub.1/2 at about 0.05 to about 5 hours after intravenous administration. In another embodiment, the pharmaceutical composition exhibits a t.sub.1/2 at about 0.01 to about 30 hours after oral administration. In one aspect, the pharmaceutical composition exhibits a t.sub.1/2 at about 1 to about 30 hours after oral administration. In another aspect, the pharmaceutical composition exhibits a t.sub.1/2 at about 5 to about 25 hours after oral administration.
[0068] One embodiment of the present invention includes a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a pharmacokinetic profile that includes a T.sub.max at about 0 to about 24 hours after administration to a mammal. In another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a T.sub.max at about 0.05 to about 10 hours after administration. In another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a T.sub.max at about 0.05 to about 4 hours after administration. In another embodiment, the pharmaceutical composition of exhibits a pharmacokinetic profile that includes a T.sub.max at about 0.05 to about 4 hours after intramuscular or subcutaneous administration. Another embodiment of the pharmaceutical composition exhibits a pharmacokinetic profile that includes a T.sub.max at about 0.05 to about 4 hours after sublingual administration. In yet another embodiment, the pharmaceutical composition exhibits a pharmacokinetic profile that includes a T.sub.max at about 0.05 to about 4 hours after intranasal administration. In another embodiment, the pharmaceutical composition of exhibits a T.sub.max at about 0.1 to about 5 hours after oral administration.
[0069] One embodiment of the present invention includes a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a pharmacokinetic profile that includes a C.sub.max at about 1 ng/mL to about 50,000 ng/mL after administration to a mammal. In another embodiment, the pharmaceutical composition provides a pharmacokinetic profile that includes a C.sub.max at about 1 ng/mL to about 5000 ng/mL after administration. In another embodiment, the pharmaceutical composition provides a pharmacokinetic profile that includes a C.sub.max at about 1 ng/mL to about 5000 ng/mL after intramuscular or subcutaneous administration. In yet another embodiment, the pharmaceutical composition provides a pharmacokinetic profile that includes a C.sub.max at about 1 ng/mL to about 5000 ng/mL after sublingual administration. In another embodiment, the pharmaceutical composition provides a pharmacokinetic profile that includes a C.sub.max at about 1 ng/mL to about 5000 ng/mL after intranasal administration. In another embodiment, the pharmaceutical composition provides a pharmacokinetic profile that includes a C.sub.max at about 1000 ng/mL. In another embodiment, the pharmaceutical composition provides a C.sub.max at about 5 ng/mL to about 50 ng/mL after oral administration.
[0070] An embodiment of the present invention further includes a pharmaceutical composition that includes (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, and provides a pharmacokinetic profile that includes a T.sub.max at about 0 to about 10 hours, a C.sub.max at about 1 ng/mL to about 50,000 ng/mL, and a t.sub.1/2 at about 0.1 to about 15 hours after administration to a mammal.
[0071] The present invention also includes embodiments of a pharmaceutical composition that includes (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a brain to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6 following administration to a mammal. Another embodiment of the present invention includes a pharmaceutical composition that includes (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a CSF to plasma ratio in a mammal for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol of about 0.1 to about 6 following administration to a mammal.
[0072] A further embodiment of the present invention includes a pharmaceutical composition that includes (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a bioavailability of about 0.1% to about 99% relative to intravenous administration. In another embodiment, the pharmaceutical composition provides an oral bioavailability of about 1% to about 40% relative to intravenous administration. In another embodiment, the pharmaceutical composition provides an oral bioavailability of about 5% to about 30% relative to intravenous administration. In yet another embodiment, the pharmaceutical composition provides a intramuscular or subcutaneous bioavailability of about 40% to about 99% relative to intravenous administration. In another embodiment, the pharmaceutical composition of claim 25 provides a sublingual or buccal bioavailability of about 20% to about 80% relative to intravenous administration. In another embodiment, the pharmaceutical composition provides an intranasal bioavailability of about 20% to about 80% relative to intravenous administration.
[0073] Embodiments of the present invention also encompass a pharmaceutical composition that includes (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and at least one pharmaceutically acceptable excipient, and provides a bioavailability of about 0.1% to about 99% relative to intramuscular or subcutaneous administration. In one embodiment, the pharmaceutical composition provides an oral bioavailability of about 1% to about 40% relative to intramuscular or subcutaneous administration. In another embodiment, the pharmaceutical composition of provides an oral bioavailability of about 5% to about 30% relative to intramuscular or subcutaneous administration. In another embodiment, the pharmaceutical composition provides a sublingual bioavailability of about 20% to about 80% relative to intramuscular or subcutaneous administration. In yet another embodiment, the pharmaceutical composition provides an intranasal bioavailability of about 20% to about 80% relative to intramuscular or subcutaneous administration.
[0074] Another embodiment includes a pharmaceutical composition having (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, and provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours. In one aspect, the pharmaceutical composition provides a provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 50 hours after administration. In another aspect, the pharmaceutical composition provides a provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 20 hours after administration. In one embodiment, the pharmaceutical composition provides a provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours after intramuscular or subcutaneous administration. In another aspect, the pharmaceutical composition provides a provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after intramuscular or subcutaneous administration. In yet another aspect, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours after sublingual or buccal administration. In other embodiments, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after sublingual or buccal administration. In another embodiment, pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours after intranasal administration. In another embodiment, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after intranasal administration. In other embodiments, the pharmaceutical composition of provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours after oral administration. In another embodiment, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.01 to about 20 hours after oral administration. In another embodiment, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 100 hours after intravenous administration. In another embodiment, the pharmaceutical composition provides an AUC.sub.last/C.sub.max ratio from about 0.0001 to about 20 hours after intravenous administration.
EXAMPLES
Example 1: Preparation of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride
[0075] The following examples contain detailed methods of preparing (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol. The general synthetic scheme is presented for illustrative purposes only and is not intended as a restriction on the scope of the invention. All parts are by weight and temperatures are in Degrees Celsius unless otherwise indicated.
##STR00002## ##STR00003##
Preparation of (R)-10-Methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-11-ol
[0076] A mixture of (5a,6a)-7,8-didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol (10.0 g, 33.4 mmol) and methanesulfonic acid (40 ml) was heated to 100° C. After 30 minutes, the temperature was lowered to 65° C. and after a further 30 minutes, the reaction was left to cool to room temperature. The mixture was diluted with water (150 ml) and then basified with ammonium hydroxide (with cooling in an ice bath to keep the temperature <50° C.). The resultant suspension was cooled to room temperature, collected by filtration and washed with water. The solid was dissolved in dichloromethane (50 ml) and separated from residual water.
[0077] The resultant solution was passed through a pad of silica eluting with 5% methanol/dichloromethane. Concentration of appropriate fractions gave the desired product as a white foam (3.8 g, 40%).
Preparation of (R)-10,11-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline
[0078] To a solution of (R)-10-Methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-11-ol (5.0 g, 17.8 mmol) in dimethylformamide (25 ml) was added 60% sodium hydride (0.75 g, 18.8 mmol). After stirring for 20 minutes, methyl iodide (2.66 g, 18.8 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred for 20 minutes and then poured onto water (70 ml) and extracted with methyl t-butyl ether (150 ml). The organic layer was separated, washed with 2M sodium hydroxide solution (3×50 ml) then brine (50 ml) and concentrated to a pink oil which was purified on a plug of silica eluted 5% methanol/dichloromethane. Concentration of appropriate fractions gave the desired product (2.43 g, 46%).
Preparation of 10,11-dimethoxy-6-methyl-5,6-dihydro-4H-dibenzo[de,g]quinoline and 10,11-dimethoxy-6-methyl-6H-dibenzo[de,g]quinoline
[0079] A solution of (R)-10,11-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline (18.47 g, 65.9 mmol) in acetonitrile (550 ml) was treated with a slurry of 10% Pd/C in acetonitrile (450 ml) and the mixture heated at reflux overnight and then allowed to cool. The catalyst was collected by filtration, washed with acetonitrile and the combined filtrate concentrated to a dark oil (10.6 g) which was used without further purification.
Preparation of (3RS)
[0080] A mixture of 10,11-dimethoxy-6-methyl-5,6-dihydro-4H-dibenzo[de,g]quinoline and 10,11-dimethoxy-6-methyl-6H-dibenzo[de,g]quinoline (10.6 g, 36.1 mmol) was dissolved in absolute ethanol (500 ml) and treated with sodium cyanoborohoydride (11 g, 175 mmol). Part of a solution of acetyl chloride (8.8 g, 0.113 mol) in absolute ethanol (100 ml) was added until the mixture became cloudy and the mixture stirred at room temperature. After stirring overnight, the reaction was found to be around 80% complete by LC-MS; stirring was continued and further aliquots of the acetyl chloride solution added at intervals. Once the reaction was found to contain <2% starting material by LCMS, the mixture was concentrated and the residue diluted with water (300 ml) and basified with potassium carbonate. After extraction with ethyl acetate (2×300 ml), the organic layer was washed with brine, concentrated and then purified by chromatography (silica gel, 3.fwdarw.10% methanol:dichloromethane) (7.53 g, 71%).
Preparation of (S)-10,11-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline (2R,3R)-2,3-bis(benzoyloxy)succinate (3S DBS salt)
[0081] To a solution of (−)-Dibenzoyl-L-tartaric acid (8.73 g, 24.4 mmol) in ethyl acetate (80 ml) was added a solution of (3RS) (7.20 g, 24.4 mmol) in ethyl acetate (30 ml) over 15 minutes. The resultant precipitate was heated to reflux and isopropanol (56 ml) added to give a dark solution. After heating for 15 minutes, the solution was allowed to slowly cool to room temperature for two hours. It was then further cooled in an ice bath for an hour and the resultant precipitate collected by filtration (2.6 g). A second crop was also collected (1.4 g). The two were combined and heated to reflux in ethyl acetate (35 ml); isopropanol (20 ml) was then added slowly until a solution was obtained. This was allowed to slowly cool and the product collected by filtration in 3 crops, all of which had >95% ee by chiral NMR (3.28 g, 21%).
Preparation of (6aS)-6-methyl-5,6,6a, 7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride
[0082] A slurry of (3S DBS salt) (4.4 g, 6.7 mmol) was treated with potassium carbonate until pH 8. The mixture was extracted with ethyl acetate (2×40 ml) and the combined extracts dried and concentrated to an orange/brown oil (1.90 g). This was dissolved in acetic acid (30 ml), treated with 48% hydrobromic acid (30 ml) and the mixture heated under reflux for 5 hours under nitrogen. After cooling to room temperature overnight, the mixture was concentrated to ca 10 ml and then carefully basified by the slow addition of saturated sodium bicarbonate solution. The product was extracted with ethyl acetate (2×60 ml), washed with brine, dried over sodium sulphate and concentrated. The resultant blue foam was dissolved in absolute ethanol (40 ml) and treated with concentrated hydrochloric acid (1.1 ml) and activated carbon (600 mg). After heating under reflux for 40 minutes, the mixture was hot filtered and the filter bed washed with boiling absolute ethanol (80 ml). The combined filtrate was concentrated and the white solid thus obtained triturated with cold acetone (15 ml) to give the desired product. (1.08 g, 60%).
[0083] In Vivo Bioactivity
[0084] The following general methods are used in order to describe and demonstrate biological activity and potential therapy usage of compounds of the present invention only, and are not to be construed in any way as limiting the scope of the invention. Final yield 210 g (92%).
Example 2: Pharmacokinetic and Brain Penetration Study Following Single Dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride Via Subcutaneous Administration in Male C57BL/6J Mouse
[0085] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol pharmacokinetic profiles were evaluated in male C57BL/6J mice (N=3). 9 male C57BL/6J mice following administration of a single subcutaneous dose of 4.40 mg/kg. The vehicle for the subcutaneous formulation contained “0.1% sodium metabisulfite solution”. During this PK study, there were no obvious drug-related changes in clinical signs and behavioral responses. There was no animal death related to drug administration.
[0086] Animals
[0087] Male C57BL/6J mice were purchased from Si Bei Fu Laboratory Animal Technology Co. Ltd (Beijing, China). The animals were about 8 weeks old with body weights of 20-30 g on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water. All animals were fasted overnight and fed after 4 hours collection.
[0088] Study Design
[0089] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered with subcutaneous dose (4.40 mg/kg, free base) at 5 mL/kg. For group 1, plasma samples were collected from dorsal metatarsal vein at each time point after subcutaneous administrations. For group 2, plasma, CSF and brain samples were collected at 0.167, 0.5 h post dose.
TABLE-US-00001 Dose Level Dose Level (mg/kg) (salt (mg/kg) (salt Dose Level Dose hemihydrate anhydrous (mg/kg) Volume Conc. No. of Group Treatment form) form) (free base) (mL/kg) (mg/mL) Route Animals 1 (6aS)-6-methyl- 5.15 5 4.40 5 0.880 Subcutaneous 3/male 5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline- 10,11-diol hydrochloride 2 (6aS)-6-methyl- 5.15 5 4.40 5 0.880 Subcutaneous 6/male 5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quinoline- 10,11-diol hydrochloride
[0090] Dosage and Administration
[0091] Preparation of dosing for subcutaneous administration (4.40 mg/kg, Group 1): 1.22 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 1.185 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 0.880 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0092] Preparation of dosing for subcutaneous administration (4.40 mg/kg, Group 2): 1.25 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 1.214 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 0.880 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0093] Dose Sample Validation (Subcutaneous):
TABLE-US-00002 Step 1 Take 20 μL subcutaneous dose to 180 μL DMSO to make first 10× dilution (S1) Step 2 Take 10 μL of solution from S1 to 990 μL 50% ACN in water to make second 100× dilution (S2) Step 3 Take 3 μL of solution from S2 to 30 μL blank Plasma to make third 10× dilution (S3)
[0094] The total dose solution was diluted 10000×. The concentrations of dose formulation were measured by LC-MS, and the results shown in the table below:
TABLE-US-00003 Dilution Nominal Measured Mean Accuracy SD CV Route Sample Name Factor (mg/mL) (mg/mL) (mg/mL) (%) (mg/mL) (%) Subcutaneous Dose_G1_1 10000 0.880 0.921 0.880 100 0.0518 5.89 G1 Dos_G1_2 10000 0.821 Dose_G1_3 10000 0.897 Subcutaneous Dose_G2_1 10000 0.880 0.979 0.953 108 0.0259 2.72 G2 Dose_G2_2 10000 0.928 Dose_G2_3 10000 0.951
[0095] Sample Collection
[0096] For Group 1 (4.40 mg/kg), blood samples were collected from each animal at 0.0833, 0.167, 0.25, 0.5, 1, 2 hr post dose. For Group 2 (4.40 mg/kg), blood, CSF and brain samples were collected from 3 mice at two time points: 0.167 hr and 0.5 hr post dose.
[0097] For plasma samples, about 30 μL or 300 μL of blood samples were collected from each animal via dorsal metatarsal vein or heart. These blood samples were placed into the tubes containing K2EDTA. And centrifugation at 3000 g for 10 minutes in a 4° C. centrifuge. The completion time from collection was less than 30 min. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min.
[0098] For brain and CSF samples, The animals were fully exsanguinated with a rising concentration of CO2 gas prior to CSF and brain collection.
[0099] For CSF collection, incision was made by scissors on the neck of the rats to expose the foramen magnum. A syringe fitted with a venoclysis needle was used to sample the CSF, the tip of the needle was inserted into the membrane of foramen magnum about 1-2 mm, and a negative pressure was applied by pulling a stopcock a small length of the syringe to extract CSF fluids into a tube. The stopcock was further actuated to until a sufficient amount of CSF was collected. There should be no blood in the CSF fluid. A stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in CSF.
[0100] For brain collection. Brain samples were collected at an adopted time point, and washed 3 times with cold saline, then dried with clean gauze and put into the weighed tube, quick frozen in an ice box and kept at −75±15° C. All brain samples were weighted and homogenized with phosphate buffered saline (PBS) by brain weight (g) to PBS volume (mL) ratio 1:3 before analysis. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in grain homogenate. The actual concentration was the detected value multiplied by the dilution factor. Brain samples were collected at designed time point, quickly homogenized and kept at −75±15° C.
Preparation of Standards
[0101] About 1 mg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate and (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in DMSO to obtain a 1 mg/mL standard stock solution (free base).
[0102] Calibration standard working solutions were prepared at concentrations of 2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL by serial dilution of the standard stock solution in 50% acetonitrile in water. Quality control working solutions at concentrations of 5, 10, 500 and 8000 ng/mL were prepared by serial dilution of the standard stock solution in 50% acetonitrile in water. These QC samples were prepared on the day of analysis in the same way as calibration standards.
[0103] Sample Treatment
[0104] For plasma and brain samples, 3 μL of each calibration standard working solution (2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL) was added to 30 μL of the blank C57BL/6J mouse plasma or brain homogenate (with 10% 200 mg/mL ascorbic acid) to achieve calibration standards of 0.2-1000 ng/mL (0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 33 μL. Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2), 50 ng/mL (mid), 800 ng/mL (high) for plasma were prepared independently for those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. The total volumes of 33 μL of standards, 33 μL of QC samples, and 33 μL of unknown samples (30 μL of unknown plasma or brain homogenate (with 10% 200 mg/mL ascorbic acid) with 3 μL 50% acetonitrile) were added to 200 μL of acetonitrile containing IS to precipitate proteins. The samples were subsequently vortexed for 30 sec. After centrifugation at 4° C., 4700 rpm for 30 min, and supernatant was diluted 2 times with water, 10μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis.
[0105] For CSF samples, 2 μL of each calibration standard working solution (2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL) was added to 20 μL of the artificial CSF (with 10% 200 mg/mL ascorbic acid) to achieve calibration standards of 0.2-1000 ng/mL (0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 22 μL. Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2), 50 ng/mL (mid), 800 ng/mL (high) for plasma were prepared independently for those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. The total volumes of 22 μL of standards, 22 μL of QC samples and 22 μL of unknown samples (20 μL of unknown CSF (with 10% 200 mg/mL ascorbic acid) with 2 μL 50% acetonitrile) were added to 200 μL of acetonitrile containing IS to precipitate proteins. Then the samples were vortexed for 30 sec. After centrifugation at 4° C., 4700 rpm for 30 min, and supernatant was diluted 2 times with water, 10 μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis.
[0106] All the samples were processed on ice.
[0107] LC-MS/MS Conditions
[0108] The LC-MS/MS system consisted of two Shimadzu LC-30AD pumps, a DGU-20A5R degasser, a Rack changer II and LCMS-8060 LC/MS/MS instrument mass spectrometer.
[0109] Chromatographic separation was performed on a DAICEL CORPORATION CHIRALCEL OD-3R Particle size 3 μm 4.6 mm×150 mm column at room temperature. The mobile phase was composed of A: Water (5 mM Ammonium Acetate); B: Ethanol (5 mM Ammonium Acetate). The flow rate was 0.7 mL/min. The injection volume was 10 μL.
[0110] Positive mode electrospray ionization (ESI) was performed to obtain a protonated ion of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions were 268.20191.10, 268.20191.10 and 393.20-373.20 for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone, respectively. The instrument parameters were set as follows: ion spray voltage: 8060; heating gas flow: 10 L/min; interface temperature: 250° C.; DL temperature: 220° C.; heat block temperature: 400° C.; driving gas flow: 10 L/min. (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone dependent parameters are listed as follows:
TABLE-US-00004 (6aR)-6-methyl- (6aS)-6-methyl- 5,6,6a,7- 5,6,6a,7- tetrahydro- tetrahydro- 4H-dibenzo 4H-dibenzo [de,g]quinoline- [de,g]quinoline- Dexamethasone Compound ID 10,11-diol 10,11-diol (IS) Transition 268.20.fwdarw.191.10 268.20.fwdarw.191.10 393.20.fwdarw.373.20 Dwell 100 100 50 Time (msec) Q1 Pre Bias (v) −10 −10 −25 CE −30 −30 −10 Q3 Pre Bias (v) −19 −19 −18
[0111] Data acquisition was performed by Lab Solutions version 5.89 Software (Shimadzu). All concentration data was reported with 3 significant figures. Data statistics were performed using Excel 2010 software. The pharmacokinetic parameters of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were calculated using a non-compartmental approach with Phoenix™ WinNonlin®.
[0112] Results
[0113] The (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations vs. time profiles are shown in
[0114] The pharmacokinetic parameters for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in mice following subcutaneous administration were calculated by non-compartmental analysis (Phoenix™ WinNonlin® 8.0), and a linear trapezoidal algorithm was used for AUC calculation. The resulting pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol are as follows:
TABLE-US-00005 t.sub.1/2 T.sub.max C.sub.max AUC.sub.last AUC.sub.∞ MRTInf AUC.sub.last/D AUC.sub.last/C.sub.max Animal (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) (h) Mouse 1 0.442 0.0833 1147 417 426 0.384 80.9 0.364 Mouse 2 0.502 0.167 931 361 375 0.434 70.1 0.388 Mouse 3 0.513 0.167 888 363 379 0.474 70.4 0.409 Mean 0.486 0.139 989 380 393 0.431 73.8 0.387 SD 0.0381 0.0483 139 31.6 28.5 0.0451 6.14 0.023
[0115] The resulting pharmacokinetic parameters for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol are as follows:
TABLE-US-00006 t.sub.1/2 T.sub.max C.sub.max AUC.sub.last AUC.sub.∞ MRTInf AUC.sub.last/D AUC.sub.last/C.sub.max Animal (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) (h) Mouse 1 0.338 0.167 8.56 5.10 5.99 0.542 139 0.596 Mouse 2 0.319 0.25 8.43 4.91 5.67 0.526 134 0.582 Mouse 3 0.312 0.167 7.78 4.80 5.47 0.515 131 0.617 Mean 0.323 0.195 8.26 4.94 5.71 0.528 135 0.598 SD 0.0133 0.0479 0.418 0.156 0.264 0.0135 4.25 0.017
[0116] The resulting ratios of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and brain following subcutaneous administration to Male C57BL/6J mice at 4.40 mg/kg are as follows:
TABLE-US-00007 Ratio Mean Ratio Time Brain Plasma (Brain/ (Brain/ (h) Animal (ng/g) (ng/mL) Plasma) Plasma) 0.167 h Mouse 4 1802 776 2.32 2.72 Mouse 5 2991 1003 2.98 Mouse 6 2446 857 2.85 0.5 h Mouse 4 1059 257 4.12 5.32 Mouse 5 1117 205 5.45 Mouse 6 975 153 6.38
[0117] The resulting ratios of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and CSF following subcutaneous administration to male C57BL/6J mice at 4.40 mg/kg are as follows:
TABLE-US-00008 Ratio Mean Ratio Time CSF Plasma (CSF/ (CSF/ (h) Animal (ng/mL) (ng/mL) Plasma) Plasma) 0.167 h Mouse 4 20.5 776 0.0264 0.0245 Mouse 5 21.3 1003 0.0213 Mouse 6 22.2 857 0.0259 0.5 h Mouse 4 9.59 257 0.0373 0.0389 Mouse 5 7.73 205 0.0377 Mouse 6 6.38 153 0.0417
[0118] The resulting ratios of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and brain following subcutaneous administration to male C57BL/6J mice at 4.40 mg/kg are as follows:
TABLE-US-00009 Ratio Mean Ratio Time Brain Plasma (Brain/ (Brain/ (h) Animal (ng/g) (ng/mL) Plasma) Plasma) 0.167 h Mouse 4 36.6 8.67 4.22 5.54 Mouse 5 57.9 10.1 5.74 Mouse 6 50.6 7.62 6.65 0.5 h Mouse 4 38.0 5.43 6.99 9.27 Mouse 5 42.1 4.60 9.15 Mouse 6 38.7 3.32 11.7
[0119] The resulting ratios of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and CSF following subcutaneous administration to male C57BL/6J mice at 4.40 mg/kg are as follows:
TABLE-US-00010 Ratio Mean Ratio Time CSF Plasma (CSF/ (CSF/ (h) Animal (ng/mL) (ng/mL) Plasma) Plasma) 0.167 h Mouse 4 BLOQ 8.67 NA NA Mouse 5 BLOQ 10.1 NA Mouse 6 BLOQ 7.62 NA 0.5 h Mouse 4 BLOQ 5.43 NA NA Mouse 5 BLOQ 4.60 NA Mouse 6 BLOQ 3.32 NA
NA=Not available; BLOQ=Below quantifiable limit of 1 ng/mL for CSF.
[0120] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following subcutaneous administration to male C57BL/6J mice at 4.40 mg/kg:
TABLE-US-00011 Time Conc. (ng/mL) Mean SD CV (h) Mouse 1 Mouse 2 Mouse 3 (ng/mL) (ng/mL) (%) 0.0833 1147 760 750 886 226 25.6 0.167 1092 931 888 970 107 11.1 0.25 721 706 648 692 38.8 5.61 0.5 169 181 211 187 21.5 11.5 1 52.0 33.7 37.2 41.0 9.70 23.7 2 14.9 18.7 22.5 18.7 3.79 20.3
[0121] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following subcutaneous administration to male C57BL/6J Mice at 0.0367 mg/kg:
TABLE-US-00012 Time Conc. (ng/mL) Mean SD CV (h) Mouse 1 Mouse 2 Mouse 3 (ng/mL) (ng/mL) (%) 0.0833 7.46 6.01 6.03 6.50 0.829 12.8 0.167 8.56 8.38 7.78 8.24 0.407 4.94 0.25 8.50 8.43 7.56 8.16 0.523 6.41 0.5 5.06 5.03 5.37 5.15 0.187 3.62 1 1.82 1.66 1.49 1.65 0.165 10.0 2 BLOQ BLOQ BLOQ NA NA NA
[0122] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following subcutaneous administration to male C57BL/6J Mice at 4.40 mg/kg:
TABLE-US-00013 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.167 776 1003 857 879 115 13.1 0.5 257 205 153 205 52.1 25.4
[0123] (6aS)-6-methyl-5,6,6a, 7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following subcutaneous administration to male C57BL/6J Mice at 4.40 mg/kg:
TABLE-US-00014 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.167 20.5 21.3 22.2 21.3 0.823 3.86 0.5 9.59 7.73 6.38 7.90 1.61 20.4
[0124] (6aS)-6-methyl-5,6,6a, 7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following subcutaneous administration to male C57BL/6J Mice at 4.40 mg/kg:
TABLE-US-00015 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.167 450 748 612 1802 2991 2446 2413 596 24.7 0.5 265 279 244 1059 1117 975 1050 71.4 6.80
[0125] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following subcutaneous administration to male C57BL/6J Mice at 0.0367 mg/kg:
TABLE-US-00016 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.167 8.67 10.1 7.62 8.79 1.24 14.1 0.5 5.43 4.60 3.32 4.45 1.07 24.0
[0126] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following subcutaneous administration to male C57BL/6J mice at 0.0367 mg/kg:
TABLE-US-00017 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.167 BLOQ BLOQ BLOQ NA NA NA 0.5 BLOQ BLOQ BLOQ NA NA NA
[0127] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following subcutaneous administration to male C57BL/6J mice at 0.0367 mg/kg:
TABLE-US-00018 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.167 7.32 11.6 10.1 36.6 57.9 50.6 48.4 10.8 22.4 0.5 7.60 8.41 7.75 38.0 42.1 38.7 39.6 2.17 5.48
Example 3: Pharmacokinetic Study Following Single Dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride Via IV, Subcutaneous, PO and IN administration at 0.880 mg/kg, 0.880 mg/kg, 4.40 mg/kg and 1.76 mg/kg in Male SD Rats
[0128] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol pharmacokinetic profiles were evaluated in male SD Rats (N=3). 12 male SD Rats were administered with single IV dose of 0.880 mg/kg, subcutaneous dose of 0.880 mg/kg, PO dose of 4.40 mg/kg, and IN dose of 1.76 mg/kg. The vehicle for the IV, subcutaneous, PO and IN formulation contained “0.1% sodium metabisulfite solution”. During this PK study, there were no obvious drug-related changes in clinical signs and behavioral responses. There was no animal death related to drug administration.
[0129] Animals
[0130] Male SD rats were purchased from Si Bei Fu Laboratory Animal Technology Co. Ltd (Beijing, China). The animals were about 8 weeks old with body weights of 200-300 g on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water. All animals were fasted overnight and fed after 4 hours collection.
[0131] Study Design
[0132] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered with IV and subcutaneous dose (0.880 mg/kg, free base) at 5 mL/kg. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered with PO dose (4.40 mg/kg, free base) at 5 mL/kg. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered with IN dose (1.76 mg/kg, free base) at 0.2 mL/kg. Plasma samples were collected from jugular vein at each time point after IV, subcutaneous, PO and IN administrations.
TABLE-US-00019 Dose Level Dose Dose Level (mg/kg) Level Dose Level (mg/kg) (salt (salt (mg/kg) (mg/kg) Dose hemihydrate anhydrous (free (anhydrous Volume Conc. No. of Group Treatment Form) form) base) salt) (mL/kg) (mg/mL) Route Animals 1 (6aS)-6-methyl- 1.03 1 0.880 1.0 5 0.176 IV 3/male 5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol hydrochloride 2 (6aS)-6-methyl- 1.03 1 0.880 1.0 5 0.176 Subcutaneous 3/male 5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol hydrochloride 3 (6aS)-6-methyl- 5.15 5 4.40 5.0 5 0.880 PO 3/male 5,6,6a,7-tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol hydrochloride 4 (6aS)-6-methyl- 2.06 2 1.76 2.0 0.2 8.80 IN (split 3/male 5,6,6a,7-tetrahydro-4H- to dose to dibenzo[de,g]quino- 2 nostrils) line-10,11-diol hydrochloride
[0133] Dosage and Administration
[0134] Preparation of dosing for IV and subcutaneous administration (0.880 mg/kg): 2.17 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 10.537 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 0.176 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0135] Preparation of dosing for PO administration (4.40 mg/kg): 5.53 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 5.371 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 0.880 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0136] Preparation of dosing for IN administration (1.76 mg/kg): 2.37 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 10.537 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 8.80 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0137] Dose Sample Validation (IV and Subcutaneous):
TABLE-US-00020 Step 1 Take 20 μL IV and subcutaneous dose to 180 μL DMSO to make first 10× dilution (S1). Step 2 Take 100 μL of solution from S1 to 900 μL 50% ACN in water to make second 10× dilution (S2). Step 3 Take 2 μL of solution from S2 to 20 μL blank Plasma to make third 10× dilution (S3).
[0138] The total dose solution was diluted 1000×.
[0139] Dose Sample Validation (IN):
TABLE-US-00021 Step 1 Take 20 μL IN dose to 180 μL DMSO to make first 10× dilution (S1). Step 2 Take 10 μL of solution from S1 to 990 μL 50% ACN in water to make second 100× dilution (S2). Step 3 Take 100 μL of solution from S2 to 900 μL 50% ACN in water to make third 10× dilution (S3). Step 4 Take 2 μL of solution from S3 to 20 μL blank Plasma to make fourth 10× dilution (S4).
[0140] Totally dose solution was diluted 100000×.
[0141] The concentrations of dose formulation were measured by LC-MS, and the results shown in the table below:
TABLE-US-00022 Dilution Nominal Measured Mean Accuracy SD CV Route Sample Name Factor (mg/mL) (mg/mL) (mg/mL) (%) (mg/mL) (%) IV&SC Dose_IV&SC_1 1000 0.176 0.125 0.129 73.1 0.00770 5.98 0.880 Dose_IV&SC_2 1000 0.123 mg/kg Dose_IV&SC_3 1000 0.138 PO Dose_PO_1 10000 0.880 0.625 0.619 70.4 0.0164 2.65 4.40 Dose_PO_2 10000 0.632 mg/kg Dose_PO_3 10000 0.601 IN Dose_IN_1 100000 8.80 6.00 6.09 69.3 0.340 5.58 1.76 Dose_IN_2 100000 6.47 mg/kg Dose_IN_3 100000 5.82
[0142] Sample Collection
[0143] For IV and subcutaneous (0.880 mg/kg) administration, blood samples were collected from each animal at 0.0333, 0.0833, 0.167, 0.25, 0.5, 1, 2, 4, 8 h post dose.
[0144] For PO (4.40 mg/kg) administration, blood samples were collected from each animal at 0.0833, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 8 h post dose.
[0145] For IN (1.76 mg/kg) administration, blood samples were collected from each animal at 0.0333, 0.0833, 0.167, 0.25, 0.5, 1, 2, 4, 8 h post dose.
[0146] About 200 μL of blood samples were collected from each animal via jugular vein. These blood samples were placed into the tubes containing K2EDTA. And centrifugation at 3000 g for 10 minutes in a 4° C. centrifuge. The completion time from collection was less than 30 min. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min.
Preparation of Standards
[0147] About 1 mg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate and (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in DMSO to obtain a 1 mg/mL standard stock solution (free base).
[0148] Calibration standard working solutions were prepared at concentrations of 2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL by serial dilution of the standard stock solution in 50% acetonitrile in water. Quality control working solutions at concentrations of 5, 10, 20, 500 and 8000 ng/mL were prepared by serial dilution of the standard stock solution in 50% acetonitrile in water. These QC samples were prepared on the day of analysis in the same way as calibration standards.
[0149] Sample Treatment
[0150] For plasma samples, 2 μL of each calibration standard working solution (2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL) was added to 20 μL of the blank SD rat plasma (with 10% 200 mg/mL ascorbic acid) to achieve calibration standards of 0.2-1000 ng/mL (0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 22 μL. Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2) and 2 ng/mL (low-3), 50 ng/mL (mid), 800 ng/mL (high) for plasma were prepared independently for those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. The total volumes of 22 μL of standards, 22 μL of QC samples and 22 μL of unknown samples (20 μL of unknown plasma (with 10% 200 mg/mL ascorbic acid) with 2 μL 50% acetonitrile) were added to 200 μL of acetonitrile containing IS to precipitate proteins. Then the samples were vortexed for 30 sec. After centrifugation at 4° C., 4700 rpm for 30 min, and supernatant was diluted 2 times with water, 10 μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis.
[0151] All the samples were processed on ice.
[0152] LC-MS/MS Conditions
[0153] The LC-MS/MS system consisted of two Shimadzu LC-30AD pumps, a DGU-20A5R degasser, a Rack changer II and LCMS-8060 LC/MS/MS instrument mass spectrometer.
[0154] Chromatographic separation was performed on a DAICEL CORPORATION CHIRALCEL OD-3R Particle size 3 μm 4.6 mm×150 mm column at room temperature. The mobile phase was composed of A: Water (5 mM Ammonium Acetate); B: Ethanol (5 mM Ammonium Acetate). The flow rate was 0.5 mL/min. The injection volume was 10 μL.
[0155] Positive mode electrospray ionization (ESI) was performed to obtain a protonated ion of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions were 268.20.fwdarw.237.15, 268.20.fwdarw.237.15 and 393.20.fwdarw.373.20 for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone, respectively. The instrument parameters were set as follows: ion spray voltage: 8060; heating gas flow: 10 L/min; interface temperature: 250° C.; DL temperature: 220° C.; heat block temperature: 400° C.; drying gas flow: 10 L/min. (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone dependent parameters are listed as follows:
TABLE-US-00023 (6aR)-6-methyl- (6aS)-6-methyl- 5,6,6a,7- 5,6,6a,7- tetrahydro- tetrahydro- 4H-dibenzo 4H-dibenzo [de,g]quinoline- [de,g]quinoline- Dexamethasone Compound ID 10,11-diol 10,1-diol (IS) Transition 268.20.fwdarw.237.15 268.20.fwdarw.237.15 393.20.fwdarw.373.20 Dwell 10.2 10.2 50 Time (msec) Q1 Pre Bias (v) −13 −13 −25 CE −18 −18 −10 Q3 Pre Bias (v) −16 −16 −18
[0156] Data acquisition was performed by Lab Solutions version 5.89 Software (Shimadzu). All concentration data was reported with 3 significant figures. Data statistics were performed using Excel 2010 software. The pharmacokinetic parameters of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were calculated using a non-compartmental approach with Phoenix™ WinNonlin®.
[0157] Results
[0158] The (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations vs. time profiles are shown in
[0159] The pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in Male SD Rats for IV at 0.880 mg/kg, subcutaneous at 0.880 mg/kg, PO at 4.40 mg/kg and IN at 1.76 mg/kg, were used to calculated by employing a non-compartmental analysis (Phoenix™ WinNonlin® 8.0). The linear trapezoidal algorithm was used for AUC calculation.
[0160] The resulting pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following IV administration to Male SD Rats at 0.880 mg/kg:
TABLE-US-00024 AUC.sub.last/ Cl t.sub.1/2 C.sub.0 AUC.sub.last AUC.sub.Inf MRT.sub.Inf AUC.sub.last/D V.sub.ss C.sub.0 Animal (mL/min/kg) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) (L/kg) (h) Rat 1 169 2.66 527 83.2 86.6 1.26 94.6 12.8 0.158 Rat 2 209 2.78 464 67.3 70.1 1.22 76.5 15.4 0.145 Rat 3 175 2.66 476 80.6 83.6 1.18 91.6 12.4 0.169 Mean 185 2.70 489 77.1 80.1 1.22 87.6 13.5 0.157 SD 21.6 0.0690 33.5 8.54 8.81 0.0401 9.70 1.63 0.012
[0161] The resulting pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following subcutaneous administration to Male SD Rats at 0.880 mg/kg:
TABLE-US-00025 t.sub.1/2 T.sub.max C.sub.max AUC.sub.last AUC.sub.Inf MRTInf AUClast/D AUC.sub.last/ F Animal (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) C.sub.max (h) (%) Rat 4 0.812 0.0833 162 70.2 71.2 0.873 79.8 0.433 89.0 Rat 5 0.733 0.167 143 66.9 67.9 0.919 76.0 0.468 84.8 Rat 6 0.839 0.167 119 67.0 68.4 1.06 76.1 0.563 85.4 Mean 0.795 0.139 141 68.0 69.2 0.951 77.3 0.488 86.4 SD 0.0552 0.0483 21.2 1.89 1.80 0.0985 2.15 0.067 2.25
[0162] The resulting ratios of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following PO administration to Male SD Rats at 4.40 mg/kg:
TABLE-US-00026 t.sub.1/2 T.sub.max C.sub.max AUC.sub.last AUC.sub.Inf MRTInf AUClast/D AUC.sub.last/ F Animal (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) C.sub.max (h) (%) Rat 7 3.93 2 23.2 88.4 127 6.26 20.1 3.810 23.0 Rat 8 4.12 0.0833 17.5 32.5 45.0 5.71 7.39 1.857 8.44 Rat 9 7.49 0.5 9.17 42.1 82.9 11.0 9.58 4.591 10.9 Mean 5.18 0.861 16.7 54.4 84.8 7.64 12.4 3.420 14.1 SD 2.00 1.01 7.08 29.9 40.8 2.88 6.79 1.408 7.76
[0163] The resulting ratios of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following IN administration to Male SD Rats at 1.76 mg/kg:
TABLE-US-00027 t.sub.1/2 T.sub.max C.sub.max AUC.sub.last AUC.sub.Inf MRTInf AUClast/D AUC.sub.last/ F Animal (h) (h) (ng/mL) (h*ng/mL) (h*ng/mL) (h) (h*mg/mL) C.sub.max (h) (%) Rat 10 1.18 0.0833 130 69.2 70.2 1.06 39.3 0.532 43.8 Rat 11 1.21 0.0833 269 116 117 1.02 66.0 0.431 73.3 Rat 12 1.34 0.0833 230 97.5 98.7 1.09 55.4 0.424 61.6 Mean 1.24 0.0833 209 94.3 95.4 1.06 53.6 0.462 59.6 SD 0.0814 0.00 71.4 23.7 23.8 0.0373 13.4 0.061 14.8
[0164] As shown in the tables below, the plasma concentrations of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following IV administration to Male SD Rats at 0.880 mg/kg (Rats 1-3) and PO administration to Male SD Rats at 4.40 mg/kg (Rats 7-9), respectively, were not suitable for calculating PK parameters.
TABLE-US-00028 Time Conc. (ng/mL) Mean SD CV (h) Rat 1 Rat 2 Rat 3 (ng/mL) (ng/mL) (%) 0.0333 3.85 3.87 3.54 3.75 0.183 4.88 0.0833 2.69 2.64 2.45 2.59 0.123 4.76 0.167 2.26 1.73 1.66 1.88 0.327 17.4 0.25 1.42 1.24 1.32 1.33 0.0903 6.79 0.5 0.582 0.520 0.575 0.559 0.0340 6.07 1 0.257 0.207 BLOQ 0.232 NA NA 2 BLOQ BLOQ BLOQ NA NA NA 4 BLOQ BLOQ BLOQ NA NA NA 8 BLOQ BLOQ BLOQ NA NA NA
TABLE-US-00029 Time Conc. (ng/mL) Mean SD CV (h) Rat 7 Rat 8 Rat 9 (ng/mL) (ng/mL) (%) 0.0333 BLOQ BLOQ BLOQ NA NA NA 0.0833 BLOQ BLOQ BLOQ NA NA NA 0.167 BLOQ BLOQ BLOQ NA NA NA 0.25 BLOQ BLOQ BLOQ NA NA NA 0.5 BLOQ BLOQ BLOQ NA NA NA 1 BLOQ BLOQ BLOQ NA NA NA 2 BLOQ BLOQ BLOQ NA NA NA 4 BLOQ BLOQ BLOQ NA NA NA 8 BLOQ BLOQ BLOQ NA NA NA NA = Not available; BLOQ = Below quantifiable limit of 0.2 or 0.5 ng/mL.
[0165] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following IV Administration to male SD Rats at 0.880 mg/kg:
TABLE-US-00030 Time Conc. (ng/mL) Mean SD CV (h) Rat 1 Rat 2 Rat 3 (ng/mL) (ng/mL) (%) 0.0333 353 319 346 339 18.1 5.33 0.0833 193 181 214 196 16.7 8.52 0.167 115 80.3 104 100 17.7 17.8 0.25 59.1 43.2 61.0 54.4 9.77 17.9 0.5 17.8 13.1 18.3 16.4 2.90 17.7 1 7.60 5.28 6.23 6.37 1.16 18.3 2 4.32 3.06 3.65 3.67 0.628 17.1 4 2.26 1.83 2.20 2.10 0.235 11.2 8 0.880 0.683 0.767 0.777 0.0989 12.7
[0166] S(6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following subcutaneous administration to male SD Rats at 0.880 mg/kg:
TABLE-US-00031 Time Conc. (ng/mL) Mean SD CV (h) Rat 4 Rat 5 Rat 6 (ng/mL) (ng/mL) (%) 0.0333 42.7 31.9 36.1 36.9 5.43 14.7 0.0833 162 91.0 104 119 37.5 31.5 0.167 151 143 119 138 16.5 12.0 0.25 107 112 113 110 3.11 2.81 0.5 40.5 43.8 44.9 43.1 2.25 5.23 1 5.76 7.07 5.44 6.09 0.860 14.1 2 1.52 1.46 1.60 1.53 0.0677 4.43 4 1.23 1.03 1.40 1.22 0.186 15.2 8 0.877 0.975 1.16 1.00 0.145 14.4
[0167] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following PO administration to male SD Rats at 4.40 mg/kg:
TABLE-US-00032 Time Conc. (ng/mL) Mean SD CV (h) Rat 7 Rat 8 Rat 9 (ng/mL) (ng/mL) (%) 0.0833 9.85 17.5 4.26 10.6 6.67 63.2 0.167 12.0 14.1 6.12 10.7 4.13 38.5 0.25 11.9 12.0 6.17 10.0 3.32 33.2 0.5 14.3 9.58 9.17 11.0 2.84 25.8 0.75 14.5 8.07 8.14 10.2 3.70 36.2 1 14.6 5.23 7.53 9.13 4.90 53.7 2 23.2 7.03 6.93 12.4 9.39 75.7 4 6.65 1.83 4.42 4.30 2.41 56.1 8 6.73 2.09 3.77 4.20 2.35 55.9
[0168] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00033 Time Conc. (ng/mL) Mean SD CV (h) Rat 10 Rat 11 Rat 12 (ng/mL) (ng/mL) (%) 0.0333 117 258 213 196 72.0 36.7 0.0833 130 269 230 209 71.4 34.1 0.167 122 210 165 166 44.1 26.7 0.25 106 129 108 114 13.0 11.4 0.5 23.9 35.7 33.3 31.0 6.25 20.2 1 5.79 12.2 8.93 8.96 3.19 35.6 2 4.82 9.16 6.64 6.87 2.18 31.7 4 2.02 3.57 3.68 3.09 0.929 30.0 8 0.607 0.706 0.640 0.651 0.0504 7.74
[0169] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following IV administration to male SD Rats at 0.880 mg/kg:
TABLE-US-00034 Time Conc. (ng/mL) Mean SD CV (h) Rat 1 Rat 2 Rat 3 (ng/mL) (ng/mL) (%) 0.0333 3.85 3.87 3.54 3.75 0.183 4.88 0.0833 2.69 2.64 2.45 2.59 0.123 4.76 0.167 2.26 1.73 1.66 1.88 0.327 17.4 0.25 1.42 1.24 1.32 1.33 0.0903 6.79 0.5 0.582 0.520 0.575 0.559 0.0340 6.07 1 0.257 0.207 BLOQ 0.232 NA NA 2 BLOQ BLOQ BLOQ NA NA NA 4 BLOQ BLOQ BLOQ NA NA NA 8 BLOQ BLOQ BLOQ NA NA NA
[0170] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following PO administration to male SD Rats at 4.40 mg/kg:
TABLE-US-00035 Time Conc. (ng/mL) Mean SD CV (h) Rat 7 Rat 8 Rat 9 (ng/mL) (ng/mL) (%) 0.0333 BLOQ BLOQ BLOQ NA NA NA 0.0833 BLOQ BLOQ BLOQ NA NA NA 0.167 BLOQ BLOQ BLOQ NA NA NA 0.25 BLOQ BLOQ BLOQ NA NA NA 0.5 BLOQ BLOQ BLOQ NA NA NA 1 BLOQ BLOQ BLOQ NA NA NA 2 BLOQ BLOQ BLOQ NA NA NA 4 BLOQ BLOQ BLOQ NA NA NA 8 BLOQ BLOQ BLOQ NA NA NA
Example 4: Pharmacokinetic and Brain Penetration Study Following Single Dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride Via IN and PO Administration at 1.76 mg/kg and 10.56 mg/kg in Male SD Rats
[0171] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol pharmacokinetic profiles were evaluated in male SD rats (N=3). 12 male SD rats were administered with single IN and PO dose of 1.76 mg/kg and 10.56 mg/kg. The vehicle for the IN and PO formulation contained “0.1% sodium metabisulfite solution”.
[0172] Animals
[0173] Male SD rats were purchased from Si Bei Fu Laboratory Animal Technology Co. Ltd (Beijing, China). The animals were about 8 weeks old with body weights of 200-300 g on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water. All animals were fasted overnight and fed after 4 hours collection.
[0174] Study Design
[0175] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered with an IN dose (1.76 mg/kg, free base) at 0.2 mL/kg, and a PO dose (10.56 mg/kg, free base) at 12 mL/kg. For group 1, plasma, CSF and brain samples were collected at 0.0833 and 0.5 h post dose. For group 2, plasma, CSF and brain samples were collected at 0.5 and 1 h post dose.
TABLE-US-00036 Dose Level Dose Level (mg/kg) (salt (mg/kg) (salt Dose Level Dose hemihydrate anhydrous (mg/kg) Volume Conc. No. of Group Treatment form) form) (free base) (mL/kg) (mg/mL) Route Animals 1 (6aS)-6-methyl- 2.06 2 1.76 0.2 8.80 IN (split 3/male/time 5,6,6a,7- to dose to point tetrahydro-4H- 2 nostril) dibenzo[de,g]quin- oline-10,11-diol hydrochloride 2 (6aS)-6-methyl- 12.36 12 10.56 12 0.880 PO 3/male/time 5,6,6a,7- point tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol hydrochloride
[0176] Dosage and Administration
[0177] Preparation of dosing for IN administration (1.76 mg/kg, Group 1): 3.86 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 0.375 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 8.80 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0178] Preparation of dosing for PO administration (10.56 mg/kg, Group 2): 24.04 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 23.347 mL of 0.1% sodium metabisulfite solution with vortexing to obtain a solution with concentration at 0.880 mg/mL of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol.
[0179] Dose Sample Validation (IN and PO):
TABLE-US-00037 Step 1 Take 20 μL IN and PO dose to 180 μL DMSO to make first 10 X dilution (S1). Step 2 Take 10 μL of solution from S1 to 990 μL 50% ACN in water to make second 100 X dilution (S2). Step 3 Take 100 μL of solution from S2 to 900 μL 50% ACN in water to make third 10 X dilution (S3). Step 4 Take 5 μL of solution from S3 to 50 μL blank Plasma to make fourth 10X dilution (S4).
[0180] Totally dose solution was diluted 100000×.
[0181] The concentrations of dose formulation were measured by LC-MS, and the results shown in the table below:
TABLE-US-00038 Dilution Nominal Measured Mean Accuracy SD CV Route Sample Name Factor (mg/mL) (mg/mL) (mg/mL) (%) (mg/mL) (%) IN IN_DOSE_1 100000 8.80 9.03 8.98 102 0.162 1.80 IN_DOSE_2 100000 8.81 IN_DOSE_3 100000 9.12 PO PO_DOSE_1 100000 0.880 0.871 0.901 102 0.0260 2.89 PO_DOSE_2 100000 0.910 PO_DOSE_3 100000 0.921
[0182] Sample Collection
[0183] For Group 1 (IN dose, 1.76 mg/kg), blood, CSF and brain samples were collected from 3 mice at two time points: 0.0833 h and 0.5 h post dose.
[0184] For Group 2 (PO dose, 10.56 mg/kg), blood, CSF and brain samples were collected from 3 mice at two time point: 0.5 h and 1 h post dose.
[0185] For plasma samples, about 200 μL of blood samples were collected from each animal via jugular vein. These blood samples were placed into the tubes containing K2EDTA, and centrifuged at 3000 g for 10 minutes in a 4° C. centrifuge. The completion time from collection was less than 30 min. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min.
[0186] For brain and CSF samples, The animals were fully exsanguinated with a rising concentration of CO2 gas prior to CSF and brain collection.
[0187] For CSF collection, an incision was made by scissors on the neck of the rats to expose the foramen magnum. A syringe fitted with a venoclysis needle was used to sample the CSF, the tip of the needle was inserted into the membrane of foramen magnum about 1-2 mm, and a negative pressure was applied by pulling a stopcock a small length of the syringe to extract CSF fluids into a tube. The stopcock was further actuated to until a sufficient amount of CSF was collected. There should be no blood in the CSF fluid. A stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in CSF.
[0188] For brain collection. Brain samples were collected at an adopted time point, and washed 3 times with cold saline, then dried with clean gauze and put into the weighed tube, quick frozen in an ice box and kept at −75±15° C. All brain samples were weighted and homogenized with phosphate buffered saline (PBS) by brain weight (g) to PBS volume (mL) ratio 1:3 before analysis. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in grain homogenate. The actual concentration was the detected value multiplied by the dilution factor. Brain samples were collected at designed time point, quickly homogenized and kept at −75±15° C.
Preparation of Standards
[0189] About 1 mg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate and (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in DMSO to obtain a 1 mg/mL standard stock solution (free base).
[0190] Calibration standard working solutions were prepared at concentrations of 2, 5, 10, 20, 50, 100, 500, 1000, 5000 and 10000 ng/mL by serial dilution of the standard stock solution in 50% acetonitrile in water. Quality control working solutions at concentrations of 5, 10, 20, 500 and 8000 ng/mL were prepared by serial dilution of the standard stock solution in 50% acetonitrile in water. These QC samples were prepared on the day of analysis in the same way as calibration standards.
[0191] Sample Treatment
[0192] For plasma, CSF, and brain samples, 5 μL of each calibration standard working solution (2, 5, 10, 50, 100, 500, 1000, 5000 and 10000 ng/mL) was added to 50 μL of the blank SD rat plasma, CSF or brain homogenate (with 10% 200 mg/mL ascorbic acid) to achieve calibration standards of 0.2-1000 ng/mL (0.2, 0.5, 1, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 55 μL. Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2), 50 ng/mL (mid), 800 ng/mL (high) for plasma, CSF or brain homogenate were prepared independently for those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. The total volumes of 55 μL of standards, 55 μL of QC samples and 55 μL of unknown samples (50 μL of unknown plasma, CSF or brain homogenate (with 10% 200 mg/mL ascorbic acid) with 5 μL 50% acetonitrile) were added to 200 μL of acetonitrile containing IS to precipitate proteins. Then the samples were vortexed for 30 sec. After centrifugation at 4° C., 4700 rpm for 30 min, and supernatant was diluted 2 times with water, 10 μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis.
[0193] All the samples were processed on ice.
[0194] LC-MS/MS Conditions
[0195] The LC-MS/MS system consisted of two Shimadzu LC-30AD pumps, a DGU-20A5R degasser, a Rack changer II and LCMS-8060 LC/MS/MS instrument mass spectrometer.
[0196] Chromatographic separation was performed on a DAICEL CORPORATION CHIRALCEL OD-3R Particle size 3 μm 4.6 mm×150 mm column at room temperature. The mobile phase was composed of A: Water (5 mM Ammonium Acetate); B: Ethanol (5 mM Ammonium Acetate). The flow rate was 0.7 mL/min. The injection volume was 10 μL.
[0197] Positive mode electrospray ionization (ESI) was performed to obtain a protonated ion of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions were 268.20191.10, 268.20191.10 and 393.20-373.20 for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone, respectively. The instrument parameters were set as follows: ion spray voltage: 8060; heating gas flow: 10 L/min; interface temperature: 250° C.; DL temperature: 220° C.; heat block temperature: 400° C.; drying gas flow: 10 L/min. (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Dexamethasone dependent parameters are listed as follows:
TABLE-US-00039 (6aR)-6-methyl- (6aS)-6-methyl- 5,6,6a,7- 5,6,6a,7-tetrahydro- tetrahydro-4H- 4H- dibenzo[de,g]quin dibenzo[de,g]quinolin Compound ID oline-10,11-diol e−10,11-diol Dexamethasone (IS) Transition 268.20.fwdarw.191.10 268.20.fwdarw.191.10 393.20.fwdarw.373.20 Dwell 100 100 50 Time(msec) Q1 Pre Bias (v) −10 −10 −25 CE −30 −30 −10 Q3 Pre Bias (v) −19 −19 −18
[0198] Data acquisition was performed by Lab Solutions version 5.89 Software (Shimadzu). All concentration data was reported with 3 significant figures. Data statistics were performed using Excel 2010 software. The pharmacokinetic parameters of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were calculated using a non-compartmental approach with Phoenix™ WinNonlin®.
[0199] Results
[0200] The plasma, CSF, and brain concentrations for (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in each animal for IN and PO dose at 1.76 mg/kg and 10.56 mg/kg, were used to calculate the ratio between brain or CSF concentrations and plasma concentrations.
[0201] The resulting of the ratio of 6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and brain following IN administration to Male SD Rats at 1.76 mg/kg:
TABLE-US-00040 Ratio (Brain/ Mean Ratio Brain Plasma Plasma) (Brain/Plasma) Time (h) Animal (ng/g) (ng/mL) (mL/g) (mL/g) 0.0833 Rat 1 1029 460 2.24 3.78 Rat 2 1995 377 5.30 Rat 3 1764 463 3.81 0.5 Rat 4 299 50.2 5.95 5.62 Rat 5 180 33.9 5.31 Rat 6 441 78.9 5.59
[0202] The resulting ratio of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and CSF following IN administration to Male SD Rats at 1.76 mg/kg:
TABLE-US-00041 CSF Plasma Ratio (CSF/ Mean Ratio Time (h) Animal (ng/mL) (ng/mL) Plasma) (CSF/ Plasma) 0.0833 Rat 1 43.0 460 0.0935 0.137 Rat 2 70.0 377 0.186 Rat 3 60.4 463 0.130 0.5 Rat 4 11.7 50.2 0.233 0.244 Rat 5 8.05 33.9 0.237 Rat 6 20.7 78.9 0.263
[0203] The resulting ratio of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and brain following PO administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00042 Mean Ratio Brain Plasma Ratio (Brain/ (Brain/Plasma) Time (h) Animal (ng/g) (ng/mL) Plasma) (mL/g) (mL/g) 0.5 Rat 7 32.2 49.3 0.654 0.689 Rat 8 39.8 44.9 0.886 Rat 9 18.8 35.6 0.528 1 Rat 10 17.1 28.9 0.590 0.391 Rat 11 12.9 43.6 0.297 Rat 12 13.6 47.8 0.285
[0204] The resulting ratio of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and CSF following PO administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00043 CSF Plasma Ratio (CSF/ Mean Ratio Time (h) Animal (ng/mL) (ng/mL) Plasma) (CSF/ Plasma) 0.5 Rat 7 1.43 49.3 0.0290 0.0419 Rat 8 2.46 44.9 0.0547 Rat 9 BLOQ 35.6 NA 1 Rat 10 BLOQ 28.9 NA NA Rat 11 BLOQ 43.6 NA Rat 12 BLOQ 47.8 NA
[0205] The resulting ratios of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and brain following IN administration to Male SD Rats at 1.76 mg/kg:
TABLE-US-00044 Ratio (Brain/ Brain Plasma Plasma) Mean Ratio (Brain/ Time (h) Animal (ng/g) (ng/mL) (mL/g) Plasma) (mL/g) 0.0833 Rat 1 18.5 4.00 4.62 7.16 Rat 2 31.8 3.38 9.43 Rat 3 28.8 3.88 7.43 0.5 Rat 4 12.7 BLOQ NA NA Rat 5 7.11 BLOQ NA Rat 6 10.7 1.23 8.67 Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for plasma.
[0206] The resulting ratios of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between plasma and CSF following IN administration to Male SD Rats at 1.76 mg/kg:
TABLE-US-00045 CSF Plasma Ratio (CSF/ Mean Ratio Time (h) Animal (ng/mL) (ng/mL) Plasma) (CSF/ Plasma) 0.0833 Rat 1 BLOQ 4.00 NA NA Rat 2 1.11 3.38 0.330 Rat 3 BLOQ 3.88 NA 0.5 Rat 4 BLOQ BLOQ NA NA Rat 5 BLOQ BLOQ NA Rat 6 BLOQ 1.23 NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for CSF and plasma.
[0207] The resulting ratios of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between Plasma and Brain Following PO Administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00046 Mean Ratio Brain Plasma Ratio (Brain/ (Brain/ Plasma) Time (h) Animal (ng/g) (ng/mL) Plasma) (mL/g) (mL/g) 0.5 Rat 7 2.46 BLOQ NA NA Rat 8 2.73 BLOQ NA Rat 9 3.10 BLOQ NA 1 Rat 10 BLOQ BLOQ NA NA Rat 11 BLOQ BLOQ NA Rat 12 BLOQ BLOQ NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for plasma and 0.5 ng/mL for brain.
[0208] The resulting ratios of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol between Plasma and CSF Following PO Administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00047 CSF Plasma Ratio (CSF/ Mean Ratio Time (h) Animal (ng/mL) (ng/mL) Plasma) (CSF/Plasma) 0.5 Rat 7 BLOQ BLOQ NA NA Rat 8 BLOQ BLOQ NA Rat 9 BLOQ BLOQ NA 1 Rat 10 BLOQ BLOQ NA NA Rat 11 BLOQ BLOQ NA Rat 12 BLOQ BLOQ NA
[0209] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00048 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.0833 460 377 463 433 48.9 11.3 0.5 50.2 33.9 78.9 54.4 22.8 42.0
[0210] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00049 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.0833 43.0 70.0 60.4 57.8 13.7 23.7 0.5 11.7 8.05 20.7 13.5 6.54 48.5
[0211] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00050 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.0833 257 499 441 1029 1995 1764 1596 505 31.6 0.5 74.7 45.1 110 299 180 441 307 131 42.6 Note: All the brain samples were added with PBS by brain weight (g) to PBS volume (mL) Ratio 1:3 for homogenizing. The actual concentration (ng/g) is the detected value (ng/mL) multiplied by 4.
[0212] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following PO administration to male SD Rats at 10.56 mg/kg:
TABLE-US-00051 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.5 49.3 44.9 35.6 43.3 7.03 16.2 1 28.9 43.6 47.8 40.1 9.90 24.7
[0213] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following PO administration to male SD Rats at 10.56 mg/kg:
TABLE-US-00052 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.5 1.43 2.46 BLOQ 1.95 NA NA 1 BLOQ BLOQ BLOQ NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for CSF.
[0214] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following PO administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00053 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.5 8.06 9.95 4.70 32.2 39.8 18.8 30.3 10.7 35.2 1 4.27 3.24 3.40 17.1 12.9 13.6 14.5 2.22 15.2 Note: All the brain samples were added with PBS by brain weight (g) to PBS volume (mL) Ratio 1:3 for homogenizing. The actual concentration (ng/g) is the detected value (ng/mL) multiplied by 4.
[0215] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00054 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.0833 4.00 3.38 3.88 3.75 0.331 8.82 0.5 BLOQ BLOQ 1.23 NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for plasma.
[0216] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00055 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.0833 BLOQ 1.11 BLOQ NA NA NA 0.5 BLOQ BLOQ BLOQ NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for CSF.
[0217] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following IN administration to male SD Rats at 1.76 mg/kg:
TABLE-US-00056 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.0833 4.62 7.96 7.20 18.5 31.8 28.8 26.4 6.99 26.5 0.5 3.19 1.78 2.68 12.7 7.11 10.7 10.2 2.85 28.0 Note: All the brain samples were added with PBS by brain weight (g) to PBS volume (mL) Ratio 1:3 for homogenizing. The actual concentration (ng/g) is the detected value (ng/mL) multiplied by 4.
[0218] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following PO administration to male SD Rats at 10.56 mg/kg:
TABLE-US-00057 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.5 BLOQ BLOQ BLOQ NA NA NA 1 BLOQ BLOQ BLOQ NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for plasma.
[0219] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following PO administration to male SD Rats at 10.56 mg/kg:
TABLE-US-00058 Time Conc. (ng/mL) Mean SD CV (h) Sort 1 Sort 2 Sort 3 (ng/mL) (ng/mL) (%) 0.5 BLOQ BLOQ BLOQ NA NA NA 1 BLOQ BLOQ BLOQ NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 1 ng/mL for CSF.
[0220] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following PO administration to Male SD Rats at 10.56 mg/kg:
TABLE-US-00059 Time Conc. (ng/mL) Conc. (ng/g) Mean SD CV (h) Sort 1 Sort 2 Sort 3 Sort 1 Sort 2 Sort 3 (ng/g) (ng/g) (%) 0.5 0.616 0.683 0.775 2.46 2.73 3.10 2.77 0.319 11.5 1 BLOQ BLOQ BLOQ NA NA NA NA NA NA Note: NA = Not available, BLOQ = Below quantifiable limit of 0.5 ng/mL for brain. All the brain samples were added with PBS by brain weight (g) to PBS volume (mL) Ratio 1:3 for homogenizing. The actual concentration (ng/g) is the detected value (ng/mL) multiplied by 4.
Example 5: Plasma and CSF Pharmacokinetic Study of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Following Single Sublingual, Oral
[0221] Administration or Subcutaneous Injection of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol to Non-Naïve Male Beagle Dogs
[0222] The pharmacokinetics of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were evaluated in 9 male non-naïve beagle dogs following single sublingual (SL) administration at 5 mg/kg, oral (PO) administration at 10 mg/kg, and subcutaneous (SC) injection at 1 mg/kg.
[0223] Animals
[0224] Nine non-naïve male beagle dogs supplied by Marshall Bioresources (Beijing, China) were used in this study. The animals were confirmed healthy before being assigned to the study. The room(s) were controlled and monitored for relative humidity (targeted mean range 40% to 70%, and were on a 12-hour light/dark cycle except when interruptions are necessitated by study activities.
[0225] Animals were fed twice daily. Stock Dogs were fed approximately 220 grams of Certified Dog Diet daily (Beijing Keao Xieli Feed Co., Ltd. Beijing, P. R. China). These amounts could be adjusted as necessary based on food consumption of the group or an individual body weight change. For SC, SL and PO groups, animals were fed at 3:30 to 4:00 pm on the day prior to oral dosing and any remaining food was removed at 7:00 pm. Food was withheld until 4-hour post-dose. Animals were fed once on the day of dosing, with the amount of approximately 220 grams.
[0226] Study Design
[0227] Nine male non-naïve beagle dogs were assigned to this study consisting of three groups. In group 1, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by single SL administration at 5 mg/kg, which was formulated in 0.83% HPMC (40˜60 cp):PEG400:VE-TPGS=60:30:10. In group 2, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by PO administration at 10 mg/kg, which was formulated in 0.5% CMC-Na in water. In group 3, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by SC administration at 1 mg/kg, which was formulated in saline. The dosing regimens and formulation (vehicle) compositions are described in the following table below:
TABLE-US-00060 Dose Dose Level Volume Conc. No. of Group Treatment (mg/kg) (mL/kg) (mg/mL) Vehicle Route Animals G1 (6aS)-6-methyl- 5 0.1 50 0.83% HPMC SL (fasted) 3 (D1001, 5,6,6a,7- (40~60 D1002, tetrahydro-4H- cp):PEG400:VE- D1003) dibenzo[de,g]quin- TPGS = 60:30:10 oline-10,11- diol hydrochloride G2 (6aS)-6-methyl- 10 5 2 0.5% CMC- PO (fasted) 3 (D2001, 5,6,6a,7- Na in water D2002, tetrahydro-4H- D2003) dibenzo[de,g]quin- oline-10,11- diol hydrochloride G3 (6aS)-6-methyl- 1 1 1 Saline SC (fasted) 3 (D0001, 5,6,6a,7- D3002, tetrahydro-4H- D3003) dibenzo[de,g]quin- oline-10,11- diol hydrochloride
[0228] The sampling regimens are described in the following table below:
TABLE-US-00061 Dose Level Sampling Time Point (hr) Group (mg/kg) Pre-dose 0.033 0.167 0.25 0.5 1 2 4 8 24 G1P 5 P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P G2P 10 P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P G3P 1 P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P + CSF P P = Plasma; CSF = Cerebral Spinal Fluid
[0229] Dose Administration
[0230] Animals were weighed prior to dose administration on the day of dosing for calculating the actual dose volume. In group 1, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by single SL administration at 5 mg/kg, which was formulated in 0.83% HPMC (40-60 cp):PEG400:VE-TPGS=60:30:10. In group 2, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by PO administration at 10 mg/kg, which was formulated in 0.5% CMC-Na in water. In group 3, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by SC administration at 1 mg/kg, which was formulated in Saline.
[0231] Sample Collection
[0232] Approximately 0.5 mL blood was collected at each time point. All blood samples were collected from a peripheral vessel. All blood samples were transferred into commercially available tubes containing Potassium (K2) EDTA on wet ice and processed for plasma. Samples were centrifuged (3,000×g for 10 minutes at 2 to 8° C.) within 30 minutes of collection. After centrifugation, the stabilizing agent was added to the harvested plasma within 60 minutes. The samples were kept on ice and protected from sunlight (under yellow light). The stabilizing agent was ascorbic acid 200 mg/mL added in a proportion of 10% in plasma. The plasma samples were divided into two equal samples with each at least 0.1 ml (0.1*2 mL/sample) and transferred into labeled polypropylene micro-centrifuge tubes and stored frozen in a freezer set to maintain −60° C. or lower until analyzed.
[0233] About 0.1 mL/time point (50 μL*2 aliquots) of CSF were collected via the catheter implanted in the Cisterna Magna of conscious beagle dogs without any sedation. For CSF collection, a polypropylene tube was first weighted to accuracy of 0.1 mg (weight #1). CSF was collected in the tube and weighted again (weight #2). Total CSF weight was calculated by subtracting weight #1 from weight #2. Samples were kept on ice and protected from sunlight. Stabilizing agent (ascorbic acid 200 mg/mL) was added within 60 minutes in a proportion of 10% in CSF. Frozen CSF samples on dry ice were stored at −60° C. or lower until bioanalysis.
Preparation of Standards
[0234] In plasma, blank male Beagle dog plasma with EDTA-K2 as anti-coagulant was used for the preparation of standards. In CSF blank artificial CSF was used for the preparation of standards.
[0235] About 1.82 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol was dissolved in DMSO to obtain a 1000 μg/mL stock solution (Stock A).
[0236] The preparation procedures of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol working solutions are detailed in the table below:
TABLE-US-00062 Source Solution Dilution Working Solution Source Conc. Volume Volume Volume Final Conc. Working Solution ID (μg/mL) (μL) (μL) (μL) (μg/mL) Solution ID Stock A 1000 30 470 500 60 WS-C1 WS-C1 60 120 240 360 20 WS-C2 WS-C2 20 160 160 320 10 WS-C3 WS-C3 10 80 320 400 2 WS-C4 WS-C4 2 160 160 320 1 WS-C5 WS-C5 1 80 320 400 0.2 WS-C6 WS-C6 0.2 80 320 400 0.04 WS-C7 WS-C7 0.04 160 160 320 0.02 WS-C8 Stock A 1000 30 595 625 48 WS-QC1 WS-QC1 48 120 240 360 16 WS-QC2 WS-QC2 16 20 380 400 0.8 WS-QC3 WS-QC3 0.8 30 370 400 0.06 WS-QC4 Stock A 1000 30 270 300 100 WS-DQC1 Dilution Solvent: ACN/water (v:v, 70:30)
[0237] Preparation details of the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol calibration and QC standards in plasma and CSF are shown in the following table:
TABLE-US-00063 Working Solution Matrix Calibration Standard Source Conc. Volume Volume Volume Final Conc. Calibration Solution ID (μg/mL) (μL) (μL) (μL) (ng/mL) Standard ID WS-C1 60 3 57 60 3000 C1 WS-C2 20 3 57 60 1000 C2 WS-C3 10 3 57 60 500 C3 WS-C4 2 3 57 60 100 C4 WS-C5 1 3 57 60 50 C5 WS-C6 0.2 3 57 60 10 C6 WS-C7 0.04 3 57 60 2 C7 WS-C8 0.02 3 57 60 1 C8 Source Conc. Volume Volume Volume Final Conc. Solution ID (μg/mL) (μL) (μL) (μL) (ng/mL) QCID WS-QC1 48 3 57 60 2400 QC1 WS-QC2 16 3 57 60 800 QC2 WS-QC3 0.8 3 57 60 40 QC3 WS-QC4 0.06 3 57 60 3 QC4 WS-DQC1 100 3 57 60 5000 DQC
[0238] The concentrations of calibration and QC samples are shown in the table below:
TABLE-US-00064 Calibration Standard Quality Control Samples Cone. Samples Cone. Compound Matrix (ng/mL) (ng/mL) (6aS)-6-methyl- Male Beagle dog 1, 2, 10, 50, 500, 3000 3, 40, 800, 2400, 5,6,6a,7-tetrahydro-4H- plasma 5000(DQC) dibenzo [de,g]quinoline- 10,11-diol group 1 (6aS)-6-methyl- Male Beagle dog 1, 2, 10, 50, 100, 500, 3, 40, 800, 2400 5,6,6a,7-tetrahydro-4H- plasma 1000, 3000 dibenzo [de,g]quinoline- 10,11-diol groups 2 and 3 (6aS)-6-methyl- Male Beagle dog CSF 1, 2, 10, 50, 100, 500, 3, 40, 800, 2400 5,6,6a,7-tetrahydro-4H- 1000, 3000 dibenzo [de,g]quinoline- 10,11-diol group 1 (6aS)-6-methyl- Male Beagle dog CSF 1, 10, 50, 100, 500, 5,6,6a,7-tetrahydro-4H- 1000, 3000 dibenzo [de,g]quinoline- 10,11-diol group 1 retest (6aS)-6-methyl- Male Beagle dog CSF 1, 2, 10, 100, 500, 1000, 3, 40, 800, 2400 5,6,6a,7-tetrahydro-4H- 3000 dibenzo [de,g]quinoline- 10,11-diol group 2 (6aS)-6-methyl- Male Beagle dog CSF 1, 2, 50, 100, 500, 1000, 3, 40, 800, 2400 5,6,6a,7-tetrahydro-4H- 3000 dibenzo [de,g]quinoline- 10,11-diol group 3
[0239] Sample Treatment
[0240] All CSF samples were added with the equal volume of plasma. An aliquot of 10 μL sample with 200 μL internal standard (100 ng/mL Labetalol & 100 ng/mL Dexamethasone & 100 ng/mL Tolbutamide & 100 ng/mL Verapamil & 100 ng/mL Glyburide & 200 ng/mL Dclofenac in ACN), the mixture was vortex-mixed well and centrifuged at 4000 rpm for 15 min, 4° C. 30 μL supernatant was then mixed with 30 μL water, vortex-mixed well and centrifuged at 4° C. 3 μL sample was injected for LC-MS/MS analysis.
[0241] Dilution procedure description: Dilution factor as 5—An aliquot of 2 μL sample was added with 8 μL blank matrix.
[0242] LC-MS/MS Conditions
[0243] The LC-MS/MS system consisted of an ACQUITY UPLC System and API 4000 mass spectrometer. Chromatographic separation was performed on a ACQUITY UPLC HSS T3 (2.1×50 mm, 1.8 μm) column. The mobile phase was composed of A: 0.1% formic acid and 2 mM HCOONH.sub.4 in Water:ACN (v:v, 95:5); and B: 0.1% formic acid & 2 mM HCOONH.sub.4 in ACN:Water (v:v, 95:5). The flow rate was 0.6 mL/min. The injection volume was 3 μL. The following gradient was implemented:
TABLE-US-00065 Flow Rate Time (min) (mL/min) A (%) B (%) Initial 0.600 100 0 0.30 0.600 100 0 1.10 0.600 5 95 1.40 0.600 5 95 1.41 0.600 100 0 1.60 0.600 100 0
[0244] Positive mode electrospray ionization (ESI) was performed to obtain a protonated ion of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Diclofenac (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Diclofenac dependent parameters are listed as follows:
TABLE-US-00066 Collision Energy Retention Compound Name Ion Transition (eV) Time (min) (6aS)-6-methyl- 268.0/219.0 32 0.85 5,6,6a,7-tetrahydro- 4H- dibenzo [de,g] quinoline- 10,11-diol Diclofenac 296.0/214.0 40 1.29
[0245] Data acquisition was performed by Analyst 1.6.2 software was used for processing the data of all samples.
[0246] Results
[0247] The individual and mean plasma concentration-time profiles are illustrated from
[0248] In group 1: For the SL dose at 5 mg/kg, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a maximum plasma concentration (C.sub.max) of 143±24.0 ng/mL, with time to reach C.sub.max (T.sub.max) at 0.583±0.382 h. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a maximum CSF concentration (C.sub.max) of 126±144 ng/mL, with time to reach C.sub.max (T.sub.max) at 1.08±0.878 h. The half-life (t.sub.1/2) of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in plasma and CSF were 2.76±2.95 h and 1.79±0.705 h, respectively. The AUC.sub.last and AUCinf values in plasma were 155±10.5 ng.Math.h/mL and 162±14.5 ng.Math.h/mL, respectively. The AUC.sub.last and AUCinf values in CSF were 162±135 ng.Math.h/mL and 170±144 ng.Math.h/mL, respectively. The AUC.sub.last ratio of CSF to total plasma was 1.08±0.964.
[0249] The table below demonstrates the individual and mean plasma PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SL administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 5 mg/kg:
TABLE-US-00067 PK Mean SL CV Parameters D01001 D01002 D01003 Plasma SD (%) No. points used for t.sub.1/2 5.00 3.00 5.00 ND — — C.sub.max (ng/mL) 162 116 151 143 24.0 16.8 T.sub.max (h) 0.500 1.00 0.250 0.583 0.382 65.5 t.sub.1/2 (h) 6.13 0.671 1.48 2.76 2.95 107 T.sub.last (h) 24.0 4.00 8.00 ND — — AUC.sub.last 165 154 144 155 10.5 6.79 (ng .Math. h/mL) AUC.sub.inf 178 159 150 162 14.5 8.92 (ng .Math. h/mL) MRT.sub.last (h) 3.39 1.19 1.29 1.96 1.24 63.3 MRT.sub.inf (h) 5.57 1.30 1.64 2.83 2.37 83.7 AUC.sub.Extra (%) 7.39 2.79 3.90 4.69 2.40 51.1 AUMC.sub.Extra 43.6 10.7 24.2 26.1 16.5 63.3 (%) AUC.sub.last/C.sub.max 1.019 1.328 0.954 1.100 0.200 18.2 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0250] The following table illustrates the individual mean CSF PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SL administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 5 mg/kg:
TABLE-US-00068 PK Mean SL CV Parameters D01001 D01002 D01003 CSF SD (%) No. points used for t.sub.1/2 3.00 3.00 5.00 ND — — C.sub.max (ng/mL) 76.2 13.8 289 126 144 114 T.sub.max (h) 2.00 1.00 0.250 1.08 0.878 81.0 t.sub.1/2 (h) 1.06 1.83 2.47 1.79 0.705 39.5 Tlast (h) 8.00 8.00 8.00 8.00 — — AUC.sub.last 122 52.0 313 162 135 83.2 (ng .Math. h/mL) AUC.sub.inf 124 55.1 332 170 144 84.6 (ng .Math. h/mL) MRT.sub.last (h) 2.43 3.05 3.04 2.84 0.353 12.4 MRTinf (h) 2.54 3.47 3.53 3.18 0.555 17.5 AUC.sub.Extra (%) 1.45 5.55 5.65 4.22 2.40 56.9 AUMC.sub.Extra 5.44 17.0 18.5 13.7 7.16 52.4 (%) AUC Ratio 0.738 0.337 2.17 1.08 0.964 89.1 AUC.sub.last/C.sub.max 1.601 3.768 1.083 2.151 1.424 66.2 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0251] In group 2: For the PO dose at 10 mg/kg, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a plasma C.sub.max of 78.4±6.21 ng/mL, with T.sub.max at 0.500±0.000 h. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a CSF C.sub.max of 30.3±16.1 ng/mL, with T.sub.max at 0.417±0.144 h. The t.sub.1/2 of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in plasma and CSF were 11.3±11.5 h and 10.1±13.3 h, respectively. The AUC.sub.last and AUCinf values in plasma were 228±74.9 ng.Math.h/mL and 322±199 ng.Math.h/mL, respectively. The AUC.sub.last and AUC.sub.inf values in CSF were 58.1±8.47 ng.Math.h/mL and 102±68.9 ng.Math.h/mL, respectively. The AUC.sub.last ratio of CSF to total plasma was 0.270±0.0663.
[0252] The table below demonstrates the individual and mean plasma PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg:
TABLE-US-00069 PK Mean PO CV Parameters D02001 D02002 D02003 Plasma SD (%) No. points used for t.sub.1/2 3.00 3.00 4.00 ND — — C.sub.max (ng/mL) 84.8 72.4 78.0 78.4 6.21 7.92 T.sub.max (h) 0.500 0.500 0.500 0.500 0.000 0.0 t.sub.1/2 (h) 2.46 7.00 24.3 11.3 11.5 102 T.sub.last (h) 8.00 24.0 24.0 ND — — AUC.sub.last 149 236 298 228 74.9 32.9 (ng .Math. h/mL) AUC.sub.inf 164 256 546 322 199 61.9 (ng .Math. h/mL) MRT.sub.last (h) 2.57 6.32 8.74 5.88 3.11 52.9 MRT.sub.inf (h) 3.42 8.44 31.6 14.5 15.0 104 AUC.sub.Extra (%) 9.43 7.63 45.4 20.8 21.3 102 AUMC.sub.Extra 31.9 30.8 84.9 49.2 30.9 62.9 (%) AUC.sub.last/C.sub.max 1.757 3.260 3.821 2.946 1.067 36.2 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0253] The following table illustrates the individual mean CSF PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg:
TABLE-US-00070 PK Mean PO CV Parameters D02001 D02002 D02003 CSF SD (%) No. points used for t.sub.1/2 3.00 4.00 4.00 ND — — C.sub.max (ng/mL) 24.7 48.5 17.8 30.3 16.1 53.1 T.sub.max (h) 0.250 0.500 0.500 0.417 0.144 34.6 t.sub.1/2 (h) 2.25 2.68 25.5 10.1 13.3 131 T.sub.last (h) 8.00 8.00 8.00 8.00 — — AUC.sub.last 49.1 66.0 59.2 58.1 8.47 14.6 (ng .Math. h/mL) AUC.sub.inf 54.1 71.3 181 102 68.9 67.4 (ng .Math. h/mL) MRT.sub.last (h) 3.09 2.74 3.94 3.26 0.615 18.9 MRT.sub.inf (h) 3.84 3.43 31.4 12.9 16.0 124 AUC.sub.Extra (%) 9.18 7.53 67.3 28.0 34.0 122 AUMC.sub.Extra 26.9 26.1 95.9 49.6 40.1 80.8 (%) AUC Ratio 0.330 0.279 0.199 0.270 0.0663 24.6 AUC.sub.last/C.sub.max 1.988 1.361 3.326 2.225 1.004 45.1 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0254] In group 3: For the SC dose at 1 mg/kg, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a plasma C.sub.max of 110±19.4 ng/mL, with T.sub.max at 0.222±0.0479 h. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a CSF C.sub.max of 28.5±14.0 ng/mL, with T.sub.max at 0.417±0.144 h. The t.sub.1/2 of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in plasma and CSF were 5.43±8.36 h and ND, respectively. The AUC.sub.last and AUC.sub.inf values in plasma were 80.1±12.3 ng.Math.h/mL and 88.4±23.8 ng.Math.h/mL, respectively. The AUC.sub.last and AUC.sub.inf values in CSF were 78.2±39.3 ng.Math.h/mL and ND, respectively. The AUC.sub.last ratio of CSF to total plasma was 0.964±0.456.
[0255] The table below demonstrates the individual and mean plasma PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SC administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 1 mg/kg:
TABLE-US-00071 PK Mean SC CV Parameters D03001 D03002 D03003 Plasma SD (%) No. points used for t.sub.1/2 3.00 5.00 4.00 ND — — C.sub.max (ng/mL) 127 88.7 113 110 19.4 17.7 T.sub.max (h) 0.250 0.167 0.250 0.222 0.0479 21.6 t.sub.1/2 (h) 15.1 0.564 0.641 5.43 8.36 154 T.sub.last (h) 24.0 4.00 4.00 ND — — AUC.sub.last 92.0 67.4 80.8 80.1 12.3 15.4 (ng .Math. h/mL) AUC.sub.inf 115 68.3 82.2 88.4 23.8 26.9 (ng .Math. h/mL) MRT.sub.last (h) 4.56 0.682 0.711 1.98 2.23 112 MRT.sub.inf (h) 12.7 0.736 0.783 4.74 6.89 145 AUC.sub.Extra (%) 19.8 1.32 1.71 7.59 10.5 139 AUMC.sub.Extra 71.2 8.64 10.8 30.2 35.5 118 (%) AUC.sub.last/C.sub.max 0.724 0.760 0.715 0.733 0.024 3.2 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0256] The following table illustrates the individual mean CSF PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SC administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 1 mg/kg:
TABLE-US-00072 PK Mean SC CV Parameters D03001 D03002 D03003 CSF SD (%) No. points used for t.sub.1/2 0.00 5.00 0.00 ND — — C.sub.max (ng/mL) 17.8 23.3 44.4 28.5 14.0 49.3 T.sub.max (h) 0.500 0.250 0.500 0.417 0.144 34.6 t.sub.1/2 (h) ND 4.79* ND ND ND ND T.sub.last (h) 8.00 8.00 8.00 8.00 — — AUC.sub.last 74.1 41.0 119 78.2 39.3 50.3 (ng .Math. h/mL) AUC.sub.inf ND 71.2 ND ND ND ND (ng .Math. h/mL) MRT.sub.last (h) 4.27 2.81 4.07 3.72 0.792 21.3 MRT.sub.inf (h) ND 7.94 ND ND ND ND AUC.sub.Extra (%) ND 42.4 ND ND ND ND AUMC.sub.Extra ND 79.6 ND ND ND ND (%) AUC Ratio 0.806 0.609 1.48 0.964 0.456 47.3 AUC.sub.last/C.sub.max 4.163 1.760 2.680 2.868 1.213 42.3 (h) “—” means not applicable “ND” means not determined “D#” means animal number
[0257] Individual and mean plasma concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SL administration of (6aS)-6-methyl-5,6,6a, 7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 5 mg/kg (ng/mL):
TABLE-US-00073 Time Mean SL CV (h) D01001 D01002 D01003 Plasma SD (%) 0 BQL BQL BQL ND ND ND 0.0330 2.26 BQL BQL ND ND ND 0.167 30.9 53.6 104 62.8 37.4 59.5 0.250 45.2 51.2 151 82.5 59.4 72.1 0.500 162 87.3 116 122 37.7 30.9 1.00 75.6 116 54.5 82.0 31.3 38.1 2.00 11.2 21.2 10.8 14.4 5.89 40.9 4.00 2.49 4.58 3.10 3.39 1.07 31.7 8.00 1.51 BQL 2.74 2.13 ND ND 24.0 1.49 BQL BQL ND ND ND
[0258] Individual and mean CSF concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SL administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 5 mg/kg (ng/mL):
TABLE-US-00074 Time Mean SL CV (h) D01001 D01002 D01003 CSF SD (%) 0 BQL BQL BQL ND ND ND 0.0330 BQL BQL 1.76 ND ND ND 0.167 1.39 2.48 4.95 2.94 1.82 62.0 0.250 1.77 2.61 289 97.8 166 169 0.500 11.8 6.23 99.0 39.0 52.0 133 1.00 17.3 13.8 54.3 28.5 22.4 78.8 2.00 76.2 9.97 8.33 31.5 38.7 123 4.00 5.73 8.75 94.2 36.2 50.2 139 8.00 1.17 1.16 5.26 2.53 2.36 93.4
[0259] Individual and mean plasma concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg (ng/mL):
TABLE-US-00075 Time Mean PO CV (h) D02001 D02002 D02003 Plasma SD (%) 0 BQL BQL BQL ND ND ND 0.0330 BQL BQL BQL ND ND ND 0.167 9.20 11.7 17.7 12.9 4.37 34.0 0.250 9.58 30.7 26.3 22.2 11.1 50.2 0.500 84.8 72.4 78.0 78.4 6.21 7.92 1.00 42.2 63.3 68.8 58.1 14.0 24.2 2.00 22.9 16.5 18.1 19.2 3.33 17.4 4.00 15.6 14.0 8.15 12.6 3.92 31.2 8.00 4.37 9.36 11.2 8.31 3.53 42.5 24.0 BQL 1.93 7.07 4.50 ND ND
[0260] Individual and mean CSF concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg (ng/mL)
TABLE-US-00076 Time Mean PO CV (h) D02001 D02002 D02003 CSF SD (%) 0 BQL BQL BQL ND ND ND 0.0330 BQL BQL BQL ND ND ND 0.167 1.33 1.93 1.13 1.46 0.416 28.5 0.250 24.7 18.7 7.78 17.1 8.58 50.3 0.500 13.0 48.5 17.8 26.4 19.3 72.9 1.00 4.85 11.8 6.46 7.70 3.64 47.2 2.00 8.70 4.63 2.28 5.20 3.25 62.4 4.00 8.15 12.4 15.8 12.1 3.83 31.6 8.00 1.53 1.39 3.32 2.08 1.08 51.7
[0261] Individual and mean plasma concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SC administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 1 mg/kg (ng/mL)
TABLE-US-00077 Time Mean SC CV (h) D03001 D03002 D03003 Plasma SD (%) 0 BQL BQL BQL ND ND ND 0.0330 8.10 4.01 25.3 12.5 11.3 90.6 0.167 89.2 88.7 102 93.3 7.54 8.08 0.250 127 84.7 113 108 21.5 19.9 0.500 54.1 76.6 73.4 68.0 12.2 17.9 1.00 11.9 22.3 27.9 20.7 8.12 39.2 2.00 3.58 2.17 3.47 3.07 0.784 25.5 4.00 1.93 1.11 1.52 1.52 0.410 27.0 8.00 BQL BQL BQL ND ND ND 24.0 1.04 BQL BQL ND ND ND
[0262] Individual and mean CSF concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single SC administration of (6aS)-6-methyl-5,6,6a, 7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 1 mg/kg (ng/mL)
TABLE-US-00078 Time Mean SC CV (h) D03001 D03002 D03003 CSF SD (%) 0 BQL BQL BQL ND ND ND 0.0330 BQL 22.6 1.73 12.2 ND ND 0.167 8.64 13.9 7.88 10.1 3.28 32.3 0.250 13.7 23.3 29.4 22.1 7.91 35.8 0.500 17.8 15.6 44.4 25.9 16.0 61.8 1.00 4.24 9.48 21.1 11.6 8.63 74.3 2.00 6.55 4.01 12.8 7.79 4.52 58.1 4.00 10.2 2.39 4.11 5.57 4.10 73.7 8.00 10.8 4.37 37.5 17.6 17.6 100
Example 6: Plasma, CSF, and Brain Pharmacokinetic Study of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride Following Single Oral Administration to Non-Naïve Male Beagle Dogs
[0263] The pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride were evaluated in plasma, cerebral spinal fluid (CSF), and brain following single oral (PO) administration at 10 mg/kg to male beagle dogs.
[0264] Animals
[0265] Three non-naïve male beagle dogs supplied by Marshall Bioresources (Beijing, China) were used in this study. The animals were confirmed healthy before being assigned to the study. Each animal was given a unique identification number which was marked on ear and written on the cage card. The room(s) were controlled and monitored for relative humidity (targeted mean range 40% to 70%, and any excursion from this range for more than 3 hours will be documented as a deviation) and temperature (targeted mean range 18° C. to 26° C., and any excursion from this range will be documented as a deviation) with 10 to 20 air changes/hour. The rooms were on a 12-hour light/dark cycle except when interruptions are necessitated by study activities.
[0266] Animals were fed twice daily. Stock Dogs were fed approximately 220 grams of Certified Dog Diet daily (Beijing Keao Xieli Feed Co., Ltd. Beijing, P. R. China). These amounts could be adjusted as necessary based on food consumption of the group or an individual body weight change. For PO groups, animals were fed at 3:30 to 4:00 pm on the day prior to oral dosing and any remaining food was removed at 7:00 pm. Food was withheld until 4-hour post-dose. Animals were fed once on the day of dosing, with the amount of approximately 220 grams.
[0267] Study Design
[0268] Three male non-naïve beagle dogs were assigned to this study. Animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by single PO administration at 10 mg/kg, which was formulated in 0.5% CMC-Na in water. The detailed dosing and sampling regimens are described in the following two tables:
TABLE-US-00079 Dosing No. of Dose Target Dose Group animals Dose Volume Concentration Dose No. (male) Test Article (mg/kg) (mL/Kg) (mg/mL) Vehicle Route G1 3 Compound 1 10 5 2 0.5% CMC-Na in water PO(fasted)
TABLE-US-00080 Dosage Sampling time point (hr) Group (mg/kg) Animal No. Pre-dose# 0.033 0.167 0.25 0.5 G1 10 D1001, D1002, P + CSF P + CSF P + CSF P + CSF P + CSF + D1003 Striatum, cortex, cerebellum
[0269] Dose Administration
[0270] PO formulation preparations were processed in study facility on the day of dosing according to instructions provided by the sponsor. The concentrations of compounds in all dosing solutions were determined by HPLC/UV. Two aliquots were taken from each clear and three aliquots were taken from suspension formulations. All formulation samples were stored at 2-8° C. until analyzed by HPLC/UV. All dose formulation samples were analyzed in duplicate using HPLC/UV.
[0271] Animals were weighed prior to dose administration on the day of dosing for calculating the actual dose volume. Animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride by PO administration at 10 mg/kg, which was formulated in 0.5% CMC-Na in water.
[0272] Sample Collection and Preparation
[0273] In plasma, approximately 0.5 mL blood was collected at each time point. All blood samples were collected from a peripheral vessel. All blood samples were transferred into commercially available tubes containing Potassium (K2) EDTA on wet ice and processed for plasma. Samples were centrifuged (3,000×g for 10 minutes at 2 to 8° C.) within 30 minutes of collection. After centrifugation, the stabilizing agent was added to the harvested plasma within 60 minutes. The samples were kept on ice and protected from sunlight (under yellow light). The stabilizing agent was ascorbic acid 200 mg/mL added in a proportion of 10% in plasma. The plasma samples were divided into two equal samples with each at least 0.2 ml (0.2*2 mL/sample) and transferred into labeled polypropylene micro-centrifuge tubes and stored frozen in a freezer set to maintain 60° C. or lower until analyzed.
[0274] In CSF, about 0.1 mL/time point (50 μL*2 aliquots) of CSF were collected via the catheter implanted in the Cisterna Magna of conscious beagle dogs without any sedation. The procedures for CSF collection and handling were as follows: a polypropylene tube was weighed to accuracy of 0.1 mg (weight #1). CSF was collected and weighed the sample tube again (weight #2). Total CSF weight=weight #2−weight #1. The samples were kept on ice and protected from sunlight (under yellow light). Stabilizing agent was added within 60 minutes. The stabilizing agent was ascorbic acid 200 mg/mL added in a proportion of 10% in CSF. CSF samples were frozen on dry ice and stored at −60° C. or lower until bio-analysis.
[0275] For brain samples, different regions of brain were collected. Dogs were anesthetized and euthanasia according to SOP and collected the brain. The striatum, cortex and cerebellum were token rapidly and homogenized immediately (within 30 min) after being harvested. All the samples were stored at −60° C. or lower freezer finally.
[0276] For tissue homogenization, the homogenization solution was methanol:10 mM PBS=1:2 and set on dry ice. An empty conical tube was weighed, recorded, and set on dry ice. The tissue pieces were picked and placed into the pre-weighed conical tubes. The tube was weighed again and recorded. 5-fold homogenization solution was added to the weighed tissue. The tissues were homogenized by a Polytron (3 strokes or more until homogenous, each 30 seconds) under cold condition (dry ice-ethanol). The sample mixture was very cold but not totally frozen. The stabilizing agent was added within 60 minutes. The stabilizing agent was ascorbic acid 200 mg/mL and added in a proportion of 10% in each homogenized tissue. All the samples were stored at −60° C. or lower freezer finally.
[0277] For blank tissue sample homogenization, the tissues were harvested under regular conditions at room temperature. The tissues were weighed and added to a pre-weighed conical tube containing methanol:10 mM PBS=1:2. The tissues were homogenized under room temperature. The stabilizing agent was added. The stabilizing agent was ascorbic acid 200 mg/mL added in a proportion of 10% in each homogenized tissue.
Preparation of Standards
[0278] In plasma, blank male Beagle dog plasma with EDTA-K2 as anti-coagulant was used for the preparation of standards. In CSF, blank artificial CSF was used for the preparation of standards. In cerebellum homogenate, blank male Beagle dog cerebellum homogenate was used for the preparation of calibration standards (C) and QC samples. For striatum homogenate, blank male Beagle dog striatum homogenate was used for the preparation of standards. For cortex homogenate, blank male Beagle dog cortex homogenate was used for the preparation of standards.
[0279] About 1.96 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride was dissolved in 1.846 mL of DMSO to obtain a 1000 ng/mL stock solution (Stock A).
[0280] The preparation procedures of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol working solutions are detailed in the table below:
TABLE-US-00081 Source Solution Dilution Working Solution Source Solution Conc. Volume Volume Volume Final Conc. Working ID (μg/mL) (μL) (μL) (μL) (μg/mL) Solution ID Stock A 1000 30 470 500 60 WS-Pre WS-Pre 60 40 360 400 6 WS-C1 WS-C1 6 120 240 360 2 WS-C2 WS-C2 2 160 160 320 1 WS-C3 WS-C3 1 80 320 400 0.2 WS-C4 WS-C4 0.2 160 160 320 0.1 WS-C5 WS-C5 0.1 80 320 400 0.02 WS-C6 WS-C6 0.02 80 320 400 0.004 WS-C7 WS-C7 0.004 160 160 320 0.002 WS-C8 Stock A 1000 30 595 625 48 WS-QC-Pre WS-QC-Pre 48 40 360 400 4.8 WS-QC1 WS-QC1 4.8 120 240 360 1.6 WS-QC2 WS-QC2 1.6 20 380 400 0.08 WS-QC3 WS-QC3 0.08 30 370 400 0.006 WS-QC4 Stock A 1000 30 270 300 100 WS-DQC1-Pre WS-DQC1-Pre 100 40 360 400 10 WS-DQC2 Dilution Solvent: 0.02% 2-mercaptoethanol in MeOH for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride
[0281] Preparation details of the (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol calibration and QC standards in plasma, CSF, and tissue homogenate are shown in the following table:
TABLE-US-00082 Working Solution Matrix Calibration Standard Source Conc. Volume Volume Volume Final Conc. Calibration Solution ID (μg/mL) (μL) (μL) (μL) (ng/mL) Standard ID WS-C1 6 2 38 40 300 C1 WS-C2 2 2 38 40 100 C2 WS-C3 1 2 38 40 50 C3 WS-C4 0.2 2 38 40 10 C4 WS-C5 0.1 2 38 40 5 C5 WS-C6 0.02 2 38 40 1 C6 WS-C7 0.004 2 38 40 0.2 C7 WS-C8 0.002 2 38 40 0.1 C8 Source Conc. Volume Volume Volume Final Conc. Solution ID (μg/mL) (μL) (μL) (μL) (ng/mL) QC ID WS-QC1 4.8 2 38 40 240 QC1 WS-QC2 1.6 2 38 40 80 QC2 WS-QC3 0.08 2 38 40 4 QC3 WS-QC4 0.006 2 38 40 0.3 QC4 WS-DQC2 10 2 38 40 500 DQC
[0282] The concentrations of calibration and QC samples are shown in the table below:
TABLE-US-00083 Calibration Standard Samples Quality Control Samples Compound Matrix Conc. (ng/mL) Conc. (ng/mL) (6aS)-6-methyl-5,6,6a,7- Male Beagle dog 0.1, 0.2, 1, 5, 10, 50, tetrahydro-4H- Cortex homogenate 100, 300 dibenzo [de,g] quinoline- 10,11-diol HCl (6aS)-6-methyl-5,6,6a,7- Male Beagle dog 1, 2, 10, 50, 100, 500, tetrahydro-4H- plasma 1000, 3000 dibenzo [de, g] quinoline- 10,11-diol HCl (6aS)-6-methyl-5,6,6a,7- Male Beagle dog 0.1, 0.2, 1, 5, 10, 50, tetrahydro-4H- Striamu homogenate 100, 300 dibenzo [de, g] quinoline- 10,11-diol HCl (6aS)-6-methyl-5,6,6a,7- Male Beagle dog 0.1, 0.2, 1, 5, 10, 50, 0.3, 4, 80, 240, 500 (DQC) tetrahydro-4H- Cerebellum 100, 300 0.3, 4, 80, 240 dibenzo [de, g] quinoline- homogenate 10,11-diol HCl (6aS)-6-methyl-5,6,6a,7- Male Beagle dog 0.1, 0.2, 1, 5, 10, 50, 0.3, 4, 80, 240 tetrahydro-4H- plasma 100, 300 dibenzo [de, g] quinoline- 10,11-diol HCl
[0283] Sample Treatment
[0284] The tissue (Cerebellum, Striatum and Cortex) was homogenized using homogenizing buffer (methanol:10 mM PB S=1:2) at the ratio of 1:5 (1 g tissue with 5 mL buffer). An aliquot of 20 μL sample with 400 μL internal standard (100 ng/mL Labetalol & 100 ng/mL Dexamethasone & 100 ng/mL Tolbutamide & 100 ng/mL Verapamil & 100 ng/mL Glyburide & 200 ng/mL Diclofenac in ACN), the mixture was vortex-mixed well and centrifuged at 13000 rpm for 10 min, 4° C. 70 μL supernatant was then mixed with 70 μL water, 3 μL sample was injected for LC-MS/MS analysis.
[0285] For the dilutions: a dilution factor as 5—an aliquot of 4 μL sample was added with 16 μL blank plasma; and a dilution factor as 10—An aliquot of 2 μL sample was added with 18 μL blank plasma.
[0286] LC-MS/MS Conditions
[0287] The LC-MS/MS system consisted of an ACQUITY UPLC System and API 4000 mass spectrometer. Chromatographic separation was performed on a ACQUITY UPLC HSS T3 (2.1×50 mm, 1.8 μm) column. The mobile phase was composed of A: 0.1% formic acid and 2 mM HCOONH.sub.4 in Water:ACN (v:v, 95:5); and B: 0.1% formic acid & 2 mM HCOONH.sub.4 in ACN:Water (v:v, 95:5). The flow rate was 0.6 mL/min. The injection volume was 3 μL. The following gradient was implemented:
TABLE-US-00084 Flow Rate Time (min) (mL/min) A (%) B (%) Initial 0.600 98.0 2.0 0.20 0.600 98.0 2.0 0.90 0.600 55.0 45.0 1.10 0.600 5.0 95.0 1.60 0.600 5.0 95.0 1.61 0.600 98.0 2.0 1.80 0.600 98.0 2.0
[0288] Positive mode electrospray ionization (ESI) was performed to obtain a protonated ion of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, and Verampil (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Verampil dependent parameters are listed as follows:
TABLE-US-00085 Ion Collision Energy Retention Time Compound Name Transition (eV) (min) (6aS)-6-methyl- 268.0/219.2 26 0.96 5,6,6a,7-tetrahydro- 4H- dibenzo[de,g] quinoline-10,11-diol Verapamil 455.2/164.9 22 1.3
[0289] Data acquisition was performed by Analyst 1.6.3 software was used for processing the data of all samples.
[0290] Results
[0291] The individual and mean plasma concentration-time profiles are illustrated from
[0292] For the PO dose at 10 mg/kg, (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a plasma C.sub.max of 257±7.21 ng/mL, with T.sub.max at 0.250±0.000 h. (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol showed a CSF C.sub.max of 2.90±1.12 ng/mL, with T.sub.max at 0.417±0.144 h. The AUC.sub.last values in plasma and CSF were 80.0±7.29 ng.Math.h/mL and 0.703±0.218 ng.Math.h/mL, respectively. The AUC.sub.last ratio of CSF to plasma was 0.00888±0.00310.
[0293] The table below demonstrates the individual and mean plasma PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg:
TABLE-US-00086 PK Parameters D1001 D1002 D1003 Mean PO SD CV (%) No. points used for t.sub.1/2 0.00 0.00 0.00 0.00 — — C.sub.max (ng/mL) 255 251 265 257 7.21 2.81 T.sub.max (h) 0.250 0.250 0.250 0.250 0.00 0.00 t.sub.1/2 (h) ND ND ND ND ND ND T.sub.last (h) 0.500 0.500 0.500 0.500 — — AUC.sub.last (ng .Math. h/mL) 72.1 81.6 86.4 80.0 7.29 9.11 AUC.sub.inf (ng .Math. h/mL) ND ND ND ND ND ND MRT.sub.last (h) 0.328 0.337 0.323 0.329 0.00717 2.18 MRT.sub.inf (h) ND ND ND ND ND ND AUC.sub.Extra (%) ND ND ND ND ND ND AUMC.sub.Extra (%) ND ND ND ND ND ND AUC.sub.last/C.sub.max (h) 0.283 0.325 0.326 0.311 0.025 7.9 “—” means not applicable “ND” means not determined “D#” means animal number
[0294] The following table illustrates the Individual and mean CSF PK parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg:
TABLE-US-00087 PK Parameters D1001 D1002 D1003 Mean PO SD CV (%) No. points used for t.sub.1/2 0.00 0.00 0.00 0.00 — — C.sub.max (ng/mL) 2.54 2.00 4.16 2.90 1.12 38.8 T.sub.max (h) 0.250 0.500 0.500 0.417 0.144 34.6 t.sub.1/2 (h) ND ND ND ND ND ND T.sub.last (h) 0.500 0.500 0.500 0.500 — — AUC.sub.last (ng .Math. h/mL) 0.840 0.451 0.817 0.703 0.218 31.0 AUC.sub.inf (ng .Math. h/mL) ND ND ND ND ND ND MRT.sub.last (h) 0.312 0.383 0.403 0.366 0.0475 13.0 MRT.sub.inf (h) ND ND ND ND ND ND AUC.sub.Extra (%) ND ND ND ND ND ND AUMC.sub.Extra (%) ND ND ND ND ND ND CSF/Plasma AUC Ratio 0.0117 0.00553 0.00945 0.00888 0.00310 35.0 AUC.sub.last/C.sub.max (h) 0.331 0.226 0.196 0.251 0.071 28.2 “—” means not applicable “ND” means not determined “D#” means animal number
[0295] The concentration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in plasma, striatum, cortex and cerebellum at 0.5 hour post-dose were 218±36.5 ng/mL, 145±22.7 ng/g, 151±36.2 ng/g and 108±19.1 ng/g, respectively. The ratios of striatum/plasma, cortex/plasma and cerebellum/plasma were 0.666±0.0544, 0.704±0.184 and 0.505±0.117, respectively.
[0296] The table below demonstrates the Individual and mean plasma concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg (ng/mL):
TABLE-US-00088 Time Mean CV (h) D1001 D1002 D1003 PO SD (%) 0.0000 BQL BQL BQL ND ND ND 0.0330 2.92 BQL 0.319 1.62 ND ND 0.167 71.0 68.8 129 89.5 34.0 38.0 0.250 255 251 265 257 7.32 2.85 0.500 178 250 227 218 36.5 16.7 “BQL” means below the quantifiable limit (1.00 ng/mL) “ND” means not determined as more than half (>50%) of the individual values are not quantifiable “D#” means animal number
[0297] The following table illustrates the individual and mean CSF concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg (ng/mL):
TABLE-US-00089 Time Mean CV (h) D1001 D1002 D1003 PO SD (%) 0.0000 BQL BQL BQL ND ND ND 0.0330 BQL BQL BQL ND ND ND 0.167 1.40 0.224 0.523 0.715 0.609 85.3 0.250 2.54 1.04 1.39 1.66 0.786 47.5 0.500 1.96 2.00 4.16 2.71 1.26 46.5 “BQL” means below the quantifiable limit (1.00 ng/mL) “ND” means not determined as more than half (>50%) of the individual values are not quantifiable “D#” means animal number
[0298] The table below demonstrates the individual and mean striatum, cortex and cerebellum concentrations of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in male Beagle dogs following single PO administration of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol at 10 mg/kg (ng/g):
TABLE-US-00090 Time Mean CV Matrix (h) D1001 D1002 D1003 PO SD (%) Striatum 0.500 127 170 137 145 22.7 15.7 Cortex 0.500 137 124 192 151 36.2 24.0 Cerebellum 0.500 102 92.4 129 108 19.1 17.7
Example 7: Plasma Pharmacokinetic Study of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Following Subcutaneous and Oral Administration in Fasted Male Cynomolgus Monkeys
[0299] The pharmacokinetic profile of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol was evaluated in male Cynomolgus Monkeys. 3 male Cynomolgus Monkeys were administrated a single Subcutaneous dose of 1.14 mg/kg and a solution formulation at a matching dose level. The (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol pharmacokinetics profile was evaluated in male Cynomolgus Monkeys. Three male Cynomolgus Monkeys were administrated a single oral dose of 11.4 mg/kg and a solution formulation at a matching dose level. The vehicle for SC formulation was 0.1% sodium metabisulfite solution and PO formulation was 0.5% CMC-Na in water.
[0300] Animals
[0301] Male Cynomolgus Monkeys were purchased from Topgene Biotechnology. The animals were 2-5 years old with body weights of 2.0-5.0 kg on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water.
[0302] All animals for SC administration had free access to food and water. All animals for PO administration were fasted overnight prior to dosing and will be fed approximately 2 hours after dosing.
[0303] Study Design
[0304] Six male Cynomolgus Monkeys were assigned to this study consisting of two groups. In group 1, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by single SC administration at 1.14 mg/kg, which was formulated in 0.1% sodium metabisulfite. In group 2, animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by PO administration at 11.4 mg/kg, which was formulated in 0.5% CMC-Na in water. The dosing regimens and formulation (vehicle) compositions are described in the following table below:
TABLE-US-00091 Dose Level Dose Volume Conc. Administration No. of Group Treatment (mg/kg) (ml/kg) (mg/ml) Route Animals 1 (6aS)-6-methyl- 1.14 1 1.14 SC 3 2 5,6,6a,7- 11.4 5 2.28 PO 3 tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol
[0305] Dose Administration
[0306] Animals were weighed prior to dose administration on the day of dosing for calculating the actual dose volume. The dosages were prepared as illustrated in the table below:
TABLE-US-00092 Free Weight Conc Volume 0.1% sodium Compound Route MW MW FW (mg) (mg/mL) (mL) metabisulfite (6aS)-6-methyl- SC 267.32 303.79 312.79 15.35 1.14 13.077 13.077 5,6,6a,7- tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol Free Weight Conc Volume 0.5% CMC- Compound Route MW MW FW (mg) (mg/mL) (mL) Na (6aS)-6-methyl- PO 267.32 303.79 312.79 119.00 2.28 50.691 50.691 5,6,6a,7- tetrahydro-4H- dibenzo[de,g]quin- oline-10,11-diol
[0307] Sample Collection
[0308] Blood samples were taken at the following times:
TABLE-US-00093 Group PK time points SC Pre-dose, 2 min, 5 min, 10 min, 15 min, 30 min, 1, 2, 4, 8 and 24 hr post dose PO Pre-dose, 5 min, 10 min, 15 min, 30 min, 1, 2, 4, 8 and 24 hr post dose
[0309] Whole blood samples were gently inverted several times to ensure anticoagulation then placed on wet ice prior to centrifugation. Within 30 minutes of collection, the samples were centrifuged at approximately 4,000×g (force) for 5 minutes at 2 to 8° C. to obtain plasma. The stabilizing agent, ascorbic acid at 200 mg/mL, was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min. The resultant plasma samples will be immediately separated divided into 2 aliquots (˜75 μL each) and transferred to cryogenic vial. Samples will be maintained on wet ice throughout processing, and stored in a freezer at −75±15° C. prior to analysis. Samples should be protected from ambient light (under sodium lamp).
Preparation of Standards
[0310] Standards were prepared with 0.96 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol added to 0.820 mL DMSO to obtain a 1 mg/mL standard stock solution (free form).
[0311] Calibration standard working solutions were prepared at concentrations of 2, 5, 10, 20, 100, 500, 1000, 5000, 10000 ng/mL by serial dilution of the standard stock solution in 1:1 acetonitrile:water. Quality control working solutions at concentrations of 5, 10, 20, 500 and 8000 ng/mL were prepared by serial dilution of the standard stock solution in 1:1 acetonitrile:water. The QC samples were prepared on the day of analysis in the same way as calibration standards.
[0312] Sample Treatment
[0313] Samples were prepared with 5 μL of each calibration standard working solution (2, 5, 10, 20, 100, 500, 1000, 5000, 10000 ng/mL) added to 50 μL of the blank Cynomolgus Monkeys plasma to achieve calibration standards concentrations of 0.2, 0.5, 1, 2, 10, 50, 100, 500, 1000 ng/mL (relative to a 50-4, plasma aliquot). Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2), 2 ng/mL (low-3), 50 ng/mL (mid) and 800 ng/mL (high) for plasma were prepared independently for those used for the calibration curves.
[0314] Total volumes of 55 μL of standards, 55 μL of QC samples and 55 μL of unknown samples (50 μL of plasma with 5 μL of 1:1 acetonitrile:water) were added to 200 μL of acetonitrile containing IS to precipitate proteins. Then the samples were vortexed for 30 sec. After centrifugation at 4° C., 3900 rpm for 15 min, supernatant was diluted 5 times with water, and 2 μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis. All of the samples were processed on ice.
[0315] LC-MS/MS Conditions
[0316] The LC-MS/MS system consisted of two Shimadzu LC-30AD pumps, a DGU-20A5R degasser, a Rack changer II and an AB Sciex Triple Quad 5500 LC/MS/MS mass spectrometer.
[0317] Chromatographic separation was performed on Agilent ZORBAX XDB-Phenyl 5 μm (50×2.1 mm) at room temperature. The mobile phase was composed of A: 5% Acetonitrile/95% water (0.1% Formic acid); B: 95% Acetonitrile/5% water (0.1% Formic acid). The flow rate was 0.6 mL/min. The injection volume was 2 μL.
[0318] Positive mode electrospray ionization (ESI) was performed on a Turbo VR® ion source to obtain a protonated ion of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Dexamethasone (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions were 268.05/237.10 Da and 393.14/373.10 Da for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Dexamethasone, respectively. The instrument parameters were set as follows: ion spray voltage: 5500 V; curtain gas: 40 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; temperature: 450° C. The compound dependent parameters are listed as follows:
TABLE-US-00094 (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H- dibenzo[de,g]quinoline- Dexamethasone 10,11-diol (IS) Precursor/ 268.05/237.10 393.14/373.10 product DP 131.00 146.00 CE 25.00 13.00 CXP 18.00 16.00
[0319] HPLC flow rate was set at 0.6 mL/min with the following conditions:
TABLE-US-00095 Time (min) A (%) B (%) 0.01 95.0 5.00 0.20 95.0 5.00 1.90 5.00 95.0 2.20 5.00 95.0 2.21 95.0 5.00 2.50 95.0 5.00
[0320] Results
[0321] The mean plasma concentration-time profiles after PO and SC administration are illustrated from
[0322] Following the SC (1.14 mg/kg) administration the t.sub.1/2 was 5.20 hr, T.sub.max was 0.195±0.048 hr, C.sub.max was 92±28 ng/mL, MRT.sub.Inf was 1.96 hr, AUC.sub.0-24 hr was 60.5±11.8 h*ng/mL, and AUC.sub.inf was 58.2 h*ng/mL in Cynomolgus Monkeys.
[0323] The table below demonstrates shows the plasma pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following SC administration to male Cynomolgus Monkeys at 1.14 mg/kg:
TABLE-US-00096 PK parameters Unit 170353C 170291C 165161C Mean SD CV(%) t.sub.1/2 h 4.68 5.72 NA 5.20 NA NA T.sub.max h 0.250 0.167 0.167 0.195 0.048 24.6 C.sub.max ng/mL 74.1 77.4 124 92 28 30.4 AUC.sub.last h*ng/mL 52.1 55.5 74.0 60.5 11.8 19.5 AUC.sub.Inf h*ng/mL 56.0 60.3 NA 58.2 NA NA AUC_% Extrap_obs % 7.05 7.98 NA 7.52 NA NA MRT.sub.Inf h 1.89 2.04 NA 1.96 NA NA AUC.sub.last/D h*mg/mL 45.7 48.7 64.9 53.1 10.4 19.5 F % NA NA NA NA NA NA AUC.sub.last/C.sub.max h 0.703 0.717 0.597 0.672 0.066 9.8
[0324] Following the PO (11.4 mg/kg) t.sub.1/2 was 2.26±0.56 hr, T.sub.max was 0.667±0.289 hr, C.sub.max was 34.7±17.9 ng/mL, MRT.sub.Inf was 2.92±0.92 hr, AUC.sub.0-24 hr was 58.1±12.4 h*ng/mL, and AUC.sub.inf was 63.5±11.9 h*ng/mL in Cynomolgus Monkeys.
[0325] The following table illustrates the plasma pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following PO administration to male Cynomolgus Monkeys at 11.4 mg/kg:
TABLE-US-00097 PK parameters Unit 170295C 174223C 174753C Mean SD CV(%) t.sub.1/2 h 1.80 2.88 2.09 2.26 0.56 24.8 T.sub.max h 0.500 1.00 0.500 0.67 0.29 43.3 C.sub.max ng/mL 27.7 21.4 55.0 34.7 17.9 51.5 AUC.sub.last h*ng/mL 54.2 48.2 72.0 58.1 12.4 21.3 AUC.sub.Inf h*ng/mL 56.6 56.6 77.3 63.5 11.9 18.8 AUC_% Extrap_obs % 4.31 14.8 6.80 8.6 5.5 63.5 MRT.sub.Inf h 2.46 3.98 2.33 2.92 0.92 31.3 AUC.sub.last/D h*mg/mL 4.75 4.23 6.32 5.10 1.09 21.3 F % NA NA NA NA NA NA AUC.sub.last/C.sub.max h 1.957 2.252 1.309 1.839 0.482 26.2
[0326] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Plasma Concentrations Following SC Administration to Male Cynomolgus Monkeys at 1.14 mg/kg:
TABLE-US-00098 Time Concentration (ng/mL) Mean SD CV (h) 170353C 170291C 165161C (ng/mL) (ng/mL) (%) 0 BLOQ BLOQ BLOQ NA NA NA 0.033 1.88 12.5 12.6 9.0 6.2 68.6 0.083 5.47 40.3 65.3 37.1 30.1 81.2 0.167 66.5 77.4 124 89 30 34.1 0.25 74.1 76.3 119 90 26 28.4 0.5 52.4 57.0 65.1 58.2 6.4 11.1 1 10.3 8.14 9.44 9.28 1.07 11.5 2 1.44 1.23 2.02 1.56 0.41 26.3 4 1.01 0.884 0.844 0.912 0.085 9.36 8 0.585 0.584 0.959 0.710 0.216 30.5 24 BLOQ BLOQ BLOQ NA NA NA
[0327] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Plasma Concentrations Following PO Administration to Male Cynomolgus Monkeys at 11.4 mg/kg
TABLE-US-00099 Time Concentration (ng/mL) Mean SD CV (h) 170295C 174223C 174753C (ng/mL) (ng/mL) (%) 0 BLOQ BLOQ BLOQ NA NA NA 0.083 2.55 0.786 10.1 4.5 5.0 111 0.167 12.3 1.26 23.1 12.2 10.9 89.4 0.25 27.2 3.90 38.1 23.0 17.5 75.7 0.5 27.7 5.15 55.0 29.3 25.0 85.2 1 10.8 21.4 18.2 16.8 5.4 32.1 2 9.42 9.30 10.8 9.8 0.8 8.33 4 4.63 3.81 2.98 3.80 0.82 21.7 8 0.941 2.02 1.74 1.57 0.56 35.8 24 BLOQ BLOQ BLOQ NA NA NA
Example 8: Pharmacokinetic and Brain Penetration Study Following Single and Repeated Dose of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Hydrochloride Via PO Administration in Male C57BL/6J Mouse
[0328] (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol pharmacokinetic profiles were evaluated in male C57BL/6J mice (N=3). 9 male C57BL/6J mice following administration of a single and repeated PO dose of 4.40 mg/kg. The vehicle for the subcutaneous formulation contained “0.1% sodium metabisulfite solution”. During this PK study, there were no obvious drug-related changes in clinical signs and behavioral responses. There was no animal death related to drug administration.
[0329] Animals
[0330] Male C57BL/6J mice were purchased from Si Bei Fu Laboratory Animal Technology Co. Ltd (Beijing, China). The animals were about 8 weeks old with body weights of 20-30 g on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water. All animals were fasted overnight and fed after 4 hours collection.
[0331] Study Design
[0332] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was administered to both groups 1 and 2 via oral administration (25 mg/kg) at 10 mL/kg. Blood, CSF, and brain samples were collected from group 1 at 5 min., 15 min., 30 min., 1 hr., 2 hrs., 3 hrs., and 4 hrs. following administration of a single dose. Blood, CSF, and brain samples were collected from group 2 at 0 min., 5 min., 15 min., 30 min., 1 hr., 2 hrs., 3 hrs., 4 hrs., 8 hrs., 12 hrs., 24 hrs., and 48 hrs. post last day (Day 4).
TABLE-US-00100 Dose Level Dose (mg/kg) (salt Volume Conc. hhemiydrate (mL/ (mg/ No. of Group Treatment form) kg) mL) Route Animals 1 (6aS)-6-methyl-5,6,6a,7- 25 10 2.5 PO (Single 3/male tetrahydro-4H- Dose) dibenzo[de,g]quinoline- 10,11-diol hydrochloride 2 (6aS)-6-methyl-5,6,6a,7- 25 10 2.5 PO (Once 6/male tetrahydro-4H- daily for 4 dibenzo[de,g]quinoline- days) 10,11-diol hydrochloride
[0333] Dosage and Administration
[0334] The formulation was freshly made prior to use. Preparation of formulation for oral dosing are as follows:
[0335] Preparation of stock solution: 1.55 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in 1.505 mL DMSO with vortex and sonification to prepare 1 mg/mL stock solution in DMSO.
[0336] Preparation of Day 1 (25 mg/kg, 10 mL/kg) formulation: 2.5 mg/mL of “10% HP-(3-CD in water” solution was prepared by dissolving 61.83 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate in 24.020 mL of 10% HP-(3-CD in water with vortex.
[0337] Preparation of Day 2 (25 mg/kg, 10 mL/kg) formulation: 2.5 mg/mL of “10% HP-β-CD in water” solution was prepared by dissolving 39.28 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate in 15.260 mL of 10% HP-(3-CD in water with vortex.
[0338] Preparation of Day 3 (25 mg/kg, 10 mL/kg) formulation: 2.5 mg/mL of “10% HP-β-CD in water” solution by dissolving 44.36 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate in 17.233 mL of 10% HP-β-CD in water with vortex.
[0339] Preparation of Day 4 (25 mg/kg, 10 mL/kg) formulation: 2.5 mg/mL of “10% HP-(3-CD in water” solution by dissolving 40.83 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate in 15.862 mL of 10% HP-β-CD in water with vortex.
[0340] Sample Collection
[0341] For Group 1 and 2, blood, CSF and brain samples were collected from 3 mice at the following time points.
TABLE-US-00101 Group PK Sampling Time Points 1 5, 15, 30 min, 1, 2, 3, and 4 hours post dose 2 0, 5, 15, 30 min, 1, 2, 3, 4, 8, 12, 24, and 48 hours post last day (Day 4) dose
[0342] Plasma samples: About 30 μL or 300 μL of blood samples were collected from each animal via dorsal metatarsal vein or heart. These blood samples were placed into the tubes containing K2EDTA. And centrifugation at 3000 g for 10 minutes in a 4° C. centrifuge. The completion time from collection was less than 30 min. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min.
[0343] Brain and CSF Samples: The animals were fully exsanguinated with a rising concentration of CO2 gas prior to CSF and brain collection.
[0344] For CSF collection, make an incision by scissors on the neck of the rats, and the foramen magnum is exposed to the sight of scientist. Use a syringe with a venoclysis needle to sample the CSF, the tip of the needle is insert into the membrane of foramen magnum about 1-2 mm, and then pull the stopcock a small length of the syringe to make a negative pressure, then the CSF fluids to the tube. Watch the fluid in the tube and pull the stopcock slowly, when the amount of the CSF is enough, stop and split the needle and the syringe to ensure there is no negative pressure in the tube. Then cut off the needle and collect the CSF fluid. If we could see the blood in the tube, stop immediately, split the needle and the syringe, and then cut off between the blood and CSF to ensure there is no blood in the CSF fluid. Then collect the CSF fluid. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in CSF. The completion time from collection was less than 60 min.
[0345] For brain collection. Brain samples were collected at adopted time point, and washed 3 times with cold saline, then dried with clean gauze and put into the weighed tube, quick frozen in ice box and kept at −75±15° C. All brain samples were weighted and homogenized with phosphate buffered saline (PBS) by brain weight (g) to PBS volume (mL) ratio 1:3 before analysis. The stabilizing agent of ascorbic acid at 200 mg/mL was added in a proportion of 10% in Brain homogenate. The actual concentration was the detected value multiplied by the dilution factor. Brain samples were collected at designed time point, quickly homogenized and kept at −75±15° C. The completion time from collection was less than 60 min.
[0346] Preparation of Standards
[0347] About 1 mg (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate and (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol hydrochloride hemihydrate was dissolved in DMSO to obtain a 1 mg/mL standard stock solution (free base).
[0348] Calibration standard working solutions were prepared at concentrations of 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL by serial dilution of the standard stock solution. These QC samples were prepared on the day of analysis in the same way as calibration standards.
[0349] Sample Treatment
[0350] Plasma: The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 10 μL of working solutions (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) were added to 10 μL of the blank C57BL/6J Mice plasma to achieve calibration standards of 0.1˜1000 ng/mL (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 20 μL. Five quality control samples at 0.2 ng/mL, 1 ng/mL, 2 ng/mL, 50 ng/mL and 800 ng/mL for plasma and brain were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. 20 μL of standards, 20 μL of QC samples and 20 μL of unknown samples (10 μL of plasma with 10 μL of blank solution) were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 4700 rpm for 15 min. The supernatant was diluted 3 times with water. 5 μL of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis.
[0351] Brain: The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 10 μL of working solutions (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) were added to 10 μL of the blank C57BL/6J Mice brain homogenate to achieve calibration standards of 0.1˜1000 ng/mL (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 20 μL. Five quality control samples at 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 50 ng/mL and 800 ng/mL for plasma and brain were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. 20 μL of standards, 20 μL of QC samples and 20 μL of unknown samples (10 μL of brain homogenate with 10 μL of blank solution) were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 4700 rpm for 15 min. The supernatant was diluted 3 times with water. 5 μL of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis.
[0352] CSF: The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 10 μL of working solutions (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) were added to 10 μL of the blank C57BL/6J Mice CSF to achieve calibration standards of 0.1˜1000 ng/mL (0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng/mL) in a total volume of 20 μL. Four quality control samples at 0.5 ng/mL, 1 ng/mL, 50 ng/mL and 800 ng/mL for plasma and brain were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. 20 μL of standards, 20 μL of QC samples and 20 μL of unknown samples (10 μL of CSF with 10 μL of blank solution) were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 4700 rpm for 15 min. The supernatant was diluted 3 times with water. 5 μL of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis.
[0353] LC-MS/MS Conditions
[0354] The LC-MS/MS system consisted of SHIMADZU (LC-30A D, Serial NO. L20555407804 AE and L20555407803 AE; DGU-20A5R Serial NO. L20705413440 IX; CBM-20A Serial NO. L20235428798 CD; SIL-30AC, Serial NO. L20565403657 AE; CTO-30A, Serial NO. L20575401041 CD), Rack Changer II (Serial No. L20585400874 SS), and LCMS-8060 instrument (Serial NO. 011105400164 AE).
[0355] Chromatographic separation was performed on a HALO 90A, C18, 2.7 μm, 2.1*50 mm, SN:USAQ001307 at room temperature. The mobile phase was composed of A: Water (5 mM Ammonium Acetate); B: Ethanol (95% methanol in water). The flow rate was 0.6 mL/min. The injection volume was 5 μL.
[0356] The calibration line used for sample quantification must consist of at least 6 accepted calibration standards. Acceptance of calibration standards requires calculated concentration within 80%-120% of the nominal concentration. 75% of the calibration standards should be within the acceptable range.
[0357] Acceptance Criteria of Quality Control Samples:
[0358] At least 3 concentrations of quality control samples (QCs) should be analyzed in a run. Each concentration should include at least 2 individual samples. Acceptance of QCs requires calculated concentration within 80%-120% of the nominal concentration. QCs should be analyzed amongst all unknown samples and ⅔ of the QCs should be within the acceptable range, including at least 1 sample at each concentration level in an analytical run.
[0359] Acceptance Criteria of Unknown Samples:
[0360] Unknown samples with normal peak shape of analytes and calculated concentration within the calibration range should be accepted. Samples with calculated concentration below LLOQ should be recorded as BLOQ. Samples with calculated concentration above 120% of ULOQ should be diluted with blank matrix and re-assayed. The re-assayed concentration should be multiplied by the dilution factor to obtain the final data. In cases of abnormality, such as equipment malfunction, power outage, sample treatment failure and/or sample injection failure, re-assay should be done in an individual analytical run.
[0361] Data acquisition was performed by Lab Solutions version 5.89 Software (Shimadzu). All concentration data was reported with 3 significant figures. Data statistics were performed using Excel 2010 software. The pharmacokinetic parameters of (6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol were calculated using a non-compartmental approach with Phoenix™ WinNonlin®.
[0362] Results
[0363] The pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol in mice following PO administration were calculated by non-compartmental analysis (Phoenix™ WinNonlin® 8.0), and a linear trapezoidal algorithm was used for AUC calculation.
[0364] Plasma pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00102 PK parameters Unit Mean T.sub.1/2 h 1.4 T.sub.max h 0.1 C.sub.max ng/mL 1153 AUC.sub.last h* ng/mL 857 AUC.sub.Inf h* ng/mL 945 AUC_.sub.%Extrap_obs % 9.34 MRT.sub.Inf_obs h 1.5 AUC.sub.last/D h*mg/mL 34.3 AUC.sub.last/C.sub.max h 0.743
[0365] Plasma pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00103 PK parameters Unit Mean T.sub.1/2 h 1.2 T.sub.max h 0.1 C.sub.max ng/mL 1363 AUC.sub.last h*ng/mL 837 AUC.sub.Inf h*ng/mL 837 AUC_.sub.%Extrap_obs % 0.0352 MRT.sub.Inf_obs h 0.9 AUC.sub.last/D h*mg/mL 33.5 AUC.sub.last/C.sub.max h 0.614
[0366] Brain pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00104 PK parameters Unit Mean T.sub.1/2 h 2.9 T.sub.max h 0.1 C.sub.max ng/mL 729 AUC.sub.last h* ng/mL 215 AUC.sub.Inf h* ng/mL 264 AUC_.sub.%Extrap_obs % 18.5 MRT.sub.Inf_obs h 2.05 AUC.sub.last/D h*mg/mL 8.61 AUC.sub.last/C.sub.max h 0.295
[0367] Brain pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00105 PK parameters Unit Mean T.sub.1/2 h 0.6 T.sub.max h 0.1 C.sub.max ng/mL 2328 AUC.sub.last h* ng/mL 556 AUC.sub.Inf h* ng/mL 558 AUC_.sub.%Extrap_obs % 0.393 MRT.sub.Inf_obs h 0.343 AUC.sub.last/D h*mg/mL 22.3 AUC.sub.last/C.sub.max h 0.239
[0368] CSF pharmacokinetic parameters for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00106 PK parameters Unit Mean T.sub.1/2 h 2.0 T.sub.max h 0.1 C.sub.max ng/mL 56.4 AUC.sub.last h* ng/mL 25.5 AUC.sub.Inf h* ng/mL 35.6 AUC_.sub.%Extrap_obs % 28.4 MRT.sub.Inf_obs h 2.82 AUC.sub.last/D h*mg/mL 1.02 AUC.sub.last/C.sub.max h 0.452
[0369] CSF pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00107 PK parameters Unit Mean T.sub.1/2 h 0.2 T.sub.max h 0.1 C.sub.max ng/mL 200 AUC.sub.last h* ng/mL 38.9 AUC.sub.Inf h* ng/mL 40.2 AUC_.sub.%Extrap_obs % 3.18 MRT.sub.Inf_obs h 0.214 AUC.sub.last/D h*mg/mL 1.56 AUC.sub.last/C.sub.max h 0.195
[0370] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00108 Time Concentration (ng/mL) Mean SD CV (h) G1 G2 G3 (ng/mL) (ng/mL) (%) 0.0833 1120 1300 1040 1153 133 11.5 0.25 524 569 658 584 68 11.7 0.5 501 701 383 528 161 30.4 1 332 162 210 235 88 37.3 2 100 138 128 122 20 16.1 3 55.5 90.3 71.5 72.4 17.4 24.0 4 89.2 58.2 30.8 44.5 29.2 65.7
[0371] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol plasma concentrations following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00109 Time Concentration (ng/mL) Mean SD CV (h) G1 G2 G3 (ng/mL) (ng/mL) (%) 0 BLOQ BLOQ BLOQ BLOQ NA NA 0.0833 1460 1520 1110 1363 221 16.2 0.25 618 1280 1070 989 338 34.2 0.5 448 599 313 453 143 31.6 1 112 202 146 153 45 29.6 2 69.0 81.8 72.5 74.4 6.6 8.89 3 35.7 35.8 73.1 48.2 21.6 44.7 4 20.1 20.8 18.2 19.7 1.3 6.83 8 0.765 0.599 1.24 0.87 0.33 38.3 12 0.198 BLOQ 0.150 0.174 NA NA 24 BLOQ BLOQ BLOQ BLOQ NA NA 48 BLOQ BLOQ BLOQ BLOQ NA NA
[0372] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00110 Time Concentration (ng/g) Mean SD CV (h) G1 G2 G3 (ng/g) (ng/g) (%) 0.0833 636 940 612 729 183 25.1 0.25 240 247 208 232 20.8 8.98 0.5 77.2 74.0 46.0 65.7 17.2 26.1 1 26.7 19.0 22.0 22.6 3.85 17.1 2 14.9 19.0 16.5 16.8 2.10 12.5 3 9.68 12.4 12.4 11.5 1.58 13.7 4 15.4 12.6 6.84 11.6 4.38 37.7
[0373] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol brain concentrations following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00111 Time Concentration (ng/g) Mean SD CV (h) G1 G2 G3 (ng/g) (ng/g) (%) 0 BLOQ BLOQ BLOQ BLOQ NA NA 0.0833 3512 2388 1084 2328 1215 52.2 0.25 756 1164 592 837 295 35.2 0.5 81.6 137 51 90 44 48.7 1 56.8 27.6 24.4 36.3 17.9 49.2 2 40.8 13.9 10.9 21.9 16.5 75.3 3 4.76 5.40 10.2 6.80 3.00 44.1 4 2.48 2.52 2.26 2.42 0.14 5.66 8 BLOQ BLOQ BLOQ BLOQ NA NA 12 BLOQ BLOQ BLOQ BLOQ NA NA 24 BLOQ BLOQ BLOQ BLOQ NA NA 48 BLOQ BLOQ BLOQ BLOQ NA NA
[0374] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following single dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00112 Time Concentration (ng/mL) Mean SD CV (h) G1 G2 G3 (ng/mL) (ng/mL) (%) 0.0833 50.2 73.1 46.1 56.4 14.6 25.8 0.25 22.7 19.8 19.0 20.5 2.0 9.53 0.5 9.76 6.83 6.50 7.70 1.79 23.3 1 3.71 3.06 3.18 3.32 0.34 10.4 2 3.08 4.37 3.22 3.56 0.71 19.9 3 BLOQ BLOQ BLOQ BLOQ NA NA 4 4.08 3.42 BLOQ 3.42 NA NA
[0375] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol CSF concentrations following last day (Day 4) repeated dose oral administration to male C57BL/6J Mice at 25 mg/kg:
TABLE-US-00113 Time Concentration (ng/mL) Mean SD CV (h) G1 G2 G3 (ng/mL) (ng/mL) (%) 0 BLOQ BLOQ BLOQ BLOQ NA NA 0.0833 344 178 79.3 200 134 66.9 0.25 41.8 67.6 41.4 50.2 15.0 29.9 0.5 6.22 9.54 4.18 6.64 2.71 40.8 1 5.22 3.47 3.07 3.92 1.14 29.2 2 4.25 BLOQ BLOQ BLOQ NA NA 3 BLOQ BLOQ BLOQ BLOQ NA NA 4 BLOQ BLOQ BLOQ BLOQ NA NA 8 BLOQ BLOQ BLOQ BLOQ NA NA 12 BLOQ BLOQ BLOQ BLOQ NA NA 24 BLOQ BLOQ BLOQ BLOQ NA NA 48 BLOO BLOO BLOO BLOQ NA NA
Example 9: Plasma Pharmacokinetic Study of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Following Single Dose Oral Administration in Fed Male
[0376] Cynomolgus Monkeys
[0377] The pharmacokinetic profile of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol was evaluated in male Cynomolgus Monkeys. Three male Cynomolgus Monkeys were administrated a single oral dose at 10 mg/kg. The oral vehicle was 10% (w/v) HP-β-CD in water.
[0378] Animals
[0379] Male Cynomolgus Monkeys were purchased from Topgene Biotechnology. The animals were 2-5 years old with body weights of 2.0-5.0 kg on the dosing date. The animals were housed in a 12-hour light/12-hour dark cycle environment and had free access to food and water.
[0380] All animals for SC administration had free access to food and water. All animals for PO administration were fasted overnight prior to dosing and will be fed approximately 2 hours after dosing.
[0381] Study Design
[0382] Three male Cynomolgus Monkeys were assigned to this study. The animals were treated with (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol by PO administration at 10 mg/kg, which was formulated in 10% (w/v) HP-β-CD in water. The dosing regimens and formulation (vehicle) compositions are described in the following table below:
TABLE-US-00114 Dose Level Dose Volume Conc. Administration No. of Group Treatment (mg/kg) (mL/kg) (mg/mL) Route Animals 1 M102 10 5 2 PO 3M
[0383] Dose Administration
[0384] Animals were weighed prior to dose administration on the day of dosing for calculating the actual dose volume. The dosages were prepared as illustrated in the table below:
TABLE-US-00115 Free Conc Compound Route MW MW FW (mg/mL) Vehicle (6aS)-6-methyl- PO 267.32 303.79 312.79 2 10% 5,6,6a,7- (w/v) tetrahydro-4H- HP-β-CD dibenzo[de,g]quin- in water oline-10,11-diol
[0385] Sample Collection
[0386] Blood samples were taken at the following times:
TABLE-US-00116 Group PK time points PO Pre-dose, 5 min, 10 min, 15 min, 30 min, 1, 2, 4, 8 and 24 hr post dose
[0387] Whole blood samples were gently inverted several times to ensure anticoagulation then placed on wet ice prior to centrifugation. Within 30 minutes of collection, the samples were centrifuged at approximately 4,000×g (force) for 5 minutes at 2 to 8° C. to obtain plasma. The stabilizing agent, ascorbic acid at 200 mg/mL, was added in a proportion of 10% in plasma. The plasma samples were stored in a freezer at −75±15° C. prior to analysis. The completion time from collection was less than 60 min. The resultant plasma samples will be immediately separated divided into 2 aliquots (˜75 μL each) and transferred to cryogenic vial. Samples will be maintained on wet ice throughout processing, and stored in a freezer at −75±15° C. prior to analysis. Samples should be protected from ambient light (under sodium lamp).
[0388] Preparation of Standards
[0389] Standards were prepared with 0.96 mg of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol added to 0.820 mL DMSO to obtain a 1 mg/mL standard stock solution (free form).
[0390] Calibration standard working solutions were prepared at concentrations of 2, 5, 10, 20, 100, 500, 1000, 5000, 10000 ng/mL by serial dilution of the standard stock solution in 1:1 acetonitrile:water. Quality control working solutions at concentrations of 5, 10, 20, 500 and 8000 ng/mL were prepared by serial dilution of the standard stock solution in 1:1 acetonitrile:water. The QC samples were prepared on the day of analysis in the same way as calibration standards.
[0391] Sample Treatment
[0392] Samples were prepared with 5 μL of each calibration standard working solution (2, 5, 10, 20, 100, 500, 1000, 5000, 10000 ng/mL) added to 50 μL of the blank Cynomolgus Monkeys plasma to achieve calibration standards concentrations of 0.2, 0.5, 1, 2, 10, 50, 100, 500, 1000 ng/mL (relative to a 50-4, plasma aliquot). Quality Control (QC) samples at 0.5 ng/mL (low-1), 1 ng/mL (low-2), 2 ng/mL (low-3), 50 ng/mL (mid) and 800 ng/mL (high) for plasma were prepared independently for those used for the calibration curves.
[0393] Total volumes of 55 μL of standards, 55 μL of QC samples and 55 μL of unknown samples (50 μL of plasma with 5 μL of 1:1 acetonitrile:water) were added to 200 μL of acetonitrile containing IS to precipitate proteins. Then the samples were vortexed for 30 sec. After centrifugation at 4° C., 3900 rpm for 15 min, supernatant was diluted 5 times with water, and 2 μL of the diluted supernatant was injected into the LC-MS/MS system for quantitative analysis. All of the samples were processed on ice.
[0394] LC-MS/MS Conditions
[0395] The LC-MS/MS system consisted of two Shimadzu LC-30AD pumps, a DGU-20A5R degasser, a Rack changer II and an AB Sciex Triple Quad 5500 LC/MS/MS mass spectrometer. Chromatographic separation was performed on Agilent ZORBAX XDB-Phenyl 5 μm (50×2.1 mm) at room temperature. The mobile phase was composed of A: 5% Acetonitrile/95% water (0.1% Formic acid); B: 95% Acetonitrile/5% water (0.1% Formic acid). The flow rate was 0.6 mL/min. The injection volume was 2 μL.
[0396] Positive mode electrospray ionization (ESI) was performed on a Turbo V® ion source to obtain a protonated ion of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Dexamethasone (IS). A multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions were 268.05/237.10 Da and 393.14/373.10 Da for (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol and Dexamethasone, respectively. The instrument parameters were set as follows: ion spray voltage: 5500 V; curtain gas: 40 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; temperature: 450° C. The compound dependent parameters are listed as follows:
TABLE-US-00117 (6aS)-6-methyl-5,6,6a,7- tetrahydro-4H- dibenzo[de,g]quinoline- Dexamethasone 10,11-diol (IS) Precursor/ 268.05/237.10 393.14/373.10 product DP 131.00 146.00 CE 25.00 13.00 CXP 18.00 16.00
[0397] HPLC flow rate was set at 0.6 mL/min with the following conditions:
TABLE-US-00118 Time (min) A (%) B (%) 0.01 95.0 5.00 0.20 95.0 5.00 1.90 5.00 95.0 2.20 5.00 95.0 2.21 95.0 5.00 2.50 95.0 5.00
[0398] Results
[0399] Following the PO (11.4 mg/kg) t.sub.1/2 was 2.26±0.56 hr, T.sub.max was 0.667±0.289 hr, C.sub.max was 34.7±17.9 ng/mL, MRT.sub.Inf was 2.92±0.92 hr, AUC.sub.0-24 hr was 58.1±12.4 h*ng/mL, and AUC.sub.inf was 63.5±11.9 h*ng/mL in Cynomolgus Monkeys.
[0400] The following table illustrates the plasma pharmacokinetic parameters of (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol following PO administration to male Cynomolgus Monkeys at 10 mg/kg:
TABLE-US-00119 PK parameters Unit 168777C 169939C 168779C Mean SD CV(%) T.sub.1/2 h 5.60 NA NA NA NA NA Tmax h 2.00 4.00 4.00 3.33 1.15 34.6 Cmax ng/mL 6.27 5.91 8.04 6.74 1.14 16.9 AUC.sub.last h*ng/mL 42.6 33.9 47.9 41.5 7.1 17.1 AUC.sub.Inf h*ng/mL 44.9 NA NA NA NA NA AUC_% Extrap_obs % 5.01 NA NA NA NA NA MRTInf_obs h 6.52 NA NA NA NA NA AUC.sub.last/D h*mg/mL 4.26 3.39 4.79 4.15 0.71 17.1 F % NA NA NA NA NA NA AUC.sub.last/C.sub.max h 6.79 5.74 5.96 6.16 0.56 9.1
[0401] (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol Plasma Concentrations Following PO Administration to Male Cynomolgus Monkeys at 10 mg/kg in fed condition:
TABLE-US-00120 Time Concentration (ng/mL) Mean SD CV (h) 168777C 169939C 168779C (ng/mL) (ng/mL) (%) 0 BLOQ BLOQ BLOQ NA NA NA 0.083 4.62 1.07 0.425 2.04 2.26 111 0.167 2.83 1.23 0.911 1.66 1.03 62.1 0.25 2.63 0.893 0.262 1.26 1.23 97.2 0.5 4.15 0.975 1.26 2.13 1.76 82.5 1 5.15 2.81 5.00 4.32 1.31 30.3 2 6.27 5.41 6.27 5.98 0.50 8.30 4 4.40 5.91 8.04 6.12 1.83 29.9 8 1.13 2.61 0.779 1.51 0.97 64.5 24 0.278 BLOQ 0.279 0.279 NA NA
[0402] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying Figures. Such modifications are intended to fall within the scope of the appended claims.
[0403] It is further to be understood that all values are approximate and are provided for description. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.