NZ624558B2 - Dosage regime for apolipoprotein formulations - Google Patents

Abstract

Disclosed herein is a method of producing a reconstituted high density lipoprotein (rHDL) formulation, which includes the steps of combining Apo A-I or a fragment thereof, one or more phospholipids and optionally a detergent at a molar ratio of Apo A-I to phospholipid of 1:20 to 1:120 and determining a fixed dosage of the rHDL formulation that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and that displays relatively reduced inter-patient variability in exposure to the Apo A-I constituent of the formulation compared to that which would be observed or associated with a weight-adjusted dosage regime. Also disclosed are formulations comprising 0.1 to 0.5 g Apo A-I or a fragment thereof, one or more phospholipids and optionally a detergent at a molar ratio of Apo A-I to phospholipid of 1:20 to 1:120 that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and displays relatively reduced inter-patient variability in exposure to the Apo A-I compared to that which would be observed or associated with a weight-adjusted dosage regime. The formulations are intended for use in therapeutic treatment of diseases or conditions including cardiovascular disease, acute coronary syndrome, atherosclerosis, unstable angina pectoris, and myocardial infarction. g a fixed dosage of the rHDL formulation that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and that displays relatively reduced inter-patient variability in exposure to the Apo A-I constituent of the formulation compared to that which would be observed or associated with a weight-adjusted dosage regime. Also disclosed are formulations comprising 0.1 to 0.5 g Apo A-I or a fragment thereof, one or more phospholipids and optionally a detergent at a molar ratio of Apo A-I to phospholipid of 1:20 to 1:120 that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and displays relatively reduced inter-patient variability in exposure to the Apo A-I compared to that which would be observed or associated with a weight-adjusted dosage regime. The formulations are intended for use in therapeutic treatment of diseases or conditions including cardiovascular disease, acute coronary syndrome, atherosclerosis, unstable angina pectoris, and myocardial infarction.

Description

DOSAGE REGIME FOR APOLIPOPROTEIN FORMULATIONS TECHNICAL FIELD THIS INVENTION relates to fixed dosing of apolipoprotein formulations. More particularly, this invention s to the delivery of fixed dosages of apolipoprotein formulations, that is, in an amount independent of t body weight.

BACKGROUND High density lipoprotein (HDL) is a class of heterogeneous lipoproteins containing lipid and n characterized by high density (>1.063 g/mL) and small size (Stoke’s diameter = 5 to 17 nm). The various HDL subclasses vary in quantitative and qualitative content of , apolipoproteins, enzymes, and lipid transfer proteins, ing in differences in shape, density, size, charge, and antigenicity. Apolipoprotein A-I (Apo-AI) is the predominant HDL protein, although other apolipoproteins such as Apo-AII and Apo»V may be present.

Epidemiological and clinical studies have established an inverse ation between levels of high-density lipoprotein cholesterol (HDL-C) and risk of cardiovascular disease (reviewed in Assmann et al., 2004, Circulation III«8). More particularly, clinical administration of apolipoprotein formulations such as in the form of reconstituted HDL (rHDL) formulations have been shown to confer beneficial effects to hypercholesterolemic patients suffering from recent acute coronary syndromes (ACS).

Dosages of apolipoprotein formulations, such as rHDL formulations, are typically calculated according to the body weight of the patient or individual to whom the formulation is stered. This approach to dosage is based on the assumption that there is a direct ation n a person’s body weight and their ability to distribute and eliminate the apolipoprotein formulation. Thus the ation is that body weight based dosing of the apolipoprotein formulation would result in each patient receiving the same exposure to the apolipoprotein with minimal variation between patients of the same or different body weights.

The t inventors have surprisingly discovered that typical dosages of apolipoprotein ations, such as reconstituted HDL (rHDL) formulations particularly when adjusted or calculated ing to patient body weight, y considerable variability between patients in terms of exposure to the oprotein (such as apoA-I) administered in the ation. More particularly, it has been shown by the inventors that inter-patient variability in exposure to apolipoprotein when dosing patients based on their body weight is greater than that observed with a fixed dosing approach.

It is an object of the invention to provide an apolipoprotein ation in a dosage which alleviates or avoids one or more of the deficiencies of prior art apolipoprotein formulations.

It is a preferred object of the invention to e an apolipoprotein formulation at a dosage that is efficacious in the prophylactic and/or therapeutic treatment of diseases or conditions including, but not limited to cardiovascular disease.

It is another preferred object of the invention to provide an apolipoprotein formulation at a dosage which displays relatively reduced inter-patient variability as measured by patient exposure to oprotein components of the apolipoprotein formulation.

In a first aspect, the invention provides a method of prophylactically or therapeutically treating a disease, disorder or condition in a human including the step of administering to the human a fixed dose oprotein formulation, to thereby treat said disease, disorder or condition in the human.

In a second aspect, the invention provides a fixed dosage apolipoprotein formulation for use in prophylactically or therapeutically treating a disease, disorder or condition in a human.

In a third aspect, the invention provides a fixed dosage apolipoprotein formulation comprising an apolipoprotein or a fragment thereof, at a therapeutically ive fixed dose.

Suitably, the fixed dosage oprotein formulation is therapeutically effective upon administration to a human of any body weight, or of any body weight in a body weight range. ly, the fixed dosage apolipoprotein formulation displays relatively reduced inter-patient variability in exposure of the apolipoprotein of the formulation compared to that which would be observed or associated with a weight-adjusted dosage regime.

In a fourth aspect, the invention provides a method of producing a fixed dosage apolipoprotein formulation comprising an apolipoprotein or a fragment thereof, including the step of producing the oprotein formulation at a fixed dosage which is therapeutically effective.

Suitably, the method includes the step of determining a fixed dosage of the apolipoprotein formulation that is therapeutically effective upon administration to a human of any body weight or of any body weight in a body weight range.

Preferably, the fixed dosage is determined as that at which the apolipoprotein formulation displays relatively reduced inter-patient variability in re over a range of t body s to the apolipoprotein constituent of the apolipoprotein formulation compared to that which would be observed or associated with weight-adjusted dosage stered to patients over the same weight range.

In a fifth , the invention provides an apolipoprotein formulation produced according to the method of the fourth aspect for lactically or therapeutically treating a disease, disorder or condition in a human.

In a sixth aspect, the invention provides a fixed dosage, apolipoprotein kit comprising one or more unit dosages of a fixed dosage apolipoprotein ation according to the second or third aspect, or produced ing to the method of the fourth aspect; and one or more other kit components.

Preferably, the disease, disorder or condition includes cardiovascular disease, hypercholesterolaemia or olesterolaemia, inclusive of acute coronary syndrome (ACS), atherosclerosis, unstable angina pectoris and dial infarction.

In a preferred form the apolipoprotein is Apo~A1 or a fragment thereof.

Suitably, the apolipoprotein formulation is a reconstituted high density lipoprotein (rHDL) formulation comprising an apolipoprotein, a lipid and, optionally, a detergent.

Throughout this specification, unless the t requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other r or group of integers.

BRIEF DESCRIPTION OF THE FIGURES Reference is made to the following Figures which assist in understanding non-limiting embodiments of the invention described in detail hereinafter wherein: Figure 1 shows median simulated ApoA-I plasma concentration vs. time profiles by dosing regimen (2 h infusion duration only) during week 4 of dose stration; Figure 2 shows projected distributions of total ApoA-I plasma concentration vs. time profiles by dosing regimen (2 h infusion duration only) during week 4 of dose administration. Line represents median profile, shaded region represents 95% prediction interval (PI); Figure 3 shows distributions of ous ApoA-I AUC0_72 for the last dose of weekly regimens infiJsed over 2 h. White line represents the median response, dark blue shaded area represents the inter-quartile range and light blue shaded area ents the 95% PI for 100 simulations. Outer solid red horizontal lines show the broader width of the exposure bands for the 40 mg/kg dose in the left panel relative to those for the equivalent fixed dose (3.6 g) in the right panel.

The inner solid red line joins the median exposure for those doses. Dashed gray lines show the comparative exposures for the 70 mg/kg dose in the left panel and the lent fixed dose (6.3 g) in the right panel; Figure 4 shows distributions of exogenous ApoA—I AUC0_163 for the last dose of weekly regimens infused over 2 h. White line represents the median response, dark blue shaded area represents the inter-quartile range and light blue shaded area represents the 95% PI for 100 simulations. Outer solid red horizontal lines show the broader width of the re bands for the 40 mg/kg dose in the left panel relative to those for the lent fixed dose (3.6 g) in the right panel.

The inner solid red line joins the median exposure for those doses. Dashed gray lines show the comparative exposures for the 70 mg/kg dose in the left panel and the equivalent fixed dose (6.3 g) in the right panel; Figure 5 shows distributions of exogenous ApoA-I Cmax for the last dose of weekly regimens infiised over 2 h. White line represents the median response, dark blue shaded area represents the inter-quartile range and light blue shaded area represents the 95% PI for 100 simulations. Outer solid red horizontal lines show the broader width of the exposure bands for the 40 mg/kg dose in the left panel relative to those for the equivalent fixed dose (3.6 g) in the right panel. The inner solid red line joins the median exposure for those doses. Dashed gray lines show the comparative exposures for the 70 mg/kg dose in the left panel and the lent fixed dose (6.3 g) in the right panel; Figure 6 shows the relationship between exogenous ApoA-I AUCO_72 (for the last dose of weekly regimens infused over 2 h) n fixed dosing and body weight dosing. White line ents the median response, dark blue shaded area represents the inter-quartile range and light blue shaded area represents the 95% PI for 100 simulations; Figure 7 shows the relationship between exogenous ApoA-I AUC0463 (for the last dose of weekly regimens infused over 2 h) and body weight. White line represents the median response, dark blue shaded area represents the inter—quartile range and light blue shaded area represents the 95% PI for 100 simulations; and Figure 8 shows the relationship between exogenous ApoA-I Cmax (for the last dose of weekly regimens infused over 2 h) and body weight. White line ents the median response, dark blue shaded area represents the inter-quartile range and light blue shaded area represents the 95% PI for 100 simulations.

ED DESCRIPTION The ion at least partly arises from the unexpected discovery that a fixed dosing regime for apolipoprotein formulations (e.g. rHDL) independent of patient body weight has less impact on ap0A~I exposure over a range of patient body weights than that imposed by body weight-adjusted dosing. Thus, there is less inter—patient variability in exposure to apo-Al associated with fixed dosing ns compared with body weight-adjusted s, particularly at the extremes ofhuman patient body weight range.

In one preferred aspect, the invention provides a fixed dosage apolipoprotein formulation at a dosage which is therapeutically effective upon administration to a human of any body weight, or of any body weight in a body weight range.

In another red aspect, the invention provides a method of producing a fixed dosage apolipoprotein formulation including the step of producing the apolipoprotein formulation at a dosage which is therapeutically effective upon administration to a human of any body weight or of any body weight in a body weight range.

It will be appreciated that the present invention relates to apolipoprotein formulations that have therapeutic efficacy in treating one or more diseases, disorders or conditions sive to therapy by said apolipoprotein formulation.

Preferably, the apolipoprotein is Apo-Al or a nt thereof The apolipoprotein formulations of the present invention may either be formulated with lipid (e.g. rHDL) or without lipid (e.g. dated apolipoprotein).

In a ular embodiment, the apolipoprotein formulation is an rHDL formulation. As used herein, a “reconstituted HDL (rHDL)” formulation may be any artificially-produced apolipoprotein formulation or ition that is onally similar to, ous to, corresponds to, or mimics, high density lipoprotein (HDL) typically present in blood . As used herein, an rHDL formulation is not a liposome preparation. rHDL formulations includes within their scope “HDL mimetics” and “synthetic HDL particles”. ly, the rHDL formulation comprises an apolipoprotein, a lipid and, optionally, a detergent.

Apolipoprotein formulations such as, but not d to, rHDL formulations may fiirther comprise terol. Apolipoprotein formulations be produced using organic solvents, which in some cases are used for dissolving the lipid component (e.g. phosphatidylcholine) when producing the formulation, such as described in US patent 5,652,339. However it is preferred that the apolipoprotein formulation is produced in the absence of organic solvent.

The apolipoprotein may be any apolipoprotein which is a functional, biologically active component of naturally-occurring HDL or of a reconstituted high density lipoprotein (rHDL). Typically, the apolipoprotein is either a plasma- derived or recombinant apolipoprotein such as Apo-AI, Apo-AII or Apo-AV, pro— apo-Al or a variant such as Apo-AI Milano. Preferably, the apolipoprotein is . Also contemplated are biologically—active fragments of the apolipoprotein. Fragments may be naturally occurring, chemical synthetic or recombinant. By way of example only, a biologically-active fragment of Apo—AI preferably has at least 50%, 60%, 70%, 80%, 90% or 95-100% or even greater than 100% of the lecithin-cholesterol acyltransferase (LCAT) stimulatory activity ofApo—AI when formulated in a rHDL preparation. ly, the apolipoprotein is at a concentration of about 5-100 g/L, preferably 10—50g/L or more ably 25—45g/L . This includes 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 g/L and any ranges between these amounts. In other embodiments, the oprotein may be at a concentration of from about 5 to 20 g/L, e.g. about 8 to 12 g/L.

The lipid may be any lipid which is a component of naturally-occurring HDL or of reconstituted high density lipoprotein (rHDL). Such lipids include phospholipids, cholesterol, cholesterol-esters, fatty acids and/or triglycerides.

Preferably, the lipid is a phospholipid. Non-limiting examples of olipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycero] (PG), phosphatidylserine (PS), atidylinositol (PI) and sphingomyelin (SM), sphingosine-l phosphate or natural or synthetic tives thereof. l derivatives include egg PC, egg PG, soy bean PC, hydrogenated soy bean PC, soy bean PG, brain PS, sphingolipids, brain SM, galactocerebroside, gangliosides, cerebrosides, in, cardiolipin, and lphosphate. Synthetic derivatives include dipalmitoylphosphatidylcho]ine (DPPC), didecanoylphosphatidylcholine (DDPC), dierucoylphosphatidylcholine (DEPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dilaurylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), oy]myristoylphosphatidylcholine (PMPC), palmitoylstearoylphosphatidylcholine (PSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylglycerol (DLPG), distearoylphosphatidylglycerol (DSPG), dimyn'stoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA), stoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylserine , dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE) dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSM) and distearoylsphingomyelin (DSSM). The phospholipid can also be a derivative or analogue of any of the above phospholipids.

In other specific ments, the lipid is, or ses, sphingomyelin in combination with a negatively charged phospholipid, such as phosphatidylglycerol (e.g. 1 ,2-dipalmitoyl-sn-g1ycero [phospho-rac—(l- glycerol». A combination of sphingomyelin and phosphatidylglycerol (particularly 1 ,2-dipalmitoyl-sn-glycero-3 -[phospho~rac-(l-glycerol)) is specifically envisaged for use as the lipid. In these embodiments, the sphingomyelin and the phosphatidylglycerol may be present in any suitable ratio, e.g. from 90:10 to 99:1 (wzw), typically 95:5 to 98:2 and most typically 97:3.

Preferably the phospholipid is, or comprises, phosphatidylcholine, alone or in combination with one or more other phospholipids. An example of another phospholipid is sphingomyelin. In some embodiments, the apolipoprotein ation may comprise a detergent.

Typically, gh not exclusively the lipid may be present at a concentration of 10—100 g/L or preferably 30-60 g/L.

The detergent may be any ionic (e.g cationic, anionic, Zwitterionic) detergent or non-ionic detergent, inclusive of bile acids and salts thereof, suitable for use in apolipoprotein (e.g. rHDL) formulations. Ionic detergents may include bile acids and salts thereof, rbates (e.g. P880), CHAPS, CHAPSO, cetyl hyl-ammoniurn bromide, lauroylsarcosine, tert-octyl phenyl esulfonic acid and 4’-amino-7~benzamido-taurocholic acid.

Bile acids are typically dihydroxylated or roxylated steroids with 24 carbons, including cholic acid, deoxycholic acid eoxycholic acid or ursodeoxycholic acid. Preferably, the detergent is a bile salt such as a cholate, deoxycholate, chenodeoxycholate or ursodeoxycholate salt. A particularly preferred detergent is sodium e.

As hereinbefore described, the fixed dosage apolipoprotein formulation is at a dosage that is therapeutically effective upon administration to human patients of any body weight or of any body weight in a body weight range. Accordingly, the apolipoprotein formulation dosage is not calculated, determined or selected according to the particular body weight of the human, such as would typically occur with “weight-adjusted dosing”.

Rather, the fixed dosage apolipoprotein formulation is determined as a dosage which when administered to human patients of any body weight or of any body weight in a body weight range, would display relatively reduced inter- patient variability in terms of exposure to the oprotein constituents of the apolipoprotein formulation. Relatively reduced patient variability is compared to that observed or ated with weight-adjusted dosing of a patient tion.

Variability of exposure may be expressed or measured in terms of the variation in exposure of ts to apolipoprotein following administration of the fixed dosage oprotein formulation. Preferably, the variability is that which would occur when the fixed dosage apolipoprotein formulation is administered to human patients over a weight range compared to the variability that would occur for weight-adjusted s administered to human patients over the same weight range as the fixed dosage patients. In some embodiments, exposure to apolipoprotein may be measured as average exposure (e.g. mean or median exposure), total exposure (e.g. amount integrated over time of exposure) or m exposure level (e.g. Cmax). Generally, the weight or weight range is , 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 kg, or any range between these values. Preferably, the weight or weight range is 20-200 kg, 2060 kg, 40-160 kg, 50-80 kg, 60-140 kg, 70-80 kg, 80~120 kg, 100—180 kg or 120-200 kg.

Suitably, the variability is less than 100% or preferably 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, or less than 90%, 85% or 80% of the variability that occurs with -adjusted dosing. Variability may be calculated and expressed by any statistical representation known in the art, including as a co- efficient of variation (e.g. %CV), standard deviation, standard error or the like, although without limitation thereto.

Notwithstanding administration of a fixed dosage apolipoprotein formulation to patients of markedly ent body weights, the exposure of the patients to apolipoprotein is surprisingly uniform. Accordingly it is proposed that the therapeutic efficacy of the fixed dosage apolipoprotein formulation will not be ntially mised or reduced compared to a weight-adjusted dosage.

By way of example only, the present ors have shown that there is no difference in total exposure to apolipoprotein upon administration of a fixed dosage apolipoprotein formulation to patients in the 60-120 kg weight range. rmore, Cmax for apolipoprotein decreased by an average of 16% over the 60- 120 kg weight range.

In comparison, for weight-adjusted dosing regimes using the same apolipoprotein formulation, a doubling of body weight from 60 kg to 120 kg requires a doubling of the dosage of apolipoprotein and increased apoA-I exposures.

Fixed dosage apolipoprotein formulations may be stered in multiple doses at any le frequency ing daily, twice weekly, weekly, fortnightly or monthly. Fixed dosage apolipoprotein formulations may be administered by any route of administration known in the art, such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time such as over 60, 90, 120 or 180 minutes), by intra-muscular, intra—peritoneal, intra-arterial including directly into coronary arteries, intra-cerebrospinal, sub-cutaneous, intra— articular, synovial, intra-thecal, oral, topical, or inhalation routes. Typically, fixed dosage apolipoprotein ations are administered parenteraliy, such as by intravenous infusion or injection.

Preferred fixed dosages include 0.1-15g, 0.5-12g, 1-10g, 2-9g, 3-8g, 4-7g or 5-6g of apolipoprotein. Particularly preferred fixed dosages include 1-2g, 3-4g, -6g or 6—7g of oprotein. Non-limiting examples of specific fixed dosages include 0.25g, 0.5g, 1g, 1.7g, 2g, 3.4g, 4g, 5.1g, 6g, 6.8g and 8g of apolipoprotein.

Non-limiting, specific examples of fixed dosage administration regimes that may be employed include 0.25g, 0.5g, lg, 1.7g, 2g, 3.4g, 4g, 5.1 g, 6g, 6.8g or 8g weekly by intravenous infusion over 90 min, 0.25g, 0.5g, 1g, 1.7g, 2g, 3.4g, 4g, 5.1g, 6g, 6.8g or 8g apolipoprotein weekly by intravenous infilsion over 120 min or 0.25g, 0.5g, 1g, 1.7g, 2g, 3.4g, 4g, 5.1g, 6g, 6.8g or 8g apolipoprotein twice weekly by intravenous infusion over 90 min or over 120 min.

In yet r aspect, the invention provides a method of prophylactically or eutically treating a disease, disorder or condition in a human including the step of administering to the human an apolipoprotein formulation disclosed ly, the disease, disorder or condition responsive to prophylactic or therapeutic administration of the fixed dosage oprotein formulation includes such diseases, disorders or conditions as cardiovascular disease (e. g. acute coronary syndrome (ACS), atherosclerosis, unstable angina pectoris and dial infarction) or diseases, disorders or conditions such as diabetes, stroke, impaired renal function, or myocardial infarction that predispose to ACS, hypercholesterolaemia (e.g. elevated serum cholesterol or elevated LDL cholesterol) and olesterolaemia resulting from reduced levels of high- y lipoprotein (HDL), such as is symptomatic of Tangier disease, although without limitation thereto.

The invention also es a fixed dosage, apolipoprotein kit comprising one or more unit doses of the fixed dosage apolipoprotein formulation disclosed 2O herein and one or more other kit components.

Suitably, the kit is for prophylactically or therapeutically treating a disease, disorder or condition in a human, as hereinbefore described.

Non-limiting examples of one or more other kit components e instructions for use; vials, containers or other storage vessels containing each of the unit doses; delivery devices such as needles, catheters, syringes, tubing and the like; and/or packaging suitable for safely and conveniently storing and/or transporting the kit. Preferably the instructions for use are a label or package , wherein the label or package insert indicates that the apolipoprotein formulation may be used to treat a disease or ion such as cardiovascular disease by administering a fixed dose amount to a human subject in need thereof.

A ‘package insert’ refers to instructions included in commercial packages of the apolipoprotein formulations, that contains information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such apolipoprotein formations.

For the purposes herein, a ‘vial’ refers to a container which holds an oprotein formulation. The via] may be sealed by a stopper pierceable by a syringe. Generally, the vial is formed from a glass al. Accordingly, a vial preferably comprises the lyophilized rHDL formulation with a protein t of 0.25g, 0.5g, 1g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 8g or 10 g per vial. More ably the apolipoprotein content is either 0.5g, ig, 2g, 4g, 6g, 8g or 10 g per vial.

The apolipoprotein formulation in the vial can be in s states including liquid, lyophilized, frozen etc. The fixed dosage oprotein ation is preferably stable as a . ity may be measured by any means known in the art, although turbidity is a preferred measure. A turbidity level of below about 10, 15, 20, or 30 NTU can generally be considered a stable fixed dosage apolipoprotein formulation. Turbidity measurements can be taken by incubating the fixed dosage apolipoprotein formulations over time periods such as 0 hr, 2 hr, 4hr, 6 hr, 12 hr, 18 hr, 24 hr, 36 hr, 72 hr, 7 days and 14 days at storage temperatures such as room temperature or 37°C. Preferably the fixed dosage apolipoprotein formulation is considered to be stable as a liquid when it is stored for 14 days at room temperature and exhibits a turbidity of less than about NTU.

The kit may facilitate administration of the fixed dosage apolipoprotein formulation by a health professional or self-administration by a patient or caregiver.

As previously described, the fixed dosage apolipoprotein formulation disclosed herein may be any apolipoprotein ation. Preferably, the apolipoprotein formulation is an rHDL formulation that comprises an apolipoprotein, lipid and, optionally, a detergent. Accordingly, the fixed dosage apolipoprotein or rHDL formulation may lly apply to any such formulation that would usually be administered by “weight-adjusted” dosing. Non-limiting examples of rHDL formulations include those described in W02003/096983; US20040266662; W02005/04l866; W02006/100567; WOO6/20069240; and W02010/093918, W02012/000048 and W02012/109162.

One particular miting example is an rHDL formulation comprising Apo-Al and one or more phospholipids including sphingomyelin and one or more negatively charged phospholipids such as phosphatidylinositol and phosphatidylglycerol.

In some embodiments, detergent is absent from the fixed dosage rHDL formulation. In other ments, ent is present in the fixed dosage rHDL formulation. In one preferred embodiment, the fixed dosage rHDL formulation comprises a detergent at a level which is not toxic, or at least displays relatively low toxicity. In this regard, reference is made to W02012/000048 which provides IO a detailed description on an rHDL formulation according to this ment.

In work leading to the present invention, it had been shown that some rHDL formulations displayed liver toxicity upon administration of the rHDL formulation to a human, such as evidenced by al or compromised liver function. Non-limiting examples of liver function(s) that may be abnormal or compromised include elevated alanine aminotransferase activity (ALT), elevated aspartate aminotransferase (AST) activity and/or elevated bilirubin levels. An example of an rHDL formulation at a level that caused liver toxicity is described in Tardif et al., 2007, xp. 297 El. Preferably the fixed dosage apolipoprotein formulation does not y liver toxicity.

Liver toxicity can be evaluated by various in vitro and in vivo models.

One example of an in vitro model uses HEP-G2 cells. This involves growing HEP-G2 cells into the log phase. The cells are then removed from the culture medium and washed in PBS prior to trypsinization and resuspension in 10 mL of culture medium (90 % DMEM, 10 % inactivated FCS, 1 % ential amino acids, 1 % Pen/Strep). Cell growth and viability are monitored using a Neubauer ytometer and trypan blue staining. Aliquots of 100 uL containing 10x104 C/mL are subsequently seeded into 96 well F-bottom plates and incubated overnight at 37°C, 5% CO2, 95% H20. Samples (700 pL) containing the test articles (6.g rHDL formulations) are prepared by addition of culture medium. The medium from the first row of wells is removed and 200 uL of the test article solution added. A serial 1:2 dilution series is ted on the plates. The plates are then incubated for 72 hours at 37°C, 5% CO2, 95% H2O. After which the cell viability is determined. This can be done by adding 50 uL of 3x Neutral Red Solution (70mg l Red in 100ml. PBS) to each well. The plates are incubated for 2 hours at 37°C, 5% C02, 95% H20 and the wells washed once with 200 uL PBS. After this 100 uL of ethanol is added to each plate and the plates shaken for 20 minutes prior to being read at 540nm.

An example of an in vivo hepatoxicity model is the conscious rabbit model. The model uses rabbits which have been placed in a restraint device (rabbit holder) and iv. catheters inserted into their car veins. Test articles are given as a 40 minute iv infusion. Blood samples are taken from the ear artery and collected into serum and streptokinase—plasma (5%) vials. Blood samples are processed to serum, stored at —20°C and to plasma and stored at — 80°C. Samples can then be assessed for the level of ALT and AST activity using enzymatic photometric test kits available commercially (Greiner Biochemica). Whilst human Apo A-I levels can be determined using a nephelometric assay. Preferably, the level of detergent when present in the fixed dosage apolipoprotein formulations is about 5- 35% of that which displays liver ty. This range includes, for example, 5%, %, 15%, 20%, 25%, 30% and 35%. More preferably, the level of detergent is about 5-20% of that which displays liver toxicity. Advantageously, the level is about 540% of that which displays liver toxicity. Preferably, these levels are sed in terms of the minimum or threshold level of detergent that displays liver toxicity.

By way of example, a level of detergent which has been shown to cause, result in or at least be associated with liver toxicity is 0.3g/g Apo-Al or 6g/L rHDL formulation (at 20g/L ). Accordingly, 5—10% of this level of detergent is 0015-003 g/g Apo-Al or 0.5~0.9 g/L rHDL ation (at 30g/L Apo-Al).

The “level” of detergent may be an te amount of detergent, a concentration of detergent (e.g. mass per unit volume of rHDL formulation) and/or a ratio of the amount or concentration of detergent ve to another amount or concentration of a ent of the rHDL formulation. By way of 3O example only, the level of detergent may be expressed in terms of the total mass of apolipoprotein (e.g. Apo-AI) present in the rHDL formulation.

A detergent concentration no less than about 0.45 g/L of rHDL formulation with 30 g/L apolipoprotein is optimal in terms of both stability and non-toxicity. Stability may advantageously be measured by any means known in the art, although turbidity of the rHDL formulation is a preferred measure.

In a further preferred embodiment, the fixed dosage rHDL formulation comprises a lipid at a level which does not cause liver toxicity. Suitably, the level of lipid is about 20~70% of that which causes, or is ated with, liver toxicity.

In particular embodiments, the level of lipid is preferably about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% of that which causes, or is associated with, liver toxicity, and any ranges between these amounts. Preferably, these levels are expressed in terms of the minimum or threshold level of lipid that displays liver toxicity.

By way of example, a level of lipid which has been shown in work leading to the present ion to cause, result in or at least be associated with liver toxicity is 84 g/L. ingly the lipid is preferably at a concentration of about —60 g/L. This includes 30, 35, 40, 45, 50, 55 and 60 g/L and any ranges between these amounts. A particularly advantageous concentration of lipid is about 30- 50g/L, or in certain embodiments about 34 or 47g/L.

The “level” of lipid may be an absolute amount of lipid, a tration of lipid (e.g. mass per unit volume of rHDL formulation) and/or a ratio of the amount or concentration of lipid relative to another amount or concentration of a component of the fixed dosage apolipoprotein formulation. By way of example only, the level of lipid may be sed in terms of a molar ratio of apolipoprotein (e.g. Apo—AI) present in the fixed dosage rHDL formulation.

In one preferred embodiment, the molar ratio of apolipoproteinzlipid is in the range 1:20 to 1:100. This range includes molar ratios such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80 and 1:90. More preferably, the molar ratio of apolipoproteinzlipid is in the range of 1:40 to 1:75. A particularly advantageous ratio of apolipoproteinzlipid is about 1:40 or 1:55.

In other ments, the molar ratio of oproteinzlipid is in the range from about 1:80 to about 1:120. For example, the ratio may be from 1:100 to 1:115, or from 1:105 to 1:110. In these embodiments, the molar ratio may be for e from 1:80 to 1:90, from 1:90 to 1:100, or from 1:100 to 1:110.

Suitably, the fixed dosage apolipoprotein formulation disclosed herein further comprises a stabilizer. In particular, the stabilizer maintains stability of the rHDL formulation during lyophilisation. Suitably the stabilizer is a carbohydrate such as a sugar or sugar l. Examples of suitable sugar or sugar alcohols are fructose, trehalose, ol and sorbitol. Preferably, the stabilizer is a haride sugar such as sucrose. A preferred concentration of sucrose is about -85 g/L (equivalent to about 10-85% w/v) of fixed dosage apolipoprotein formulation. Preferably, the concentration of sucrose is about 46-48 g/L (equivalent to about 4.6-4.8% w/v) or about 75 g/L (equivalent to about 7.5% w/v), which is relatively reduced compared to rHDL formulations such as CSLl l 1. It is proposed that this relatively reduced sucrose may allow for a faster infusion rate of the rHDL formulation of the invention. Other izers may be or include amino acids (e.g. glycine, proline), antioxidants, emulsifiers, surfactants, chelating agents, gelatine, synthetic oils, polyols, alginate or any aceutically acceptable carriers and/or excipients, although without limitation thereto. In this regard, reference is made by way of example to "Pharmaceutical Formulation Development of es and Proteins", Frokjaer et al., Taylor &; Francis (2000), "Handbook of Pharmaceutical Bxcipients", 3rd edition, Kibbe et al., ceutical Press (2000) and International Publication /025754.

Reduction or removal of detergent may be performed by any means known in the art including filtration, hydrophobic adsorption or hydrophobic interaction chromatography, dialysis, ion-exchange adsorption and ion-exchange chromatography, for example.

In some ments, non-polar polystyrene resins may be suitable for reducing detergent levels. Such resins preferably are in the form of a linked copolymer (e.g. a cross-linked styrene and divinylbenzene mer). Non- limiting examples include Amberlite XAD-Z and Bio Beads SM.

Filtration es gel filtration, gel permeation, ration and ultrafiltration, although without limitation thereto, as are well understood in the art. A non-limiting example of gel permeation may utilize porous, cross-linked dextran such as Sephadex resins.

In a particularly preferred embodiment particularly suitable for large scale manufacture, the detergent level is reduced by diafiltration.

So that preferred embodiments of the invention may be fully tood and put into practical effect, reference is made to the following non-limiting INTRODUCTION tituted high density lipoprotein (HDL) consists of purified human apolipoprotein A-I (ApoA-I), the primary protein constituent of HDL. Low plasma HDL is associated with an increased risk of coronary artery disease, one of the principal causes of death in developed countries. uently, it has been postulated that HDL may be protective of atherosclerosis. The mechanism by which HDL exerts its atheroprotective effect is complex and incompletely understood, but is believed to act at various steps involved in the efflux of cholesterol from peripheral cells (including arterial cells) to the liver.

The rHDL ation described in this example is a novel therapeutic agent that mimics endogenous HDL. Proof of concept was demonstrated for an older formulation of reconstituted HDL, CSLl 1 1, in the ERASE trial, a Phase 2a study of short term CSLl 11 infusions in 183 patients. However, development of CSLlll was ceased due to hepatotoxicity (Tardiff et al., 2010, supra and also described in more detail in W02012/000048).

The rHDL formulation bed in this example consists of purified human oprotein A-l (ApoA—I), the primary protein constituent of HDL (as hereinbefore described and also described in more detail in W02012/000048).

The primary objective was to e a justification for an appropriate dosing gy (weight-adjusted or fixed dosing) for the rHDL formulation using a model-based approach and Monte Carlo simulations.

METHODS GeneralApproach Monte Carlo simulations were performed using interim population PK models for ApoA—I developed in this analysis. The simulations used all components of the pharmacokinetic (PK) model including important covariates ined to influence ApoA—l disposition. The estimated population means (typical values) and intersubject variability values were used to define distributions for the interim PK parameters and simulations used random sampling from those distributions. The randomness described by this ng process reflected the intersubject variability in the model ters.

The randomly generated PK parameters were used to simulate plasma concentrations of ApoA~l for each virtual patient in the simulations. Estimates of al error, from the PK model, were used to introduce measurement error into the concentration predictions. Using the concentration predictions (that now incorporated both intersubj ect variability and measurement error), ed ApoA- I exposure metrics were calculated for each virtual patient. s are summarized graphically and in tabular form.

Simulation Specifications ApoA-I exposure was simulated separately for each of the following dosing regimens: - 40, 70 and 100 mg/kg weekly by intravenous infusion (IVI) over 90 min and 120 min ' 1.7, 3.4 and 5.1 g weekly by IVI over 90 min and 1.7, 3.4, 5.1 and 6.8 g weekly by IVI over 120 min - 1.7, 3.4 and 5.1 g twice weekly by IVI over 90 min and over 120 min Dosing was assumed to be continued for a period of 4 weeks. For the twice weekly ns, the second weekly dose was administered 72 hours after the start of the first dose administration.

Since body weight, creatinine clearance (CLCR) and sex were identified as significant sources of intersubject variability for ApoA~I, clinically relevant ranges of these patient covariates were included in the simulations: 0 Body weight: 60, 80, 100 and 120 kg for ApoA-I o CLCR: 50, 80 and 140 ml/min 0 Age: 20, 60 and 80 yrs 0 Males and females in equal proportions.

Each tion set included each relevant patient covariate and dosing regimen combination. ore, there were 456 virtual subjects (Lee 4 body weight values x 3 CLCR values x 2 sex x 19 dosing regimens) in the ApoA-I simulation set. 100 simulations were performed.

Plasma concentration measurements were assigned to occur at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 and 168 h from the start of the first dose for the weekly regimens and at 0, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, 72, 73, 73.5, 74, 75, 76, 78, 80, 90, 96, 108, 120, I44 and 168 h from the start of the first dose for the twice weekly regimens. ng was conducted during the first and fourth weeks of dosing.

Endpoints of interest were total plasma concentration vs. time profiles and ous maximum plasma concentration (Cmax), area under the curve from 0 to 72 hr (AUCO-72) and area under the curve from O to 168 hr (AUC0.;68) following each dosing regimen. Cmax and AUC0_72 were calculated for the first and last doses while 63 was calculated for the first and last weeks of dosing.

Exogenous plasma concentrations for exposure determinations were calculated by subtracting the simulated baseline plasma concentration from the simulated total plasma concentration at each time point.

RESULTS ProjectedApoA-IExposures by Dosing Regimen Projected median ( and distributions ( of ApoA-I plasma concentration vs. time profiles are shown by dosing regimen (2 h infusion ons shown). Given a median body weight of 90 kg, median exposure for the 40 mg/kg doses reflects a 3.6 g dose and is comparable to the 3.4 g fixed dose (see . Similarly, median re for the 70 mg/kg dose s 6.3 g and is approximately comparable to the 6.8 g fixed dose.

Table 1 summarizes projected ApoA-I exposures during the fourth week of dose administration by dosing regimen. Despite similar mean exposures for the 40 mg/kg and 3.4 g dosing regimens and for the 70 mg/kg and 6.8 g dosing regimens, there was imately 8-10% greater variability (as measured by %CV in exposure metrics) associated with the weight~adjusted dosing regimens compared with the fixed dosing weekly regimens, regardless of infilsion duration.

FIGS 3-5 show modestly broader exposure bands for the 40 mg/kg and 70 mg/kg -adjusted dosing regimens relative to the equivalent fixed doses (3.6 g and 6.3g, respectively) infused over 2 hours weekly.

Effect ofBody Weight on ApoA-I Exposures by Dosing Regimen For fixed dosing regimens, body weight did not influence total exposure (AUC) while Cmax decreased by an average of 16% with a doubling of body weight from 60 kg to 120 kg (to right panel). In comparison, for weight-adjusted dosing regimens, a doubling of body weight from 60 kg to 120 kg results in a ng of dose and, consistent with linear pharmacokinetic theory, exposure is projected to increase accordingly (to left panel). Thus, body weight had a smaller impact on ApoA-I exposure than that imposed by body weight-adjusted dosing which was more evident at the extremes of the body weight range.

DISCUSSION The results of the simulation analysis suggest that body weight had a smaller impact on ApoA~l exposures than that imposed by body weight-adjusted dosing. Thus, there is expected to be less variability in exposures associated with fixed dosing regimens ed with body weight-adjusted regimens at the extremes of the body weight range.

The modeling and simulation s described herein t the administration of fixed dosing regimens of the rHDL ation in order to reduce variability in ApoA—I exposures over a broad range ofbody weights.

Throughout the cation, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or c collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present ion.

The disclosure of each patent and scientific document, computer program and thm referred to in this specification is incorporated by reference in its entirety.

Table 1: Projected exogenous ApoA—I exposures by dosing regimen in week 4.

Dose Infusion AUCMZ AUCorsa Cmax Accumulation ratio based on firsttlast Dose Unit on(h) Frequency (mg.h/dL) (mg.h/dL) (mg/dL) AUCW; AUCmss Cmax 1437(24) 1952.4(343) 38.8(1s.7) 2732.9(24.9) 3787.5(35.1) 79.1(15) 4102.5(25.2) 5804.3(351) 320.4(154) ; 5371.8(25.6) 7608.1(3s.6) 16.2) i 2859.4 (34.1) 3977.5 (43.7) 82.2 (25.1) 4845.1(34) 6770.2(43.1) 144.1(24.7) . (35.1) 9638.6(44.6] 49) f 1423.2(24.9) 1892.6(35.4) 40.1(16) ' 2757(242) 3840.9(33.9) 81.5(163) 4023.4(25.4) 56721657) 123.2(15.9) » 2810.9(33.4) 3896.2(42.8) 84.6 (24.1) 4900.7(342) 6910.7 (43.2) 148.3(24.4) 6937.5(34.9) 9743.4(44.6) 214.8(24.1) : 184594334) 3885.1(35) 473(139) 3575.7 (34) 7498.4 (35.8) 96.8 (19.4) 5320.6(33.5 11127.8 35.3) 18453834) 3884.6(35) 48.6(19.6) (34) 7497.7(35.8) 99.3(19.1) 5320.9 (33.5) 11125.5(3s.3) lSO.4(18.7| Values are mean (%CV) and reflect a typical subject (body weight = 90 kg, CLCR = 80 ml/min) and ed across males and females. QW : weekly dosing, BW = twice weekly dosing

Claims (19)

1. A method of producing a reconstituted high density lipoprotein (rHDL) formulation, which includes the steps of combining Apo A—l or a fragment thereof, one or more olipids and optionally a detergent at a molar ratio of Apo A-I to phospholipid of 1:20 to 1:120 and determining a fixed dosage of the rHDL formulation that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and that displays relatively reduced inter~patient variability in exposure to the Apo A-I constituent of the formulation compared to that which would be observed or associated with a 10 weight-adjusted dosage regime.

2. An rHDL formulation produced according to the method of Claim 1.

3. The rHDL formulation according to Claim 2, which ses an amount of apolipoptotein in a range selected from: 0.1-15g; 1-10g; 2-9g; 3-8g; 4-7g; and 5-6g apolipoprotein. 15

4. The rHDL formulation of Claim 3, which comprises an amount of apolipoprotein selected from: 0.25g; 0.5g; lg; 1.7g; 2g; 3.4g; 4g; 5.1g; 6g; 6.8g; and 8g apolipoprotein.

5. An rHDL formulation comprising 0.1 to 0.5 g Apo A-I or a fragment f, one or more phospholipids and optionally a detergent at a molar ratio of 20 Apo A~I to phospholipid of 1:20 to 1:120 that is therapeutically effective upon administration to a human of any body weight in a body weight range 60-140 kg and ys relatively reduced inter-patient ility in exposure to the Apo A-I compared to that which would be observed or ated with a weight-adjusted dosage . 25

6. The rl-IDL formulation of any one of Claims 2-5, wherein the variability is less than 90% of that observed or associated with the weight-adjusted dosage regime.

7. The rHDL formulation according to any one of Claims 2-6, n the amount or concentration of one or more phospholipids and detergent is ed to 30 reduce liver toxicity.

8. The rHDL formulation according to any one of Claims 2—7, wherein the Apo A-I is purified from plasma, the phospholipid is phosphatidylcholine, and the rHDL formulation further comprises sodium cholate detergent.

9. The rHDL formulation according to Claim 8, wherein the ratio between the Apo A—1 and the one or more phospholipids is from 1:40 to 1:75 (molzmol), and the sodium e is present at a concentration of 0.5 to 0.9 g/L.

10. The rHDL formulation according to any one of Claims 2—7, wherein the one or more phospholipids is a mixture of sphingomyelin and phosphatidylglycerol.

11. The rHDL formulation according to Claim 10, wherein the ratio between the Apo A-I and the one or more phospholipids is between 1:80 and 1:120 (molzmol) and the sphingomyelin and the phosphatidylglycerol are present in a 10 ratio from 90:10 to 99:1 (w:w).

12. A kit comprising one or more unit doses of an rHDL formulation according to any one of Claims 2-1 1; and one or more other kit components.

13. The kit of Claim 12, wherein said one or more other kit components e instructions for use; vials, containers or other storage vessels containing 15 each of the unit doses; one or more delivery devices such as needles, catheters, syringes, tubing and the like; and/or packaging suitable for safely and conveniently storing and/or transporting the kit.

14. The kit of Claim 12 or Claim 13, for use in therapeutically or prophylactically treating a e, disorder or ion including cardiovascular 20 disease, holesterolaemia or hypocholesterolaemia.

15. The kit of Claim 14, wherein the disease, disorder or condition includes acute coronary syndrome (ACS), atherosclerosis, unstable angina pectoris, and myocardial tion.

16. An rHDL formulation according to any one of Claims 2-11 for use in 25 therapeutically or prophylactically treating a disease, disorder or condition including cardiovascular disease, hypercholesterolaemia or hypocholesterolaemia.

17. The rHDL formulation for use according to Claim 16, wherein the disease, disorder or condition es acute coronary syndrome (ACS), sclerosis, unstable angina pectoris, and dial infarction. 30

18. Use of Apo A-I or a nt thereof, one or more phospholipids and optionally a detergent in the manufacture of an rHDL formulation according to any one of Claims 2-11, for therapeutically or prophylactically ng a disease, disorder or ion including cardiovascular disease, hypercholesterolaemia or hypocholesterolaemia.

19. Use ing to Claim 18, wherein the disease, disorder or condition includes acute coronary syndrome (ACS), atherosclerosis, unstable angina pectoris, and myocardial infarction. 1I9 , June 8n Emma 8~ Time after Dose, h June 8». .250 8“ 8.85 8w .50... a: time after Dose. h '