WO2016059149A1 - Procédé de test d'une libération systémique d'azd1152 hqpa ou de ses bioéquivalents - Google Patents

Procédé de test d'une libération systémique d'azd1152 hqpa ou de ses bioéquivalents Download PDF

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Publication number
WO2016059149A1
WO2016059149A1 PCT/EP2015/073865 EP2015073865W WO2016059149A1 WO 2016059149 A1 WO2016059149 A1 WO 2016059149A1 EP 2015073865 W EP2015073865 W EP 2015073865W WO 2016059149 A1 WO2016059149 A1 WO 2016059149A1
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azd1152 hqpa
sample
nanoparticle
metabolite
borne
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PCT/EP2015/073865
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English (en)
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Michael Walker
Colin Howes
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Astrazeneca Ab
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Publication of WO2016059149A1 publication Critical patent/WO2016059149A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Definitions

  • therapeutics that include an active agent distributed preferentially to a specific diseased tissue more than to normal tissue, may increase the exposure of the drug in those tissues over others in the body. This is particularly important when treating a condition such as cancer where it is desirable that a cytotoxic dose of the drug is delivered to cancer cells without killing the surrounding non-cancerous tissue. Effective drug distribution may reduce the undesirable and sometimes life threatening side effects common in anticancer therapy. In addition, such therapeutics may allow drugs to reach certain tissues they would otherwise be unable to reach.
  • Therapeutics offering controlled release of the active ingredient can also be advantageous.
  • One way of achieving targeted tissue distribution and controlled release can be by formulating the active ingredient as a nanoparticulate formulation, although the relative contributions of differential distribution and controlled release, the administration routes and the specifics of any particular formulation may vary between different tumour types.
  • Cancer (and other hyperproliferative disease) is characterised by uncontrolled cellular proliferation. This loss of the normal regulation of cell proliferation often appears to occur as the result of genetic damage to cellular pathways that control progress through the cell cycle.
  • Aurora Kinases encode cell cycle regulated serine-threonine protein kinases (summarised in Adams et al., 2001, Trends in Cell Biology.11(2): 49-54). These show a peak of expression and kinase activity through G2 and mitosis and a role for human Aurora kinases in cancer has long been implicated.
  • the Aurora Kinase inhibitor known as AZD1152 (2-(ethyl(3-((4-((5-(2-((3- fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)amino)quinazolin-7- yl)oxy)propyl)amino)ethyl dihydrogen phosphate), pictured below, was first disclosed in International Patent Application WO2004/058781 (Example 39) and has been studied by AstraZeneca as a potential treatment for various cancers.
  • AZD1152 is metabolized in vivo to a compound known as
  • AZD1152 hqpa (2-(3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4- yl)amino)-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide), also disclosed in
  • AZD1152 hqpa is in fact, to a large extent, the moiety exerting the biological effect when AZD1152 itself is administered.
  • the therapeutic nanoparticles described in this PCT application comprise about 50 to about 99.75 weight percent of a diblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly(ethylene)glycol, and about 0.2 to about 30 weight percent of AZD1152 hqpa or a pharmaceutically acceptable salt thereof, particularly AZD1152 hqpa.
  • a poly(lactic) acid- poly(ethylene)glycol copolymer having a number average molecular weight of about 15kDa to about 20kDa poly(lactic acid) and a number average molecular weight of about 4kDa to about 6kDa poly(ethylene)glycol is described, for example, a poly(lactic) acid- poly(ethylene)glycol copolymer having a number average molecular weight of about 16kDa poly(lactic acid) and a number average molecular weight of about 5kDa
  • poly(ethylene)glycol is exemplified.
  • the nanoparticles exemplified in the PCT application also include a substantially hydrophobic acid, which may form a hydrophobic ion pair in the nanoparticle with the AZD1152 hqpa.
  • a substantially hydrophobic acid which may form a hydrophobic ion pair in the nanoparticle with the AZD1152 hqpa.
  • Many suitable hydrophobic acids are described, with exemplified formulations including deoxycholic acid, cholic acid, dioctyl sulfosuccinic acid (i.e., docusate acid), oleic acid and pamoic acid.
  • One of the approaches to be used has been to provide drug-bearing nanoparticles to a subject, and then, after some time has elapsed, to attempt to measure the concentration of “free” drug in a sample from the subject.
  • Such release may either occur gradually, for example through diffusion of the drug molecule out of the nanoparticle, or through degradation which causes“leaks” from nanoparticles, or suddenly, if conditions cause numerous nanoparticles to rupture. Release of this sort may occur within a sample waiting to be analysed or during the process of analysis.
  • An additional factor may be the low solubility of AZD1152 hqpa.
  • Pluim et al J Chromatography B, 877 (2009), 3549-3555 describes a reverse phase liquid chromatography method for simultaneous determination of AZD1152 and AZD1152 hqpa in human and mouse plasma and mouse tissues.
  • Stern et al J Controlled Release, 172 (2013), 558-567) describes a comparative pharmacokinetic study of three docetaxel (including prodrug) related formulations, including a nanoscale micellar formulation, in bile duct cannulated rats.
  • a method of measuring the amount of AZD1152 hqpa released systemically within a subject after administration of nanoparticles containing AZD1152 hqpa comprising:
  • a release profile for nanoparticle-borne AZD1152 hqpa in a biological system with capacity to metabolise the AZD1152 hqpa comprising:
  • a method of assessing bioequivalence for nanoparticle-borne AZD1152 hqpa administered to a subject comprising: ⁇ assaying a blood-derived sample from the subject to determine an amount of a metabolite of the nanoparticle-borne AZD1152 hqpa present in the sample;
  • ⁇ comparing a value based upon the amount of the nanoparticle-borne AZD1152 hqpa released systemically within the subject with a comparison value in respect of AZD1152 hqpa released from a second drug product and, optionally in combination with other comparative experimental parameters, thereby assessing the bioequivalence of the nanoparticle-borne AZD1152 hqpa and the second drug.
  • determination of the levels of the metabolite of AZD1152 hqpa at various time points provides an exposure profile of the metabolite. Direct comparison of such an exposure profile with the metabolite exposure profile from another drug product could be used as part of a bioequivalence assessment, without the need to correlate the metabolite levels with levels of released AZD1152 hqpa.
  • a method of assessing bioequivalence for nanoparticle borne AZD1152 hqpa administered to a subject comprising: ⁇ assaying a blood-derived sample from the subject to determine an amount of a metabolite of the nanoparticle-borne AZD1152 hqpa present in the sample;
  • a method of determining an exposure profile for a metabolite of AZD1152 hqpa in nanoparticle-borne AZD1152 hqpa in a biological system with capacity to metabolise the AZD1152 hqpa comprising:
  • Figure 1 is a graph setting out the observed and predicted plasma concentrations of prodrug in plasma samples produced in an experimental rat model following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h.
  • Figure 2 is a graph setting out the observed and predicted plasma concentrations of active drug in plasma samples produced in an experimental rat model following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h.
  • Figure 3 is a graph setting out the observed and predicted plasma concentrations of active drug within the nanoparticles in plasma samples produced in an experimental rat model following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h.
  • Figure 4 is a graph setting out the observed and predicted plasma concentrations of metabolite in plasma samples produced in an experimental rat model following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h.
  • the disclosure herein is, at least in part, based upon the inventors’ finding that the difficulties associated with measuring the systemic release of drugs, such as AZD1152 hqpa, borne in nanoparticles, may be overcome by measuring the level of a metabolite of the drug in a sample from a subject to whom the drug has been administered. This method of indirect measurement provides a great number of benefits over the techniques that have previously been described.
  • Methods described herein take into consideration the amount of AZD1152 hqpa which was actually released systemically by the nanoparticles within a subject, since it is only this portion of the drug that is able to be processed to yield the metabolite that is assessed in the methods of the invention.
  • the pharmacokinetic model applied takes this into consideration, and thereby allows an accurate determination of the AZD1152 hqpa that has been systemically released (released and become systemically available).
  • the methods described herein also avoid the problems associated with handling of nanoparticles, or sensitivity of measurement techniques, that have caused difficulties in previous approaches. Even in the event that the AZD1152 hqpa bearing nanoparticles ruptures or leak this will not cause contamination leading to inaccuracies, since is not the drug itself that is being measured, but its metabolite. Similarly, there is no need to identify measurement techniques that provide suitable accuracy in respect of both high and low concentrations of a compound, since it is only necessary to use a technique that is able to accurately measure relevant concentrations of the metabolite.
  • the methods described herein are particularly convenient for use in assessing bioequivalence as they may be carried out on biological samples such as blood or plasma, as described herein, which are easily (and repeatedly) obtained from a patient. It will be understood by the skilled person, as described hereinafter, that additional tests which compare other aspects of a nanoparticle- borne AZD1152 hqpa formulation with a potentially bioequivalent formulation, may be required to demonstrate bio-equivalence to the standard required by regulatory agencies controlling registration of medicines, such as the US Food and Drug Administration.
  • references herein to a“bio-equivalent version” of a composition is intended to refer to a version of the composition which has, or could be, granted marketing authorization by a regulatory body on the basis of being bio-equivalent (ie having the same biological effect) to the composition.
  • a nanoparticle may be considered to be any particle, with a size between 1 and 150nm, such as between 1 and 130, such as between 50 and 130nm, such as between 1 and 100 nm, that may be used to encapsulate AZD1152 hqpa.
  • a nanoparticle may be considered to be any particle which is capable of behaving in a bioequivalent fashion to the nanoparticles exemplified herein.
  • potentially bioequivalent particles may release AZD1152 hqpa at a release rate comparable to the nanoparticles exemplified herein.
  • a nanoparticle for the purposes of the present disclosure may be formed of a material selected from the group consisting of: a diblock poly(lactic) acid-poly(ethylene)glycol copolymer; and a diblock poly(lactic acid-co-glycolic acid)- poly(ethylene)glycol copolymer.
  • a nanoparticle encompassing AZD1152 hqpa may encompass the drug in combination with another compound or compounds, or substantially on its own.
  • AZD1152 hqpa may be combined with another compound that forms a hydrophobic ion pair with the AZD1152 hqpa.
  • the AZD1152 hqpa may be combined with a substantially hydrophobic acid within the nanoparticles.
  • a hydrophobic acid may be selected from the group consisting of: deoxycholic acid; cholic acid; dioctyl sulfosuccinic acid; oleic acid; and pamoic acid.
  • a method of the invention comprises a step of administering nanoparticles containing AZD1152 hqpa to a subject. Such administration may use any suitable route.
  • nanoparticles containing AZD1152 hqpa are administered intravenously to a subject.
  • suitable nanoparticles containing AZD1152 hqpa are those described in WO2015/036792, particularly those formulations exemplified in WO2015/036792, and in particular, formulations containing pamoic acid exemplified in WO2015/036792.
  • suitable nanoparticles include a therapeutic nanoparticle comprising AZD1152 hqpa, pamoic acid, and a diblock poly(lactic) acid-poly(ethylene)glycol copolymer (wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly(ethylene)glycol and the poly(lactic) acid-poly(ethylene)glycol copolymer has a number average molecular weight of about 16kDa poly(lactic acid) and a number average molecular weight of about 5kDa poly(ethylene)glycol).
  • suitable nanoparticles include a therapeutic nanoparticle comprising about 12 to about 25 weight percent of AZD1152 hqpa, about 7 to about 15 weight percent of pamoic acid, and a diblock poly(lactic) acid-poly(ethylene)glycol copolymer (wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent
  • poly(ethylene)glycol and the poly(lactic) acid-poly(ethylene)glycol copolymer has a number average molecular weight of about 16kDa poly(lactic acid) and a number average molecular weight of about 5kDa poly(ethylene)glycol).
  • suitable nanoparticles include a therapeutic nanoparticle comprising about 15 to about 25 weight percent of AZD1152 hqpa, about 7 to about 15 weight percent of pamoic acid, and a diblock poly(lactic) acid-poly(ethylene)glycol copolymer (wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly(ethylene)glycol and the poly(lactic) acid-poly(ethylene)glycol copolymer has a number average molecular weight of about 16kDa poly(lactic acid) and a number average molecular weight of about 5kDa poly(ethylene)glycol).
  • the term“about” means +/- 0.5 for figures ⁇ 10, and means +/- 1 for figures of 10 or above.
  • AZD1152 hqpa is used throughout the present disclosure to refer to the compound (2-(3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5- yl)-N-(3-fluorophenyl)acetamide).
  • a second (or Another) AZD1152 hqpa containing drug (or product) is one that can act as a source of AZD1152 hqpa and is not necessarily restricted to another
  • nanoparticulate formulation provided that it releases AZD1152 hqpa and which may spontaneously leak AZD1152 hqpa during sample handling or analysis for example polymer conjugates or dendrimer.
  • Nanoparticle-borne AZD1152 hqpa may be said to be“released systemically” for the purposes of the present disclosure if the AZD1152 hqpa has been released from nanoparticles within the subject, so that the AZD1152 hqpa is then capable of metabolism by the subject. It will be appreciated that AZD1152 hqpa that has not been released from nanoparticles cannot be said to have been systemically released for the purposes of this disclosure. Nor can AZD1152 hqpa that has been released from nanoparticles, but in circumstances, such as in a location, where it is not available to be metabolised by the subject. “A subject”
  • a subject for the purposes of the present disclosure, may be any animal to whom nanoparticle-borne AZD1152 hqpa has been administered.
  • the subject is a human.
  • a suitable human subject may be an individual taking part in clinical trials investigating properties, such as bioavailability, of the nanoparticle-borne AZD1152 hqpa.
  • a suitable human subject may be an individual undergoing treatment with the nanoparticle-borne AZD1152 hqpa.
  • the subject is a non-human animal.
  • the non-human animal may be an animal used in pre-clinical investigation of the nanoparticle-borne AZD1152 hqpa.
  • the non-human animal may be a rodent, such as a rat.
  • the sample from the subject is a sample that provides information representative of metabolism in the subject.
  • the sample will be one capable of retaining a metabolite of AZD1152 hqpa.
  • the sample may be one capable of retaining a metabolite of AZD1152 hqpa such as hqpa desfluoraniline (described further herein, and also referred to as AZ12102238).
  • the sample is a sample taken from the subject.
  • samples may be suited to in vitro or ex vivo analysis.
  • the sample may suitably be selected from the group consisting of: a tissue sample; and a body fluid sample.
  • a suitable example of a body fluid sample may be selected from the group consisting of: a blood sample; a serum sample; a plasma sample; a urine sample; a lymph sample; an interstitial fluid sample; a bile sample; a saliva sample; and a faeces sample.
  • a number of different sample types may be derived from the blood, including blood samples, serum samples, and plasma samples. Each of these sample types may be considered a“blood-derived sample” for the purposes of the present disclosure.
  • the sample is a blood sample.
  • the sample is a plasma sample.
  • the sample is a serum sample.
  • the sample is a urine sample. In a further embodiment, the sample is a faeces sample.
  • a method of the invention may comprise a step of taking one or more samples to be assayed.
  • a first sample may generally be taken after a period has elapsed which is sufficient to allow release and metabolism of the AZD1152 hqpa to an extent which is accurately quantifiable.
  • samples may be taken at one or more of the time points following administration of nanoparticles containing AZD1152 hqpa considered below.
  • AZD1152 hqpa and particular nanoparticle formulation in the particular species as well as the time that AZD1152 hqpa is administered over.
  • AZD1152 hqpa dosed by intravenous bolus to rats or mice as an example a suitable sampling schedule would utilise the following timepoints: 5, 10 and 30 minutes and 1, 3, 6, 24, 30 and 48 hours following dosing of the prodrug, active drug or metabolite drug and 5, 10 and 30 minutes and 1, 6, 24, 72, 168, 240, 336, 432 and 504 hours following dosing of a nanoparticle formulation.
  • Similar time courses for sampling may apply to administration to humans, although sampling may be continued for slightly longer.
  • a suitable sampling schedule may utilise the following timepoints: 5, 10 and 30 minutes and 1, 3, 6, 24, 30, 48 and 72 hours following dosing of the prodrug, active drug or metabolite drug and 5, 10 and 30 minutes and 1, 6, 24, 72, 168, 240, 336, 432, 504 and 672 hours following dosing of a nanoparticle formulation.
  • the skilled person will understand that the above exemplary sampling schedules may be adapted (more / fewer / different times) for convenience and that such variation is within the scope of the methods described herein.
  • a metabolite for the purposes of the present disclosure, should be considered to encompass any compound produced on metabolism of nanoparticle-borne AZD1152 hqpa.
  • any metabolite of AZD1152 hqpa could be used.
  • the inventors have found that certain metabolites have properties that confer notable advantages in methods utilising these metabolites.
  • the metabolite is selected from the group consisting of: 2- (3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5- yl)acetic acid (also referred to as AZD1152 hqpa desfluoroaniline, or AZ12102238);
  • AZD1152 hqpa desfluoroaniline N-acetic acid AZD1152 hqpa N-acetic acid
  • AZD1152 hqpa N-oxide AZD1152 hqpa N-desethyl.
  • the inventors have found that these metabolites of AZD1152 hqpa are formed in quantities that should generally be sufficient for the metabolites to be accurately measured in the methods of the invention.
  • AZD1152 hqpa desfluoroaniline (AZ12102238): 2-(3-((7-(3-(ethyl(2- hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5-yl)acetic acid:
  • AZD1152-hqpa N-oxide N-ethyl-3-[4-[[3-[2-(3-fluoroanilino)-2-oxo-ethyl]-1H-pyrazol-5- yl]amino]quinazolin-7-yl]oxy-N-(2-hydroxyethyl)propan-1-amine oxide:
  • the metabolite is selected from the group consisting of: AZD1152 hqpa desfluoroaniline; and AZD1152 hqpa desfluoroaniline acetic acid.
  • AZD1152 hqpa is formed by either or both human and rat subjects, allowing ready modelling between these important clinical and pre-clinical species.
  • the selected metabolite is one the formation of which is consistent within different patient populations. This provides a notable advantage in terms of the confidence that can be placed in results generated in within patient populations.
  • the selected metabolite is one that is only formed by action of the body on released active AZD1152 hqpa, and so the presence of the metabolite only arises in respect of drug that has been released systemically and metabolised.
  • the selected metabolite is one which exhibits clearance rates within biological systems, and volumes of distribution, that facilitate their collection and accurate analysis.
  • the selected metabolite is one that is readily synthesised, allowing sufficient quantities to be manufactured to produce standard curves that facilitate quantification of the metabolite in a sample.
  • a suitable metabolite for use in the methods of the invention should not be a significant breakdown product of AZD1152 hqpa which may be formed within
  • a suitable metabolite for use in the methods of the invention should not be a significant impurity within the AZD1152 hqpa formulation, otherwise it may be detected without the need for AZD1152 hqpa to be released systemically and metabolised.
  • the metabolite used for the measurement is 2-(3-((7-(3- (ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5-yl)acetic acid, which is also referred to as AZ12102238 or AZD1152 hqpa desfluoroaniline.
  • Suitable processes for making AZD1152 hqpa desfluoroaniline and AZD1152-hqpa N-oxide are set out in the Examples hereinafter. Suitable processes for making the other metabolites described in this section will be readily understood by the skilled person to be analogous to the processes known for making AZD1152 hqpa or AZD1152 hqpa desfluoroaniline, or other similar compounds known in the art, with the application of routine chemistry where required.
  • an isolated synthetic sample of a metabolite of AZD1152 hqpa is a sample of 2-(3-((7- (3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5-yl)acetic acid.
  • said isolated sample is a sample of 2-[5-[[7-[3- [carboxymethyl(ethyl)amino]propoxy]quinazolin-4-yl]amino]-1H-pyrazol-3-yl]acetic acid.
  • said isolated sample is a sample of 2-[ethyl-[3-[4-[[3-[2-(3- fluoroanilino)-2-oxo-ethyl]-1H-pyrazol-5-yl]amino]quinazolin-7- yl]oxypropyl]amino]acetic acid.
  • said isolated sample is a sample of N-(3-fluorophenyl)-2-[5-[[7-[3-(2-hydroxyethylamino)propoxy]quinazolin-4-yl]amino]- 1H-pyrazol-3-yl]acetamide.
  • said isolated sample is a sample of N- ethyl-3-[4-[[3-[2-(3-fluoroanilino)-2-oxo-ethyl]-1H-pyrazol-5-yl]amino]quinazolin-7- yl]oxy-N-(2-hydroxyethyl)propan-1-amine oxide. It will be understood that an isolated synthetic sample is one which has been made outside the human or animal body.
  • a metabolite of AZD1152-hqpa in an analytical method for the measurement of AZD1152 hqpa in a biological sample.
  • the analytical method is an HPLC method as described hereinafter.
  • the metabolite is selected from the group consisting of: AZD1152 hqpa desfluoroaniline; and AZD1152 hqpa desfluoroaniline acetic acid.
  • a metabolite of AZD1152 hqpa in determining an in-vivo release profile of AZD1152 hqpa from a drug product containing AZD1152 hqpa, in a human or non-human animal which has been administered said drug product.
  • the metabolite is selected from the group consisting of: AZD1152 hqpa desfluoroaniline; and AZD1152 hqpa desfluoroaniline acetic acid.
  • an isolated synthetic sample of a metabolite of AZD1152 hqpa in determining an in-vivo release profile of said metabolite from a drug product containing AZD1152 hqpa, in a human or non-human animal which has been administered said drug product.
  • the metabolite is selected from the group consisting of: AZD1152 hqpa desfluoroaniline; and AZD1152 hqpa desfluoroaniline acetic acid. “Assaying a sample for nanoparticle-borne AZD1152 hqpa” or“assaying a sample for a metabolite”
  • the method used in assaying a sample for nanoparticle-borne AZD1152 hqpa, or assaying a sample for a metabolite is a liquid
  • the method used in assaying a sample for nanoparticle- borne AZD1152 hqpa, or assaying a sample for a metabolite is a liquid chromatography-mass spectroscopy (LC-MS) technique.
  • LC-MS techniques may advantageously be used because they are able to distinguish closely related compounds, such as metabolites and the drugs from which they are derived, from one another.
  • LC-MS techniques are also very well suited to the discrimination of compounds such as nanoparticle-borne AZD1152 hqpa or metabolites in complex mixtures of the sort found in samples derived from animal subjects. Details of suitable parameters that may be used in such LC-MS techniques when assaying a sample for AZ12102238 or for AZD1152 hqpa are described further in the Examples of this specification.
  • the method used in assaying a sample for nanoparticle- borne AZD1152 hqpa or assaying a sample for a metabolite is mass spectroscopy (MS) imaging technique.
  • MS imaging techniques to assay a sample for a metabolite or nanoparticle-borne AZD1152 hqpa may be of particular utility in methods of the invention in which the sample is a tissue sample.
  • Methods of the invention may utilise any suitable technique that allows a determination to be made of the amount of nanoparticle-borne AZD1152 hqpa, or a metabolite (such as AZ12102238), within a sample.
  • a method of the invention allows the amount of nanoparticle-borne AZD1152 hqpa or a metabolite in a sample to be determined by a technique in which the sample is compared with a standard curve prepared using a range of concentrations of the AZD1152 hqpa or metabolite.
  • a method of the first aspect may also include a step of assaying a sample from the subject to determine the amount of AZD1152 hqpa present in the sample.
  • the AZD1152 hqpa may be retained within nanoparticles.
  • the amount thus determined may be used in a pharmacokinetic model applied to derive an amount of AZD1152 hqpa released systemically within the subject.
  • the amount thus determined may be used in a population pharmacokinetic model.
  • the sample from which the metabolite is determined and the sample from which the amount of AZD1152 hqpa is determined are derived from the same sample.
  • the sample from which the metabolite is determined and the sample from which the amount of AZD1152 hqpa is determined are different samples.
  • a method of the second or third aspect may also include a step of assaying a sample from the subject to determine the amount of nanoparticle-borne AZD1152 hqpa present in the sample.
  • the nanoparticle-borne AZD1152 hqpa may be retained within nanoparticles.
  • the amount thus determined may be used in a pharmacokinetic model applied to derive an amount of the nanoparticle-borne AZD1152 hqpa that has been released systemically within the subject.
  • the amount thus determined may be used in a population pharmacokinetic model.
  • the second aspect provides a method of determining a release profile for nanoparticle-borne AZD1152 hqpa, the method comprising assaying samples from the biological system at first and second time points to determine the amount of a metabolite present in the samples, and from this deriving first and second values representative of the amount of the nanoparticle-borne AZD1152 hqpa released at the respective time points. These values are then used to produce a release profile.
  • exposure profile means a graph of the amount of metabolite present in a sample over a number of time points.
  • a method of this second aspect may further involve assaying a sample from the biological system at a third, or subsequent, time point to determine an amount of a metabolite of the nanoparticle-borne AZD1152 hqpa present in the sample at this third or subsequent time point.
  • a pharmacokinetic model is then applied to derive a third value representative of an amount of the nanoparticle-borne AZD1152 hqpa released at the third time point, and this third value used in the production of a release profile for the nanoparticle-borne AZD1152 hqpa.
  • Such methods may include fourth, fifth, sixth, seventh, eighth, ninth, tenth, and further samples from corresponding time points, and that these may be used to derive fourth, fifth, sixth, seventh, eighth, ninth, tenth, and further values that may be used in the production of a release profile.
  • a method of the second or fifth aspect may comprise a step of introducing a quantity of nanoparticles containing AZD1152 hqpa to the biological system.
  • the biological system with the capacity to metabolise AZD1152 hqpa may be an in vivo system.
  • an in vivo system will utilise an animal subject, either human or otherwise, as considered elsewhere in the present disclosure.
  • the samples taken from such an in vivo system may be of the sorts, including blood, serum, or plasma samples, already contemplated herein.
  • the biological system with capacity to metabolise AZD1152 hqpa may be an in vitro system.
  • Such an in vitro system may utilise biological cells maintained in culture.
  • the biological cells may be hepatocytes.
  • an in vitro system may use cell fragments, such as liver microsomes; cell fractions, such as the liver S9 fraction; or incubation with enzymes, such as specific enzyme isoforms.
  • a suitable sample taken from the system may comprise medium in which the biological cells are cultured.
  • Methods of the second or fifth aspect may suitably be employed in quality assurance regimes.
  • methods in accordance with the second or fifth aspect of the invention may be used to ensure consistency between different batches of AZD1152 hqpa-bearing nanoparticles.
  • a method of the third aspect may make use of multiple time points upon which samples are taken.
  • the multiple samples generated this way may be used to derive amounts of nanoparticle-borne AZD1152 hqpa released systemically at these various time points, and these amounts used to generate a profile, such as a concentration curve, in respect of the amount of AZD1152 hqpa released.
  • This information regarding the concentration of AZD1152 hqpa over these time points may be used in assessing the bioequivalence of the nanoparticle-borne AZD1152 hqpa and the second drug.
  • a method of the fourth aspect may make use of multiple time points upon which samples are taken, and these amounts used to generate a profile, such as a concentration curve / exposure profile, in respect of the amount of metabolite of AZD1152 hqpa formed.
  • the value based upon the amount of nanoparticle-borne AZD1152 hqpa systemically released in the subject or the amount of metabolite of AZD1152 hqpa, which is compared with the comparison value in respect of the second drug may be selected from the group consisting of: area under the curve (AUC); peak concentration (C max ); and time to peak concentration (T max ).
  • such methods of the invention are performed in respect nanoparticle-borne AZD1152 hqpa that has been administered to one or more subjects at different doses.
  • Comparison values may be generated by practicing the same steps (including assaying a sample from the subject to determine an amount of a metabolite of the nanoparticle-borne AZD1152 hqpa present in the sample, and , for the third aspect, applying a pharmacokinetic model to derive an amount of the nanoparticle-borne
  • AZD1152 hqpa systemically released based upon the determined amount of the metabolite) in respect of the second drug as have been performed in respect of the nanoparticle-borne AZD1152 hqpa.
  • the second drug is also a nanoparticle-borne drug.
  • Subjects for use in methods in accordance with the third or fourth aspect of the invention may be selected with reference to criteria that are conventional in the field of assessing bioequivalence.
  • subjects may be selected with respect to specific groupings on the basis of gender, age, or ethnicity.
  • Suitable approaches may use groups of subjects that are the same as one another with reference to one or more of the criteria considered above, or may use groups that are selected to include a desired level of diversity with respect to the different criteria.
  • any of the first, second, or third aspects, in which the amount of nanoparticle-borne AZD1152 hqpa is determined it may be preferred that relevant amounts are determined in respect of samples collected at two or more time points.
  • Such embodiments of the methods of the invention allow a profile of the concentration over time of the nanoparticle-borne AZD1152 hqpa to be produced. A profile of this sort can then be utilised in the pharmacokinetic model.
  • Such embodiments are useful in that they are able to provide additional information about the release of AZD1152 hqpa prior to metabolism.
  • relevant amounts of metabolite may be determined in respect of samples collected at two or more time points.
  • Such embodiments of the methods of the invention allow a profile of the concentration over time of the metabolite to be produced.
  • the assessment of bioequivalence according to the third or fourth aspect of the invention may require measurement of additional comparative parameters, potentially depending on the type of cancer to be treated. For example by measurement of biomarkers (eg pHH3, see examples in WO2015/036792), exposure levels in target tissue (such as levels in tumour or bone marrow etc), efficacy (response rate), imaging (for example by mass spec), or by measurement of attributes of the nanoparticles (for example particle size by dynamic light scattering, in vitro release (or dissolution) of drug from the nanoparticle).
  • biomarkers eg pHH3, see examples in WO2015/036792
  • exposure levels in target tissue such as levels in tumour or bone marrow etc
  • efficacy response rate
  • imaging for example by mass spec
  • attributes of the nanoparticles for example particle size by dynamic light scattering, in vitro release (or dissolution) of drug from the nanoparticle.
  • Regulatory bodies such as the US Food and Drug Administration may issue guidance on how to assess bioequi
  • nanoparticulate docetaxel which is reproduced in the Reference Example hereinafter.
  • a method of assessing bioequivalence for nanoparticle-borne AZD1152 hqpa administered to a subject suffering from a solid tumour disease comprising:
  • a method of assessing bioequivalence for nanoparticle-borne AZD1152 hqpa administered to a subject suffering from a haematological cancer comprising:
  • ⁇ comparing a value based upon the amount of the nanoparticle-borne AZD1152 hqpa released systemically within the subject with a comparison value in respect of AZD1152 hqpa released from a second drug product and, optionally in combination with one or more additional comparative experimental parameters, thereby assessing the bioequivalence of the nanoparticle-borne AZD1152 hqpa and the second drug.
  • a fourth aspect there is provided a method of assessing bioequivalence for nanoparticle borne AZD1152 hqpa administered to a subject suffering from a solid tumour disease, the method comprising:
  • a method of assessing bioequivalence for nanoparticle borne AZD1152 hqpa administered to a subject suffering from a haematological cancer comprising:
  • a solid tumour disease examples include prostate cancer, gastric cancer, colorectal cancer, skin cancer, e.g., melanomas or basal cell carcinomas, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC)), breast cancer, ovarian cancer, cancers of the head and neck, bronchus cancer, pancreatic cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer of the oral cavity or pharynx, liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), testicular cancer, biliary tract cancer, small bowel or appendix cancer, gastrointestinal stromal tumor, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and the like.
  • a solid tumour disease is selected from NSCLC, SCLC, ovarian cancer and colorectal cancer.
  • haematological cancers include chronic myelogenous leukemia, chronic myelomonocytic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia, mantle cell lymphoma, acute myeloid leukemia, diffuse large B cell lymphoma, myeloma, peripheral T-cell lymphoma and myelodysplastic syndrome.
  • a haemotological cancer is selected from AML, DLBCL and
  • the encapsulated drug in the nanoparticles in the body is treated as though it is a small molecule.
  • the amount of drug that is both in the body and contained within the nanoparticles is treated in the same way a small molecule would be.
  • the clearance of the encapsulated drug that is both in the body and contained within the nanoparticles relates to any process that reduces the amount of active drug that is both in the body and contained within the nanoparticles, this clearance can be thought of as consisting of two groups of mechanisms.
  • the first group of clearance mechanisms are those that result in active drug being released into the body and could include mechanisms such as diffusion from an intact nanoparticle which would leave an intact nanoparticle containing less drug, or break-up of an individual nanoparticle with some or all of the active drug that was inside the nanoparticle being released in to the body leaving fewer drug containing nanoparticles within the body.
  • the second group of clearance mechanisms are those where no active drug is released into the body and could include the loss of an intact drug containing nanoparticles from the body, or a chemical change in intact active drug either while it is contained within a nanoparticle or while the nanoparticle is destroyed.
  • the exemplified combined pharmacokinetic model for the release of the active drug, such as AZD1152 hqpa, from nanoparticles is made up from four sub-models. These sub-models are all standard pharmacokinetic mammillary compartmental models, each of which is composed of a central compartment and a number of peripheral compartments. The central compartments are linked such that a proportion of the clearance of drug that is both in the body and contained within the nanoparticles results in an increase in the amount of active drug within the active drugs central compartment.
  • AZD1152 is a prodrug
  • the prodrug central compartment is linked to the active drug central compartment such that a proportion of the clearance of the prodrug results in an increase in the amount of active drug within the active drugs central compartment.
  • prodrug part of the model is only required for drugs dosed as a prodrug. It will be understood that references in this section to“prodrug” should be taken to mean AZD1152. Similarly a proportion of the clearance of the active drug (AZD1152 hqpa) results in an increase in the amount of metabolite within the central compartment for the metabolite. It will be understood that references in this section to“active drug” should be taken to mean
  • AZD1152 has been included in the model used below because it provides additional data to increase robustness of the model, but in general is not required in order to analyse release of AZD1152 hqpa from the nanoparticles by the methods described herein.
  • the number of peripheral compartments is one for the prodrug, the drug contained in the nanoparticle and the metabolite and two for the active drug, however the exact number of peripheral compartments for each component will depend on the compounds and metabolites in question, the species used and the amount and quality of the data available. The skilled person will be able to determine a suitable number of peripheral compartments without any undue need for experimentation or the application of inventive activity.
  • a suitable model that may be used for AZD1152hqpa in the rat is expressed mathematically below.
  • V n1 is the apparent volume of distribution of the nanoparticle central compartment
  • V n2 is the apparent volume of distribution of the nanoparticle peripheral compartment
  • C n1 is the concentration of drug that is both in the body and contained within the nanoparticles and in the nanoparticle central compartment
  • C n2 is the concentration of drug that is both in the body and contained within the nanoparticles and in the nanoparticle peripheral compartment;
  • CL n is the clearance of drug that is both in the body and contained within the nanoparticles from the nanoparticle central compartment;
  • Q n is the inter-compartmental clearance or flow of drug that is both in the body and contained within the nanoparticles between the nanoparticle central and peripheral compartments;
  • input n is a function describing the input of drug that is both in the body and contained within the nanoparticles resulting from dosing of the nanoparticle formulation;
  • dC n1 /dt and dC n2 /dt are the rates of change with time of C n1 and C n2 respectively.
  • V p1 is the apparent volume of distribution of the prodrug central compartment
  • V p2 is the apparent volume of distribution of the prodrug peripheral compartment
  • C p1 is the concentration of prodrug in the prodrug central compartment
  • C p2 is the concentration of prodrug in the prodrug peripheral compartment
  • CL p is the clearance of prodrug from the prodrug central compartment
  • Q p is the inter-compartmental clearance or flow of prodrug between its central and peripheral compartments
  • Input p is a function describing the input of prodrug resulting from dosing of the prodrug; dC p1 /dt and dC p2 /dt are the rates of change with time of C p1 and C p2 respectively.
  • V a1 is the apparent volume of distribution of the active drug central compartment
  • V a2 is the apparent volume of distribution of the active drug peripheral compartment number 2;
  • V a3 is the apparent volume of distribution of the active drug peripheral compartment number 3;
  • C a1 is the concentration of active drug in the active drug central compartment
  • C a2 is the concentration of active drug in the active drug peripheral compartment number 2
  • C a3 is the concentration of active drug in the active drug peripheral compartment number 3
  • M na is the proportion of the clearance of drug that is both in the body and contained within the nanoparticles that results in active drug being released into the body;
  • M pa is the proportion of the clearance of prodrug that results in active drug being released into the body
  • CL a is the clearance of active drug from the active drug central compartment
  • Q a2 is the inter-compartmental clearance or flow of active drug between its central and the peripheral compartment number 2;
  • Q a3 is the inter-compartmental clearance or flow of active drug between its central and the peripheral compartment number 3;
  • Input a is a function describing the input of active drug resulting from dosing of the active drug
  • dC a1 /dt, dC a2 /dt and dC a3 /dt are the rates of change with time of C a1 , C a2 and C a3 respectively.
  • V m1 is the apparent volume of distribution of the metabolite central compartment
  • V m2 is the apparent volume of distribution of the metabolite peripheral compartment
  • C m1 is the concentration of metabolite in the metabolite central compartment
  • C m2 is the concentration of metabolite in the metabolite peripheral compartment
  • M am is the proportion of the clearance of active drug that results in metabolite being released into the body
  • CL m is the clearance of metabolite from the metabolite central compartment
  • Q m is the inter-compartmental clearance or flow of metabolite between its central and peripheral compartments
  • Input m is a function describing the input of metabolite resulting from dosing of metabolite; dC m1 /dt, and dC m2 /dt are the rates of change with time of C m1 and C m2 respectively.
  • concentration-time data for drug that is both in the body and contained within the nanoparticles together with concentration-time data for at least one of the active drug (AZD1152 hqpa) or the metabolite following intravenous dosing of the nanoparticle;
  • concentration-time data for the prodrug together with concentration-time data for at least one of the active drug or the metabolite following intravenous dosing of the prodrug;
  • concentration-time data for the active drug ie AZD1152 hqpa
  • V m1 /M am is the apparent volume of distribution of the metabolite central compartment over the proportion of the clearance of active drug that results in metabolite being released into the systemic circulation;
  • V m2 /M am is the apparent volume of distribution of the metabolite peripheral compartment over the proportion of the clearance of active drug that results in metabolite being released into the systemic circulation;
  • CL m /M am is the clearance of metabolite from the metabolite central compartment over the fraction of active drug that is metabolised to the metabolite;
  • Q m /M am is the inter-compartmental clearance or flow of metabolite between its central and peripheral compartments over the fraction of active drug that is metabolised to the metabolite.
  • a population modelling approach is used.
  • the parameters for each individual are assumed to come from a statistical distribution which is determined from the data from a large group of individuals.
  • the modelling approach uses information both from the individuals own data set and from the wider population to estimate the most likely set of parameters for that individual, these parameters can then be used to estimate the concentrations of all of the relevant compounds in that individual for that dosing period.
  • the model can be thought of as using the individuals own data to determine the pharmacokinetics of the drug that is both in the body and contained within the nanoparticles. This is then combined with the rates of elimination of drug from the nanoparticles and the rate of appearance of the metabolite, together with the population data for the active drug to estimate the most likely parameter set for the drug that is both in the body and contained within the nanoparticles, the active drug which has been released in the body and the metabolite in that individual.
  • the following provides an exemplary method by which nanoparticle-borne AZD1152 hqpa, or a metabolite of nanoparticle-borne AZD1152 hqpa, may be extracted from a sample, assayed, and the amount of AZD1152 hqpa or metabolite present determined.
  • the method described is a multi step process which should be carried out on ice wherever possible to halt spontaneous release of AZD1152 hqpa drug from the nanoparticles.
  • the production of standard curves is of use in the determination of amounts of the drug or metabolite present in a sample.
  • the metabolite 2-(3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4- yl)amino)-1H-pyrazol-5-yl)acetic acid, also referred to as AZ12102238, can be synthesised in order to yield sufficient quantities for the ready production of standard curves.
  • the starting material (2-(3-((7-(3-chloropropoxy)quinazolin-4-yl)amino)-1H- pyrazol-5-yl)acetic acid) has been described in the literature: Mortlock et al, Discovery, Synthesis, and in Vivo Activity of a New Class of Pyrazolylamino Quinazolines as Selective Inhibitors of Aurora B Kinase; Journal of Medicinal Chemistry (2007), 50(9), 2213-2224.
  • the metabolite may be synthesised by the following method:
  • the product was purified by preparative HPLC ( Column: Phenomenex luna C18, 250 x 77mm x 10 ⁇ m, using decreasingly polar mixtures of water (containing 0.1% TFA) and MeCN as eluents). Most of the water was removed under vacuum. To the solution in H 2 O (70 mL) was added MeCN (200mL), then the mixture was stirred at 0°C for 1h. The resulting precipitate was filtered and washed with MeCN (100mL), then dried under vacuum. Product AZ12102238 (5.10 g, 12.23 mmol, 18.73% yield, 99.4% purity) was obtained as light yellow solid.
  • Standard curves may be produced by automated equipment.
  • the robot will first add suitable diluent into the microplate for the dilutions before serially diluting the stocks from right to left in the microplate, one row per compound (Table 1)
  • LC-MS or MS approaches may be used in the assessment of nanoparticle-borne drugs or their metabolites, and determination of the amounts of these compounds present.
  • the following parameters set out in Tables 3 and 4 may be used in embodiments utilising AZD1152 hqpa, or its metabolite as AZ12102238. 4
  • Example model fit
  • This example model fit is for a rat which was given a single intravenous bolus dose of the active drug, AZD1152 hqpa, at 0 h, a single intravenous bolus dose of the prodrug, AZD1152, at 168 h, and a single intravenous bolus dose of the active drug in a
  • nanoparticle formulation at 336 h.
  • the plasma concentrations of the active drug and of the metabolite were measured at a number of time points.
  • the plasma concentrations of the prodrug were measured at a number of time points.
  • the plasma concentrations of the active drug in the nanoparticle and of the metabolite were measured at a number of time points.
  • the parameters shown are for an individual rat, but the parameters were derived from a population pharmacokinetic model built using 3600 data points from 850 rats with a variety of dosing regimen for each of the prodrug, the active drug, the metabolite and the active drug encapsulated in a nanoparticle formulation.
  • Brij®100 Brij®S 100 surfactant is a commercially available polyoxyethylene
  • Tween®80 A commercially available polyoxyethylene sorbitan monooleate, also known as polysorbate 80, CAS number 9005-65-6
  • Span®80 A commercially available sorbitan monooleate, CAS number 1338- 43-8 “polymer-PEG” means PLA-PEG co-polymer where the co-polymer has a number average molecular weight of about 16kDa poly(lactic acid) and a number average molecular weight of about 5kDa poly(ethylene)glycol.
  • polymers are commercially available or may be made by methods known in the art. Such polymers are used for example in
  • Nanoparticles of AZD1152 hqpa with pamoic acid were prepared having the composition as set out in Table 5. 4.2.1 Preparation of pamoic acid solution.
  • a 29% (w/w) solution of pamoic acid in DMSO was prepared by mixing 2.9 g of pamoic acid with 7.1 g of DMSO in a container. The container was heated in a heating oven at 70-80 o C until all of the pamoic acid was dissolved.
  • Trifluoroacetic acid (TFA) (3.2 g), deionized (DI) water (3.0 g), and benzyl alcohol (BA) (33.8 g) were combined to prepare the 8% TFA/7.5% water/84.5% benzyl alcohol (wt%) solution.
  • Diafilter ⁇ 20 diavolumes (4 liter) using cold DI water.
  • formulation G2 Another pamoic acid formulation, referred to hereinafter as formulation G2 was prepared as follows, using a nominal 1g batch, and with the composition as shown in Table 5a:
  • formulation G2 was prepared as follows, using a nominal 1g batch, and with the composition as shown in Table 5a:
  • Example 4.2b
  • pamoic acid solution A 29% (w/w) solution of pamoic acid in DMSO was prepared by mixing 2.9 g of pamoic acid with 7.1 g of DMSO in a container. The container was heated in a heating oven at 70-80 o C until all of the pamoic acid was dissolved.
  • Trifluoroacetic acid (TFA) (3.2 g), deionized (DI) water (3.0 g), and benzyl alcohol (BA) (33.8 g) were combined to prepare the 8% TFA/7.5% water/84.5% benzyl alcohol (wt%) solution.
  • Diafilter ⁇ 20 diavolumes (4 liter) using cold DI water.
  • the rat was dosed by intravenous bolus with the following compounds at the times listed in Table 7.
  • the observed and predicted plasma concentrations of active drug within the nanoparticles following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h are shown in Figure 3.
  • the observed and predicted plasma concentrations of metabolite following an active drug dose at 0 h, a prodrug dose at 168 h and an active drug in nanoparticle formulation at 336 h are shown in Figure 4.
  • the pharmacokinetic behaviour of the active drug is assumed to be unaltered by the route of administration (i.e. active drug dosed by an intravenous bolus is taken to behave in the same way as drug formed from the prodrug or released from the nanoparticle formulation).
  • the pharmacokinetic behaviour of the metabolite is assumed to be unaltered by the route of administration (i.e. metabolite formed from active drug which has been given by an intravenous bolus behaves in the same way as metabolite formed from active drug formed from the prodrug or formed from active drug released from the nanoparticle formulation).
  • the concentration-time profile for the active drug following nanoparticle dosing without having measurements of the released active drug, as long as the input profile of the released active drug into the body is known.
  • the input profile is assumed to be by first order release from the active drug contained in the nanoparticle (i.e. the rate is proportional to the amount of active drug still in the nanoparticles).
  • the metabolite concentration-time profile observed after dosing the nanoparticle formulation provides information about the extent of the release of active drug from the nanoparticle combined with the extent of the formation of the metabolite from the active drug. Since information about the extent of formation of the metabolite from the active drug has been obtained from both the concentration-time profiles following dosing of the active drug and of the prodrug, it is then possible to infer the extent of release of the active drug from the nanoparticle formulation.
  • concentration of the prodrug over time is not needed (although it can provide useful information for the model). It is, however, necessary to know how the plasma concentrations of the active drug and metabolite change following dosing of the active drug. As discussed elsewhere in this specification, having information regarding how plasma concentration changes with time following dosing of the metabolite may be helpful to accurate determination of the amount of active drug released systemically within the subject, but this is not essential.
  • Information regarding the concentrations of the drug in the nanoparticles is necessary in order to provide a rate of input.
  • An advantage of using all the other information in a population model is that the parameters that the model uses for the fit of the data generated from an individual rat uses information both from this individual as well as a much larger data set from a number of individuals. In the present example it is not overly important to have the information from the whole population as information following dosing of both the active drug and the nanoparticle formulation is available for this individual. That said, normally a rat would only be dosed with the nanoparticle formulation.
  • the predicted concentrations of active drug released can then be used to build pharmacokinetic-pharmacodynamic (pkpd) models to link the dosing of the nanoparticle-encapsulated drug with resultant biological changes (such as changes in biomarker levels, or toxicity, or changes in tumour growth inhibition).
  • Table 1 showing the 11 dilutions from right to left (columns 12-1) of the dilution plate for a 2mM starting stock in column 12 of the dilution microplate.
  • Table 2 Table demonstrating the calibration curve generated following the spiking of the robot-generated dilution series.
  • AZD1152 hqpa N-oxide N-ethyl-3-[4-[[3-[2-(3-fluoroanilino)-2-oxo-ethyl]-1H-pyrazol-5- yl]amino]quinazolin-7-yl]oxy-N-(2-hydroxyethyl)propan-1-amine oxide
  • N-ethyl-3-[4-[[3-[2-(3-fluoroanilino)-2-oxo-ethyl]-1H-pyrazol-5- yl]amino]quinazolin-7-yl]oxy-N-(2-hydroxyethyl)propan-1-amine oxide may be made as follows:
  • ⁇ are manufactured by an active liposome loading process with an ammonium sulfate gradient and
  • ⁇ have equivalent liposome characteristics including liposome composition, state of encapsulated drug, internal environment of liposome, liposome size distribution, number of lamellar, grafted PEG at the liposome surface, electrical surface potential or charge, and in vitro leakage rates. The following clinical and in vitro studies are recommended to demonstrate
  • Subjects Ovarian cancer patients whose disease has progressed or recurred after platinum- based chemotherapy and who are already receiving or scheduled to start therapy with the reference listed drug (RLD) or the reference standard product.
  • RLD reference listed drug
  • Doxorubicin is a cytotoxic drug. Therefore, a Bio-IND is required for
  • the two arms of the crossover study are to be conducted on two of the days when the patients are scheduled to receive their usual therapy so that the treatment regimen is not altered or delayed.
  • the standard of care treatment regimen should not be altered except to randomize the patients to the test or reference therapy on the specified dosing days.
  • ⁇ Patient is ⁇ 18 years of age or > 75 years of age.
  • the sponsor can provide a non- high-fat diet during the proposed study.
  • the treatment can be initiated 2 hours after a standard (non-high-fat) breakfast.
  • a generic doxorubicin HCl liposome injection must be qualitatively and quantitatively the same as the RLD or reference standard, except differences in buffers, preservatives and antioxidants provided that the applicant identifies and characterizes these differences and demonstrates that the differences do not impact the safety/efficacy profile of the drug product.
  • FDA has no recommendations for the type of studies that would be needed to demonstrate that differences in buffers, preservatives and antioxidants do not impact the safety/efficacy profile of the drug product.
  • Lipid excipients are critical in the liposome formulation. ANDA sponsors should obtain lipids from the same category of synthesis route (natural or synthetic) as found in the RLD or reference standard. Information concerning the chemistry, manufacturing and control of the lipid components should be provided at the same level of detail expected for a drug
  • ANDA sponsors should have specification on lipid excipients that are similar to those used to produce the RLD or reference standard. Additional comparative characterization (beyond meeting specifications) of lipid excipients including the distribution of the molecular species should be provided.
  • an ANDA sponsor would be expected to use an active loading process with an ammonium sulfate gradient.
  • the major steps include 1) formation of liposomes containing ammonium sulfate, 2)) liposome size reduction, 3) creation of ammonium sulfate gradient, and 4) active drug loading.
  • An active loading process uses an ammonium sulfate concentration gradient between the liposome interior and the exterior environment to drive the diffusion of doxorubicin into the liposomes 2, 3 .
  • in vitro liposome characterization should be conducted on at least three batches of the ANDA and the RLD or reference standard products (at least one ANDA batch should be produced by the commercial scale process and used in the in vivo bioequivalence study). Attributes that should be included in the characterization of ANDAs claiming equivalence to the RLD or reference standard are:
  • Liposome composition including lipid content, free and encapsulated drug, internal and total sulfate and ammonium concentration, histidine concentration, and sucrose concentration should be measured.
  • the drug- to-lipid ratio and the percentage of drug encapsulation can be calculated from liposome composition values.
  • the doxorubicin in the RLD or reference standard is largely in the form of a doxorubicin sulfate precipitate inside the liposome.
  • the generic doxorubicin HCl liposome must contain an equivalent doxorubicin precipitate inside the liposome.
  • the internal environment of the liposome including its volume, pH, sulfate and ammonium concentration, maintains the precipitated doxorubicin.
  • the measurements of total and free concentrations of components (including sulfate ions) described in liposome composition section allow the inference of the internal concentration inside the liposome.
  • Liposome morphology and number of lamellae Liposome morphology and number of lamellae
  • lamellarity should be determined as drug loading, drug retention, and the rate of drug release from the liposomes are likely influenced by the degree of lamellarity.
  • ⁇ Lipid bilayer phase transitions Equivalence in lipid bilayer phase transitions will contribute to demonstrating equivalence in bilayer fluidity and uniformity.
  • the phase transition profiles of the raw lipid excipients and liposomes should be comparable to those of the RLD or reference standard.
  • Liposome size distribution is critical to ensuring
  • the ANDA sponsor should select the most appropriate particle size analysis method to determine the particle size
  • the number of liposome product vials to be studied should not be fewer than 30 for each of the test and reference products (i.e., no fewer than 10 from each of three batches). See recommended study 2 (above) for details of the recommended statistical equivalence tests.
  • the surface-bound methoxypolyethylene glycol (MPEG) polymer coating protects liposomes from clearance by the mononuclear phagocyte system (MPS) and increases blood circulation time.
  • MPEG mononuclear phagocyte system
  • the PEG layer thickness is known to be thermodynamically limited and estimated to be in the order of several nanometers. The PEG layer thickness should be determined.
  • Liposome surface charge can affect the clearance, tissue distribution, and cellular uptake. Liposome surface charge should be measured.
  • a Bio-IND is required to conduct bioequivalence studies of doxorubicin liposome injection in humans since doxorubicin is a cytotoxic drug.
  • Sponsors should measure both liposome-encapsulated and free doxorubicin to demonstrate the same in vivo stability of generic liposome formulation and the RLD or reference standard. The studies may be conducted under either fasted or standard diet conditions depending on patient needs. See recommended study 1 (above) for details of the recommended statistical equivalence tests.
  • a method of measuring the amount of AZD1152 hqpa released systemically within a subject after administration of nanoparticles containing AZD1152 hqpa comprising:
  • a method of determining a release profile for nanoparticle-borne AZD1152 hqpa in a biological system with capacity to metabolise the AZD1152 hqpa comprising: ⁇ assaying a sample from the biological system at a first time point to determine a first amount of a metabolite of the nanoparticle-borne AZD1152 hqpa present in the sample;
  • a method of assessing bioequivalence for nanoparticle-borne AZD1152 hqpa administered to a subject comprising:
  • AZD1152 hqpa desfluoroaniline also referred to as AZ12102238
  • AZD1152 hqpa desfluoroaniline N-acetic acid AZD1152 hqpa N-acetic acid
  • AZD1152 hqpa N-oxide AZD1152 hqpa N-desethyl.
  • nanoparticle is formed of a material selected from the group consisting of: a diblock poly(lactic) acid- poly(ethylene)glycol copolymer; and a diblock poly(lactic acid-co-glycolic acid)- poly(ethylene)glycol copolymer.
  • hydrophobic acid is selected from the group consisting of: deoxycholic acid; cholic acid; dioctyl sulfosuccinic acid; oleic acid; and pamoic acid.
  • the sample is selected from the group consisting of: a tissue sample; and a body fluid sample.
  • the body fluid sample is selected from the group consisting of: a blood sample; a serum sample; a plasma sample; a urine sample; a lymph sample; an interstitial fluid sample; a bile sample; a saliva sample; and a faeces sample.
  • a method according to any preceding aspect further comprising a step of assaying a sample from the subject to determine the amount of nanoparticle-borne AZD1152 hqpa in the sample.
  • the method comprising a further step of assaying one or more further samples from the subject and thereby deriving an amount of the nanoparticle-borne AZD1152 hqpa present in the further samples.
  • the value in respect of the amount of the nanoparticle-borne AZD1152 hqpa compared with the comparison value in respect of the second drug is selected from the group consisting of: area under the curve (AUC); peak concentration (C max ); and time to peak concentration (T max ).

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Abstract

La présente invention concerne des procédés de mesure de la libération systémique ou du profil de libération in vivo d'un médicament actif, par exemple AZD1152 (barasertib), à partir d'une nanoparticule, et leur utilisation, par exemple dans l'évaluation de la bioéquivalence. Ledit procédé comprend la détection d'un métabolite d'AZDI 152 hqpa (par exemple : l'AZD1152 hqpa desfluoroaniline (également appelé AZ12102238); l'acide AZD1152 hqpa desfluoroaniline N-acétique; l'acide AZD1152 hqpa N-acétique; l'AZD-1152 hqpa N-oxyde; et l'AZD 1152 hqpa N-déséthyline) dans un échantillon au moyen de la spectrométrie de masse (LC-MS) et par l'application d'un modèle pharmacocinétique.
PCT/EP2015/073865 2014-10-17 2015-10-15 Procédé de test d'une libération systémique d'azd1152 hqpa ou de ses bioéquivalents WO2016059149A1 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
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BARRY SIMON T ET AL: "AZD1152-hQPA Accurins: Nanoparticle formulations showing extended release and the potential for improved therapeutic index", CANCER RESEARCH, vol. 74, no. 19, Suppl. S, 5409, 1 October 2014 (2014-10-01), & 105TH ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-CANCER-RESEARCH (AACR); SAN DIEGO, CA, USA; APRIL 05 -09, 2014, XP055238595, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2014-5409 *
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