Pharmacokinetics and metabolism in early phase clinical trials · 2014-04-01 · • 5F203 (Pro-drug Phortress) • Novel cancer drug • Induces CYP1A family of enzymes • Activates

Newcastle Cancer Centre at the Northern Institute for Cancer Research

Pharmacokinetics and metabolism in early phase clinical trials

Alan Boddy

Phase I studies in oncology

•  Key stage between pre-clinical and therapeutic use

•  Endpoints – Safety – Phase II dose – PK/PD –  (Response)

Context - Phase I study

•  Informed by pre-clinical data •  First in human, in patients •  Inclusion criteria

– No appropriate treatment – Life expectancy – Performance status – Organ function

•  Exclusion criteria –  Interacting drugs.

Phase I case study •  5F203 (Pro-drug Phortress) •  Novel cancer drug •  Induces CYP1A family of enzymes •  Activates own metabolism to form DNA-adducts •  Mechanism of action informed design of toxicokinetic studies

–  IV administration on days 1 and 8

6 Current Medicinal Chemistry, 2004, Vol. 11, No. 1 Bradshaw and Westwell

Fig. (10). DNA adducts analyses of Phortress in vitro and in vivo.

No Net Uptake

InsensitiveTumour Cell

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AhR ligand

Fig. (11). Summary of Phortress mode of action.

CYP1A1 expression was not induced in mice exposed to thesame dose of Phortress.

In mice bearing MCF-7 and MDA-MB-435 tumours inopposite flanks, only MCF-7 tumours demonstratedinducible CYP1A1 protein and Phortress-derived adductgeneration. These observations raise the possibility thatmonitoring CYP1A1 expression and DNA adduct formationin human tumours is achievable and may provide abiomarker for the identification of sensitive tumourphenotypes, since generation of Phortress-derived adducts invitro concurs with sensitivity of xenografts to Phortress(Figure (10)). Co-elution of DNA extracted from MCF-7cells exposed to 5F 203 (1 µM) and MCF-7 xenograft tissuerecovered from mice exposed to Phortress (20 mg/kg)demonstrated generation of identical adduct species in vitro

and in vivo [18].

Clinical evaluation of Phortress, which demonstratespotent, selective antitumour activity via a novel mechanismof action, provides the laboratory investigator with anexcellent opportunity to examine whether the biochemicalmechanisms underlying sensitivity in experimental tumourstranslates to tumours endured by humans. Examination ofCYP1A1 protein expression and Phortress derived DNAstrand breaks and adduct formation in tumour biopsysamples may reveal correlations between these putativepharmacodynamic endpoints and tumour response and thuswhether sensitivity to Phortress may be predicted.

Our understanding of the mechanism of action ofPhortress is summarised in Figure (11).

Preclinical PK •  Prodrug and “active” metabolite •  Mouse data, 20mg/kg

Preclinical Toxicokinetic Evaluation of Phortress

Pharmacology 468 5

ter drug administration on days 1 and 8. There is no evi-dence of Phortress accumulation; indeed, the measured plasma concentrations can be considered approximately equivalent on the 2 days, taking into consideration inter-individual biological variability. The comparisons be-tween plasma concentrations of 5F 203 on days 1 and 8 of the study are illustrated in figure 2 c. No accumulation of either Phortress or 5F 203 takes place between days 1 and 8. PK parameters, measured on days 1 and 8 of the study, are compared in table 1 .

By reference to predose and 24-hour sample analyses, it is possible to observe 13 additional peaks in HPLC chromatograms as a result of Phortress intravenous dose. Chromatograms of a predose sample and 1-hour samples on days 1 and 8 are shown ( fig. 2 d). These peaks represent putative metabolic or degradation products of 5F 203 possessing similar fluorescence characteristics as Phor-tress or 5F 203. Non-fluorescent metabolites may also be produced, but would remain undetected by this tech-nique. The time course for detection of metabolites in

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Fig. 2. a Plasma concentrations of Phortress and 5F 203 following intravenous administration of Phortress to mice. Blood plasma samples, collected after the initial Phortress dose (day 1; 20 mg/kg) are shown. b Plasma concentrations of Phortress after intra-venous administration of Phortress (20 mg/kg) on days 1 and 8. c Plasma concentrations of 5F 203 after intravenous administra-

tion of Phortress (20 mg/kg) to mice on days 1 and 8. d Compar-ison of HPLC chromatograms of plasma samples taken from mice dosed with Phortress (20 mg/kg, intravenously): samples were collected before and 1 h after injection on days 1 and 8. Phortress, 5F 203, internal standard (IS) and putative metabolite peaks are illustrated.

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Pharmacology 468 5



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Bradshaw PHARMACOLOGY Volume: 83 Issue: 2 Pages: 99-109

Enzyme induction? •  Prodrug and “active” form

–  Mouse data 20mg/kg •  Induction on day 8


Pharmacology 468 5



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Bradshaw PHARMACOLOGY Volume: 83 Issue: 2 Pages: 99-109

Toxicokinetic study •  PK at doses known to be toxic •  Reproduces proposed clinical schedule •  Dose, 20mg/kg IV day 1 and day 8

CONFIDENTIAL

Pharmacokinetics In addition to the toxicology studies, a toxicokinetic study in male mice was also commissioned by Cancer Research UK. The results of a basic pharmacokinetic analysis of the plasma concentration-time data are shown in Table 5. It is difficult to make firm conclusions from this data, particularly when comparing Day 1 and Day 8 of the study, because of the limited number of time points (3 or 4) available to calculate AUCs on Day 8. A substantial portion of the final AUC value was dependent on whether or not a 24 hour plasma concentration was measurable. A more accurate comparison can be obtained by the use of AUC0-4 h and these values support a small reduction in AUC for the active metabolite on Day 8 compared to Day 1. However, it is clear that no accumulation of either the parent drug or active metabolite takes place between Day 1 and Day 8 of the toxicokinetic study. Table 5. Basic Pharmacokinetic Comparison between Day 1 and Day 8 of the Toxicokinetic Study in Mice (mean data used)

Phortress 5F203 active metabolite DAY 1 DAY 8 DAY 1 DAY 8 AUC0-24 (ȝmol/L.h) 3.239 5.415 5.577 5.735 AUC0-4 (ȝmol/L.h) 1.754 1.735 3.679 1.995 Cmax (ȝmol/L) 2.31 1.27 1.39 0.962 Tmax(h) 0.333 0.333 1.0 0.333

In preliminary studies conducted by the NCI, 70 Pmol Phortress (equivalent to 32.13 mg/kg, 96.39 mg/m2) was administered by intravenous injection to CD2F1 mice. Plasma concentrations of Phortress declined in a biexponential manner with half-lives for the initial and terminal phase of 2 and 43 minutes respectively. The total rate of clearance was 191 mL/min/kg. Concomitant with declining levels of the prodrug, plasma concentrations of 5F203 increased to between 2 and 4µM by 13 minutes, and remained in that range until 120 minutes, when they decreased with a half life of 110 minutes. Dogs were administered with 14.3 mg/kg Phortress as a one hour infusion. In both the male and female Phortress was quickly converted to 5F 203, with half-lives of 17 and 30 minutes respectively. Post infusion plasma concentrations of 5F 203 ranged from 2 to slightly over 5µM from 180 to 300 minutes, then declined with half lives of 1.5 to 2 hours. The areas under the plasma concentration-time profile, extrapolated to infinity, were 1637 and 1112 µM x min, for the male and female dog respectively. The pharmacokinetic data for 5F 203 from the first primate toxicokinetic study performed by the NCI – see Table 4 above for toxicity results – are summarised in Table 6. The data have been taken from the poster presented by the NCI in at the AACR meeting in Boston, November 2003.

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Revised 23/07/07 to include Substantial Amendment 2

CONFIDENTIAL

Pharmacokinetics In addition to the toxicology studies, a toxicokinetic study in male mice was also commissioned by Cancer Research UK. The results of a basic pharmacokinetic analysis of the plasma concentration-time data are shown in Table 5. It is difficult to make firm conclusions from this data, particularly when comparing Day 1 and Day 8 of the study, because of the limited number of time points (3 or 4) available to calculate AUCs on Day 8. A substantial portion of the final AUC value was dependent on whether or not a 24 hour plasma concentration was measurable. A more accurate comparison can be obtained by the use of AUC0-4 h and these values support a small reduction in AUC for the active metabolite on Day 8 compared to Day 1. However, it is clear that no accumulation of either the parent drug or active metabolite takes place between Day 1 and Day 8 of the toxicokinetic study. Table 5. Basic Pharmacokinetic Comparison between Day 1 and Day 8 of the Toxicokinetic Study in Mice (mean data used)

Phortress 5F203 active metabolite DAY 1 DAY 8 DAY 1 DAY 8 AUC0-24 (ȝmol/L.h) 3.239 5.415 5.577 5.735 AUC0-4 (ȝmol/L.h) 1.754 1.735 3.679 1.995 Cmax (ȝmol/L) 2.31 1.27 1.39 0.962 Tmax(h) 0.333 0.333 1.0 0.333

In preliminary studies conducted by the NCI, 70 Pmol Phortress (equivalent to 32.13 mg/kg, 96.39 mg/m2) was administered by intravenous injection to CD2F1 mice. Plasma concentrations of Phortress declined in a biexponential manner with half-lives for the initial and terminal phase of 2 and 43 minutes respectively. The total rate of clearance was 191 mL/min/kg. Concomitant with declining levels of the prodrug, plasma concentrations of 5F203 increased to between 2 and 4µM by 13 minutes, and remained in that range until 120 minutes, when they decreased with a half life of 110 minutes. Dogs were administered with 14.3 mg/kg Phortress as a one hour infusion. In both the male and female Phortress was quickly converted to 5F 203, with half-lives of 17 and 30 minutes respectively. Post infusion plasma concentrations of 5F 203 ranged from 2 to slightly over 5µM from 180 to 300 minutes, then declined with half lives of 1.5 to 2 hours. The areas under the plasma concentration-time profile, extrapolated to infinity, were 1637 and 1112 µM x min, for the male and female dog respectively. The pharmacokinetic data for 5F 203 from the first primate toxicokinetic study performed by the NCI – see Table 4 above for toxicity results – are summarised in Table 6. The data have been taken from the poster presented by the NCI in at the AACR meeting in Boston, November 2003.

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Revised 23/07/07 to include Substantial Amendment 2

PK in weekly schedule •  3mg/m2

•  Hepatotoxicity problem •  Little evidence of enzyme induction

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Comparison of clinical and mouse PK

•  45 patients – 3 to 40 mg/m2 (1 dose q 3 weeks) •  Relatively little 5F203 compared to preclinical PK/TK

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Pharmacology 468 5



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Comparison of preclinical and clinical PK •  Relevance of preclinical PK for Phase I dose?

– Change of schedule – Species variation in metabolism

TL310 •  Paclitaxel analogue •  Not a p-Glycoprotein substrate •  Can be given orally •  Interesting pre-clinical activity •  Molecular wt = 865 (ESI-LCMS M/z=888)

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Metabolism of paclitaxel

CYP3A4 CYP2C8

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Preclinical metabolism data

•  Data from company •  Human, mouse and rat liver microsomes •  LC-TOF

Proposed metabolite structures •  M1-M4, m/z=904 •  Side chain hydroxylation •  Positional and stereoisomers

Side chain and ring oxidation •  Loss of hydrogen •  Expected 6-hydroxy metabolite

Preclinical PK data •  Rat PK •  Dog bioavailability study <10%, but females>males

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Preclinical toxicology

•  Repeat dose study in rats –  Oral 15-60mg/kg weekly x 4 for males,

10-40mg/kg weekly x 4 for females –  All animals in the 60(m) and 40(f) mg/kg groups died on study –  Weight loss significant in high and medium dose groups –  NAOEL dose was 15mg/kg (m), 10 mg/kg (f), equivalent to

90mg/m2 or 60mg/m2. •  Repeat dose in beagles

–  Oral 0.625-2.5mg/kg dose weekly x 4 –  3 of 8 dogs at highest dose died on days 11-29 of administration –  Toxicity included diarrhoea and emesis –  Neutropenia observed in 50% of animals at 1.25-2.5mg/kg

Phase I trial

•  28 patients •  TL310, 5-160mg/m2 orally •  Days 1, 8 and 15 of 28 day cycle •  Cremophor-based formulation (no IV) •  9 patients given novel formulation at

120-160mg/m2

•  Terminated due to acute toxicity

•  Metabolite peaks present in patient samples •  Assignment of peaks by mass and Rt •  Additional peak, time-course mirrors parent drug

Patient trace

Side chain +OH

Baccatin Ring + OH TL310

Additional Peak – TL310 + 32

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In vivo metabolism (clinical)

Metabolite profiles – repeated doses

•  Patient 20

In Vitro trace

Side chain +OH 1

Side chain +OH 2

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TL310

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§ Incubation with human liver microsomes § LC-MS/MS, assignment of structure by mass and Rt § No additional peak at Rt=3.5 min

In vitro metabolism

“Dihydroxy metabolite”

•  Rt, M/z and time profile indicate metabolite •  Possible Potassium adduct? (K=39, Na=23) •  Much higher concentrations than for

corresponding paclitaxel metabolite •  Poor UV absorbance •  Couldn’t be formed in vitro •  Did not fragment •  Likely a constituent of formulation

PK data with metabolites

Patient 16 (160mg/m2)

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Female Male

Metabolite profiles and toxicity •  Patients 17 and 30 experienced severe toxicity

–  Highest day 1 Cmax for TL310

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PK in early phase studies

•  Oncology is particular setting – Patients as first in human

•  Guided by preclinical PK and toxicokinetics •  Metabolite PK frequently of equal importan •  Data often explain, but don’t often forewarn

§  Acknowledgements

§ Nicola Harris

§ Julieann Sludden

§ Melanie Griffin

§ Kellie Turner

§ Taxolog

§ Cancer Research UK

Toxicokinetic – toxicity data •  Toxicity alongside PK and PD •  PD

–  DNA adducts –  CYP induction (lung only)

•  Spot the significant toxicity? CONFIDENTIAL

Page 17 of 74 PH1/090. Phortress ¤Cancer Research UK Final 16/06/03 Revised 12/02/04 to include Amendment 1 Revised 11/05/06 to include Substantial Amendment 1 Revised 23/07/07 to include Substantial Amendment 2

Table 1. CR UK Study in Mice: Phortress or Vehicle only Intravenously on Days 1 and 8 Phortress dose in mg/kg 0 (Mannitol only) 10 20 Phortress dose in mg/m2 0 30 60 Animals per group 20 males 20 males 20 males Toxicology Findings Clinical observations Nil Lethargy, laboured respiration, loss of righting

reflex (2/20, resolved 30 mins post dose) Lethargy, laboured respiration, loss of righting reflex

(1/20, resolved 30 min post dose) 5/20 premature sacrifice due to trauma at the injection

site (tail). Various degrees of trauma to the tail Haematololgy Nil � total leukocyte count (Day 11, less marked

on Day 22) � total leukocyte count (Day 11, less marked on Day

22) Biochemistry Nil cholesterol (Day 11) � ALT, AST, AP, plasma bilirubin, albumin,

cholesterol (Day 11) Organ weight Nil in liver weight, (Day 11) Slight � in spleen weight (Day 11 and Day 36)

in liver, kidney weight (Day 11) Histology: Injection site Nil Subepithelial inflammation Oedema, subepithelial inflammation Histology: spleen Nil � severity of extramedullary haemopoiesis

(likely to be a secondary response to tail injury)

� severity of extramedullary haemopoiesis (likely to be a secondary response to tail injury)

Histology: Liver Nil Nil Single cell hepatocyte necrosis associated with proliferation of sinusoidal lining cells, hepatocyte

enlargement (Day 11), single cell hepatocyte necrosis (Day 36)

PD Measurements DNA Adducts: Liver Negligible adducts

detected (2/2 animals)

Adducts detected 2/2 animals (Day 11) and 1/2 animals (Day 36)


DNA Adducts: Lung N/A Adducts detected 2/2 animals (Day 11) and 1/2 animals (Day 36)


Western Blots: Liver N/A No observable Phortress induced changes No observable Phortress induced changes Western Blots: Lung N/A Induction of CYP1A1 (Day 11), significant

recovery by Day 36 Induction of CYP1A1 (Day 11), significant recovery by

Day 36

Comparative PD data •  Measurement of DNA adduct formation, as proof of target

interaction •  Matches induction of CYP1A1 in liver and lung

Toxicology in dogs •  Fewer animals – greater flexibility, no PK •  BNF as potential CYP1A inducer

In a separate study, summarized in Table 1, the efficiencyof conversion, or bioavailable fraction of prodrug to parent,was 87% with 1a and 106% with 1b, far greater than ob-served in mice.

Dogs. Prodrug 2b (14.3 mg/kg) was rapidly converted toparent amine 2 with t1/2! of 17 and 30 min in one male andone female dog (Fig. 2B), respectively. Plasma concentra-tions of 2 were between 2 and 5 "M between 60 and 300 minafter the start of the infusion. At doses of 2 and 4 mg/kg 2b,peak plasma concentrations of 2 were 0.5 and 0.9 "M, re-spectively.

Toxicity Results. Gastrointestinal toxicity (emesis, diar-rhea, and low food consumption) and lethargy were exhibitedby two dogs for 5–7 days after treatment with 14.3 mg/kg 2b,and by day 7, the dogs had lost between 9 and 14% bodyweight. Increased levels of serum bile acids, # glutamyltrans-ferase, aspartate aminotransferase, alanine aminotrans-ferase, and alkaline phosphatase were measured. On day 5,the male dog that received a 24-h infusion exhibited laboredrespiration and was sacrificed because of moribund condi-tion. Histopathology examination revealed depletion/atrophyin the bone marrow, lymphoid tissues and gut-associatedlymphoid tissue, intestinal lesions, and extensive acute in-flammation in the alveolar spaces with edema and hemor-rhage; pulmonary toxicity was dose limiting. At doses of 2and 4 mg/kg 2b (1-h civ), emesis and soft stool occurred onthe day of dosing, but during days 1–5, no effects on clinicalpathology parameters and no lesions in lung or liver tissueswere observed. Moreover, plasma concentrations of 2reached a peak of 0.9 "M, an efficacious concentration invitro against sensitive breast cancer cell lines (MCF-7, ZR 75,T47D; 8).

In vivo antitumor activity MTDs of 1a and 1b, established infemale B6D2F1 mice after i.v. injection, was 25 mg/kg and

Fig. 2. Plasma concentrations of 2b (!) and 2 (F) after i.v. infusion of32.2 mg/kg 2b in mice (A) and 14.3 mg/kg 2b in a male dog (B).

Table 1 A summary of the pharmacokinetic data generated after i.v. administration of prodrugs to mice, rats, and dogs

Species Dose(mg/kg: i.v.) Prodrug Prodrug

(t1⁄2 !: min)

Total rate plasmaclearance:ml/min/kg

Max. plasmaamine conc;

"M

Duration:min

Amine(t1⁄2 !: min)

Mouse 26.5 1a 3 241 3–6 7–90 7975

31.3 1b 1 160 3–5 10–90 671488

25.6 2a 2.4 190 2–3 9–120 101115

32.2 2ba 2 191 2–4 13–120 11043

26.6 3a 4 261 2 60 12370

32.2 3b 1.4 177 3–4 15–90 6630

Rat 25 1a 2 77 5–6 30–45 14329

24931 1b 3 188 6–12 10–90 83

2897

DogMale 14.3 2bb 17 17.7 2–5 55–300 127Female 14.3 2b 30 28.0 2–5 62–180 91

a,b These data are represented graphically in Figs. 2, A and B, respectively.

242 Preclinical Evaluation of Benzothiazole Prodrugs

Mass spectra for side-chain hydroxy metabolites

Structure from fragmentation MS

•  Fragment analysis gives structural info

•  Limited by fragment resolution

Cytochrome P450 Metabolism

• Click to add text

33

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