The Open Drug Metabolism Journal, 2009, 3, 43-55
`
`43
`
`Open Access
`
`Pharmacokinetics and Pharmacodynamics of Hyaluronan Infused into
`Healthy Human Volunteers
`
`Sara Rae Hamilton#,1, Mandana Veiseh#,2, Cornelia Tölg#,3, Rommel Tirona4, Jakob Richardson3,
`Richard Brown5, Mario Gonzalez6, Michael Vanzieleghem7, Patricia Anderson8, Samuel Asculai9,
`Françoise Winnik10, Rashmin Savani11, David Freeman12, Leonard Luyt13, James Koropatnick*,14
`and Eva Ann Turley*,15
`
`1London Regional Cancer Program, Cancer Research Laboratories, London, Ontario, Canada, and Department of Bio-
`chemistry, University of Western Ontario, London, Ontario, Canada; 2Life Sciences Division, Lawrence Berkeley Na-
`tional Laboratories, Berkeley CA; 3London Regional Cancer Program, Cancer Research Laboratories, London, On-
`tario, Canada; 4 Department of Physiology & Pharmacology and Division of Clinical Pharmacology, Department of
`Medicine, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada;
`5BioMS Medical Corporation, Edmonton, Alberta, Canada; 6P-kinetics Corp., Miami, Florida, USA; 7MV Pharmaceuti-
`cal Consultants, Mississauga, Ontario, Canada; 8CanReg Inc., Dundas, Ontario, Canada; 9Bio-Strategies Consulting
`Group, Toronto, Ontario, Canada; 10Département de Chimie et Faculté de Pharmacie, Université de Montréal, Pavillon
`J.A. Bombardier, CP 6128 Succursale Centre Ville, Montréal, QC, Canada; 11William Buchanan Chair in Pediatrics,
`Director, Division of Neonatal-Perinatal Medicine, Associate Director, Division of Pulmonary & Vascular Biology Uni-
`versity of Texas Southwestern Medical Center, Dallas, TX; 12Faculty of Medicine, University of Western Ontario, Lon-
`don, Ontario, Canada;13London Regional Cancer Program, Cancer Research Laboratories and Department of Chemis-
`try; 14London Regional Cancer Program, Cancer Research Laboratories and Departments of Oncology, Microbiology
`and Immunology, Pathology, and Physiology and Pharmacology, University of Western Ontario and Lawson Health
`Research Institute, London Health Sciences Centre, London, Ontario, Canada; 15London Regional Cancer Program,
`Cancer Research Laboratories (London, Ontario, Canada) and Departments of Biochemistry and Oncology, University
`of Western Ontario, London, Ontario, Canada
`
`Abstract: The pharmacodynamics and elimination kinetics of escalating doses (1.5-12 mg/kg) of hyaluronan (HA) infu-
`sions were studied in healthy human volunteers. Metabolic breakdown of serum HA and associated adverse events were
`monitored throughout the study. The HA-binding capacities of circulating CD4+ and CD8+ T lymphocytes, CD19+ B-
`lymphocytes and CD14+ peripheral blood monocytes (PBMC) were also quantified. Breakdown of infused HA into small
`fragments (<37 kDa) were not detected and adverse events related to HA infusions were infrequent and non-serious in na-
`ture. Binding of FITC-HA was greatest to CD14+ monocytes and the binding capacity of these cells for FITC-HA was
`significantly increased by the final HA infusion. At that time, binding to CD14+ monocytes was related to serum HA lev-
`els suggesting a close relationship between PK and PD of serum HA. Drug level analysis demonstrated a disproportional
`increase in the area under the serum concentration vs. time curve with increasing HA dose. The observed non-linear HA
`kinetics appears to result from a saturable elimination process as revealed by pharmacokinetic modeling. These results
`have implications for the use of injected HA for drug delivery or in imaging applications.
`
`INTRODUCTION
`
` HA is an ubiquitous glycosaminoglycan produced by
`three distinct but homologous HA synthases (HAS1-3) and is
`degraded by five known hyaluronidases (HYAL1-5) most of
`which are lysosomal. HA is retained in tissues as a result of
`specific interactions with extracellular and cellular HA-
`binding proteins defined as hyaladherins and amounts are
`regulated during morphogenesis, wound repair, chronic in-
`flammatory disorders, and neoplasia [1, 2]. Altered tissue
`HA results from changes in the activity and expression of
`
`*Address correspondence to these authors at the London Regional Cancer
`Program, 790 Commissioners Rd E, London, Ontario, N6A4L6. Business,
`Canada; Tel: 519-685-8600 ext 53677; Fax: 519-685-6816; E-mail:
`eva.turley@lhsc.on.ca; jkoropat@uwo.ca
`#These authors contributed equally.
`
`HAS and hyaluronidases as well as rate of HA uptake into
`the cell [3-6]. HA modifies the physico-chemical nature of
`the extracellular matrix within tissues and contributes to both
`homeostasis and response-to-injury processes [1, 4, 5, 7-10].
`For example, interactions with cell receptors activates signal-
`ing cascades that promote migration and proliferation, which
`consequently influence both differentiation and immune traf-
`ficking/function during tissue homeostasis and response-to-
`injury [2, 11]. HA is metabolized in multiple compartments
`including tissues, lymphatics and vasculature [6] and altera-
`tion of this metabolism can have serious consequences to
`homeostasis. For example, modification of tissue HA me-
`tabolism during response-to-injury promotes unremitting,
`non-resolving inflammation [4, 12]. A proportion of tissue
`HA escapes into the lymphatics, is processed in lymph nodes
`and from there gains entry into the blood vasculature [13].
`
`1874-0731/09
`
`2009 Bentham Open
`
`APOTEX EXHIBIT 1056
`Apotex v. Alkermes
`IPR2025-00514
`
`

`

`44 The Open Drug Metabolism Journal, 2009, Volume 3
`
`Hamilton et al.
`
`Uptake and metabolism of HA occurs in each of these com-
`partments as well. Endocytosis of HA by liver, and to a
`lesser extent kidney endothelium, removes most serum HA
`[14]. Ultimately HA is degraded in lysosomes by hyaluroni-
`dases [15, 16].
`
`In spite of this limited knowledge of both the metabolic
`
`fate and functions of HA, it is currently extensively used in
`humans (e.g. for joint supplementation and tissue replace-
`ment, surgical adhesion prevention, regulation of inflamma-
`tory diseases, tissue healing/regeneration and tissue engi-
`neering) [17-20]. Future products of HA or modified HA are
`also being developed to enhance targeted drug delivery into
`traditionally inaccessible compartments such as tumors, pe-
`ripheral lymph nodes, and bone marrow [21-23]. HA is also
`being investigated as an imaging agent. For example, in-
`creased HA accumulation at the periphery of invading breast
`tumors enhances ultrasound detection of the margins of the
`invading tumor [24]. HA can be readily modified with non-
`specific contrast agents such as metals, and these complexes
`are being studied for their ability to detect areas of altered
`HA metabolism [25].
`
` The current clinical uses of HA often result in repeated
`exposure of patients to high levels of exogenous HA in se-
`
`Table 1. Patient Demographics and Exclusion Criteria
`
`A. Patient Demographics
`
`12 males, 12 females
`
`rum, far exceeding normal serum concentrations, which are
`in the order of ug/L [26]. Surprisingly few studies have ad-
`dressed the pharmacokinetics or dynamics of the exogenous
`HA polymer itself or in combination with other drugs [27,
`28] and to our knowledge there are no reports of the pharma-
`codynamics of chronically administered, high dose (mg/kg)
`exogenous HA under conditions that patients might experi-
`ence when HA is used as a delivery vehicle for therapeutic
`reagents. Since quantifying the pharmacokinetics of adminis-
`tered HA is an essential first step to characterize its pharma-
`codynamics for identifying potential deleterious side effects
`of exogenous HA, and for further refinement of HA as a
`drug delivery and imaging agent, we assessed the elimina-
`tion kinetics and toxicity of multiple, escalating doses of HA
`from 1.5 mg/kg-12 mg/kg administered to healthy male and
`female human volunteers over a 4 week period.
`
`SUBJECTS, MATERIALS AND METHODS
`
`Patient Eligibility
`
` Twelve non-smoking male and 12 non-smoking female
`volunteers, 30-50 years of age, weighing at least 60 kg
`(males) or 45 kg (females), and who were within 15% of
`their optimum weight [29] were enrolled in this study by
`
`aNon-Smokers
`
`Age (30 – 50 yrs)
`bWeight (avg. 60 kg for males, 45 kg for females)
`
`Height (avg. 178 cm for males, 160 cm for females)
`
`B. Medical Histories
`
`Significant Cardiovascular disease
`
`(for exclusion from study)
`
`Significant hepatic disease
`
`Significant renal disease
`
`Significant CNS disease
`
`Significant hematological disease
`
`Significant gastrointestinal disease
`
`Clinically significant illness 4 weeks prior to study entry
`
`Alcoholism or drug abuse in previous year before study entry
`
`Hypersensitivity or idiosyncratic reaction to HA or other GAG
`
`Pregnancy
`
`Nursing
`
`Inadequate contraception
`
`cPersonality disorders
`
`Conditions interfering with drug absorption, distribution, metabolism or excretion
`
`Use of enzyme inducing drugs within 30 days of study entry
`
`Treatment drugs toxic to major organs within 3 months of study entry
`
`Abnormal diet during 4 weeks preceding study
`
`Through completion of study, donation of >900 mL of blood over 20 weeks
`
`Participation a drug study 4 weeks prior to study entry
`
`C. Prohibited (for duration of study)
`
`dMedication (including over-the-counter products)
`
`Use of alcohol-containing or xanthine-containing beverages through sample collection
`
`See Methods and Results sections for descriptions of procedures.
`aSubjects could not have used nicotine within 3 months of study entry.
`bPatients were within 15% of their ideal weight (“Table of Desirable Weights of Adults”, Metropolitan Life Insurance Company, 1983).
`cIncludes disorders that would preclude informed consent or compliance with protocol requirements.
`dIf medication other than that specified in the protocol was required, decision to continue subject was made by L.A.B. Medical Personnel.
`
`

`

`Pharmacodynamics of Escalating i.v. Infusions of Hyaluronan
`
`The Open Drug Metabolism Journal, 2009, Volume 3 45
`
`L.A.B. Pharmacological Research International Clinical Re-
`search Center (Montreal, Canada). Subjects were further
`screened for medical histories/demographic data (Table 1).
`All underwent a complete physical examination and standard
`laboratory tests to detect medical abnormalities (Table 2).
`to non-smoking, medically
`Participants were restricted
`healthy subjects with clinically acceptable laboratory pro-
`files. Exclusion criteria are listed in Table 1.
`
`Ethical Approval
`
`The protocol was reviewed and approved (IND#BV5200-
`02) by TPD (formerly HPB, Regulatory Body of Canada).
`The protocol was also internally reviewed by the L.A.B.
`
`Table 2. Clinical Laboratory Tests Performed on Subjects
`
`Pharmacological International Institution Review Board and
`was carried out in accordance with established clinical re-
`search guidelines [30, 31]; and the principles enunciated in
`the Declaration of Helsinki [32]. The purpose of the study,
`the procedures that were carried out, and the potential haz-
`ards were described to the subjects in non-technical terms in
`conformity with regulatory requirements.
`
`Study Design
`
`This was a single-dose, dose escalation, pharmacokinetic
`study. The primary objective was to evaluate the pharma-
`cokinetic profile of HA after 120 min intravenous infusion of
`a sterile 1% HA solution at escalating doses of 1.5 mg/kg,
`
`A. Hematologya
`
`B. Serum Chemistrya
`
`C. Urinalysisa
`
`hemoglobind
`
`hematocritd
`
`total and differential leukocyte count
`
`red blood cell count
`
`platelet count
`
`calculated indices
`
`sedimentation rate
`
`PT/PTT
`
`BUN
`
`creatine
`
`total bilirubin
`alkaline phosphatasee
`SGOT e
`
`SGPT e
`
`LDH
`
`sodium
`
`potassium
`
`calcium
`
`phosphorus
`
`glucose
`
`B-HCG (for female subjects only)
`
`pH
`
`specific gravity
`
`protein
`
`glucose
`
`ketones
`
`bilirubin
`
`blood
`
`nitrate
`
`urobilinogen-microscopic examination
`
`B-HCG (for female subjects only)a
`
`D. Urine drugs-of-abuse screenc
`
`E. Otherc
`
`cocaine
`
`cannabis
`
`HIV
`
`See Methods and Results sections for descriptions of procedures.
`aConducted at screening and post study.
`bConducted prior to entry into each section.
`cConducted at screening only.
`dConducted prior to and following period 4.
`eConducted at 72 hrs. post-dose in periods 1, 2, and 3.
`
`

`

`46 The Open Drug Metabolism Journal, 2009, Volume 3
`
`Hamilton et al.
`
`3.0 mg/kg, 6.0 mg/kg and 12 mg/kg. Each dose subsequent
`to the 1.5 mg/kg dose was administered following a 7-day
`washout period. In each case a total volume of 250 ml of
`stock solution of HA, diluted appropriately with 0.9% so-
`dium chloride, was administered over a period of 120 min.
`Blood was sampled from all volunteers at 2, 1.5, 1, and 0.5 h
`before administration and at 2, 4, 6, 10, 14, 18, 22, 32, 38, 50
`and 74 h after administration of the HA solution. Blood
`samples were collected and serum HA levels were measured
`on site by L.A.B., Inc.. Blood collected from a subset of six
`subjects (3 females and 3 males) at 0, 1, 4, 12, 24 and 72 h
`post-infusion of each HA dose were analyzed to determine
`the molecular weight profile of serum HA and to assess HA
`binding by T cells, B cells and blood monocytes. Measure-
`ment of these parameters was the first step in assessing
`pharmacodynamic disposition of HA and as described be-
`low. Samples were not segregated according to gender.
`
`Antibodies and Reagents
`
`FMC63 (CD19) antibody was conjugated to PE. PE con-
`jugated CD4 and CD8 antibodies were purchased from Bec-
`ton Dickinson (San Jose, CA). Isotype matched control
`monoclonal antibodies (mAb) were from Southern Biotech
`(Birmingham, AL). For infusions, lyophilized HA (medical
`grade, lyophilized HA form prepared by Hyal Pharma, Mis-
`sissauga ON from original fermented HA product purchased
`from Kyowa Hakkos, Japan) was dissolved in phosphate
`buffered saline (PBS) and autoclaved to sterilize and reduce
`molecular weight. HA samples from this commercial source
`were analyzed with respect to MW using HPLC/SEC multi-
`angle laser light scattering (LifeCore Biomedical, MS). For
`cell binding assays, HA (Healon, medical grade) was ob-
`tained from Pharmacia (Dorval ZB) and conjugated to fluo-
`rescein (FITC) as described previously [33].
`
`Assessment of HA Purity
`
` HA preparations were tested for the presence of protein,
`DNA and endotoxin. Protein content was assayed by absorp-
`tion at 280 nm. DNA content was determined by electropho-
`resis of 10-100 μg HA in agarose gels containing 0.7% aga-
`rose and 0.5 mg/ml ethidium bromide at 100 V for 3 h. DNA
`was visualized using a UV transilluminator (wavelength:
`302nm) [34]. Endotoxin was detected using a colorimetric
`Limulus ameobocyte lysate assay with a sensitivity of 0.01
`endotoxin units/ml [35].
`
`Injections of HA, Sampling and Pharmacokinetic
`Assessment
`
` Each subject received four escalating doses of 1% HA
`(1.5, 3.0, 6.0, and 12 mg/kg) one week apart as a 2 h infu-
`sion. Between 8-10 a.m. on day one of the study, blood was
`drawn from volunteers 2 h prior to HA infusion, to deter-
`mine baseline serum HA. Blood was collected in 1 X 1.8 ml
`citrated vacu-containers (BD Canada, Oakville, On). On the
`first day, 24 volunteers were infused i.v. with 1.5 mg/kg HA
`(1%) over a 2 h period (period 1). Blood was sampled at the
`times listed above in “Study Design”. Subsequently, at 7-day
`intervals, volunteers were infused with 3.0 mg/kg (period 2),
`6.0 mg/kg (period 3) and 12 mg/kg (period 4) and blood
`samples taken as described for period 1.
`
` Baseline, endogenous serum HA was 35-40 ng/ml, con-
`sistent with that reported in healthy persons 18-65 years old
`
`[26]. HA serum levels had dropped to these baseline levels
`in all subjects during the washout period between dose peri-
`ods. These values were remarkably consistent and were 0.1-
`0.4% of serum HA at the earliest time point (0.5 h) after in-
`jection of the lowest amount of HA (1.5 mg/kg). For simplic-
`ity of analysis, baseline serum HA levels were subtracted
`prior to analysis of the data.
`
` Maximum serum concentrations (Cmax) were the values
`obtained 2 h after HA infusion and AUC (“area-under-
`curve”, in a plot of HA concentration vs time infusion [0-
`75]) was determined using a linear trapezoid method. Model-
`dependent analysis of the serum concentration-time profiles
`was used to estimate zero-order endogenous HA synthesis
`rate (ksyn), saturable elimination process (Km, serum concen-
`tration at half-maximal elimination rate; Vmax, maximum
`elimination rate) and one-compartment volume of distribu-
`tion (Vd) (Fig. 1). Hence, the rate of change in HA mass in
`serum as a function of time was described as:
`Dose
`
`ksyn
`
`Vd
`
`Km
`Vmax
`
`Fig. (1). Hyaluronan Pharmacokinetics Model. The pharmacoki-
`netics of HA was modeled with a one-compartment distribution
`scheme to which the dose is delivered as a 2-hour infusion. En-
`dogenous HA synthesis is described as a zero-order process (ksyn)
`while the rate of saturable HA elimination is governed by the pa-
`rameters Km and Vmax.
`
`dHA
`
`dT
`
`= ksyn -
`
`Vmax x [HA]
`Km + [HA]
`
`where the concentration of HA is:
`
`[HA] =
`
`HA
`Vd
`
` Eq. I
`
`Eq. II
`
` The initial HA concentration was set as the pre-infusion
`level. It was assumed that the rate of HA appearance in se-
`rum (ksyn) remained constant over the sampling period. The
`entire mean data set over time 0 to 75 h for each dose regi-
`men (1.5, 3, 6, and 12 mg/kg administered over 2 h) was
`fitted to the model to estimate parameter values using a least-
`squares minimization procedure with optimized weighting
`determined by visual inspection of the fitted result (SCIEN-
`TIST, MicroMath Scientific Software, St. Louis, MO). The
`present model was employed because preliminary kinetic
`analyses determined that simpler models describing elimina-
`tion as a single linear function, or more complex models
`such as those including a combination of saturable and linear
`clearance processes, did not yield improved fits of the data
`after comparisons of sums of squared deviation, residual
`plots, standard deviation of estimates and values for a modi-
`fied Akaike criterion.
`
`

`

`Pharmacodynamics of Escalating i.v. Infusions of Hyaluronan
`
`The Open Drug Metabolism Journal, 2009, Volume 3 47
`
`Clinical Tests Performed to Identify Serious Adverse
`Reactions
`
`Measurement of HA Binding by Subsets of Peripheral
`Blood Mononuclear Cells ( PBMC)
`
` Clinical laboratory tests (Table 2) were conducted at
`screening and post-study. B-HCG urine levels were assessed
`in female subjects at screening, prior to entry into each dos-
`ing period and post-study (Table 1). Hemoglobin and hema-
`tocrit were both tested on screening, prior to entry into the
`study, prior to and following period 4 dosing, and at the end
`of the study. Serum alkaline phosphatase, SGOT, and SGPT
`levels were determined at screening, prior to entry into the
`study and 72h post-dose in periods 1, 2 and 3, and at the end
`of the study.
`
`Measurement of Serum HA
`
` The analysis for HA levels in serum samples from 23
`healthy volunteers for each time point described above was
`performed using a Pharmacia HA Test (Kabi Pharma, Up-
`sala, Sweden) based upon the same principles as the pres-
`ently available HA Test Kit (Echelon). The HA of serum
`samples reacts with a specific HA binding protein labeled
`with 125I. The limit of quantification (LOQ) of the assay was
`10 ng/ml, and the limit of detection (LOD) was 4.73 ng/ml.
`Inter and intra-day variation of the assay was controlled
`through incorporation of an analysis of HA analysis stan-
`dards, provided with the kit.
`
`HA Molecular Weight Analysis
`
` HA and other glycosaminoglycans were precipitated us-
`ing cetylpyridinium chloride treatment of serum samples
`from 6 subjects using the 0.5hrs and 48 hrs sampling times
`[35]. Equal amounts of precipitated glycosaminoglycans,
`detected with a modified anthrone assay, which detects
`uronic acid, were used for molecular weight analyses [36].
`The heterogeneity of HA size range was determined using
`non-denaturing gel filtration. Precipitates were dissolved in
`PBS and then passed through a 1.6 x 61.5 cm Sephacryl 500
`column, previously calibrated using dextran standards (MW
`11.3-2,000 kDa). One ml fractions from a total 120 ml
`volume were analyzed for uronic acid. Inter- and intra-day
`variations in amounts of HA detected with the modified an-
`throne assay were determined and adjusted for by using HA
`standards to generate concentration curves for each assay.
`Elution profile curves were plotted and molecular weight
`categories of >2 x 106, 1.9 x 105-2.0 x 106; and <1.9 x 105
`were quantified by measuring AUC using Adobe Photoshop
`to obtain pixel number/unit area.
`
`Table 3. Frequency of Adverse Effects
`
`PBMC were purified by centrifugation over Ficoll
`
`PAQUE (Pharmacia, Dorval, QB), washed and stained for
`surface marker expression as well as for the ability to bind
`FITC-HA as previously described [33]. To determine the
`ability of PBMC subsets to bind HA, PBMC were stained in
`two-color immunofluorescence with PE-coupled mAb to a
`defined surface marker and HA-FITC. The optimum amount
`of mAb-PE and of FITC-HA was determined by titration
`using PBMC from untreated normal donors. HA binding by
`T cells (CD4+ or CD8+), B cells (CD19+) or monocytes
`(CD14+) was analyzed in replicate aliquots of each PBMC
`sample. Isotype-matched Ab binding was also assessed as a
`control for background staining.
`
`PBMC stained with mAb-PE and FITC-HA were ana-
`
`lyzed by FACS (Becton Dickinson). Data from 10,000 cells
`(after exclusion of erythrocytes and dead cells) were ana-
`lyzed using Lysis II software. Cells expressing CD4+,
`CD8+, CD19+ and CD14+, (depending on the mAb used)
`were assessed for staining with FITC-HA in each PBMC
`subset. Auto-fluorescence of PBMC and PBMC stained with
`IgG-FITC or avidin-FITC were used as negative controls in
`evaluation of staining by FITC-HA. Staining was moderately
`bright for FITC-HA for 13 untreated normal donors, with
`mean fluorescence intensity reported in arbitrary units. HA
`binding by subsets of PBMC after infusion of HA was com-
`pared to the normal values. Assay variability was compen-
`sated through assessment of HA binding to CD14+ mono-
`cytes (collected at the 20 hr time point from each period) in
`each FACS analysis.
`
`RESULTS
`
`Characterization of HA Used for Study
`
` The average MW of HA used in this study was 276.6
`(hereafter referred to as 280) kDa after heat sterilization
`(which fragments HA slightly), and 519.7 kDa prior to heat
`exposure. Protein contamination of less than 0.001 μg/mg
`was detected (although values obtained were at the lower
`limit of detection of the assays). DNA contamination in
`commercial samples of HA, which has the capacity to induce
`cytokines in monocytes in vitro has been reported [34].
`Thus, we assessed samples for DNA contamination but none
`was detected. Endotoxin was also not detected. These results
`indicate that contaminants common to commercial HA
`batches were below the detection limits of our assays.
`
`Adverse Effects
`
`Frequency
`
`Related to Study
`
`Severity
`
`Subject Continued or Discontinued in Study
`
`Influenza
`
`Lower Arm Rash
`
`Headache
`
`Pro-thrombin Time
`
`Practical Thromboplastic Time
`
`Others
`
`1
`
`1
`
`4
`
`2
`
`2
`
`7
`
`Unrelated
`
`Possibly related
`
`Possibly related
`
`aUnrelated
`
`aUnrelated
`
`Possibly related
`
`Mild
`
`Mild
`
`Mild
`
`Mild
`
`Mild
`
`Mild
`
`Discontinued
`
`Continued
`
`Continued
`
`Continued
`
`Continued
`
`Continued
`
`See Methods and Results sections for descriptions of procedures.
`aLevels were altered at normal, endogenous HA levels.
`
`

`

`48 The Open Drug Metabolism Journal, 2009, Volume 3
`
`Hamilton et al.
`
`Toxicity Profile of HA Infusions
`
` A number of standard toxicity parameters were moni-
`tored in this study to aid in identification of any adverse ef-
`fects (Table 2). A listing of adverse events noted in this
`study is reported in Table 3. One subject left the study due to
`a minor illness (influenza) unrelated to the treatment. In gen-
`eral HA was well tolerated by all subjects and there were no
`withdrawals due to study drug effects. Seven subjects expe-
`rienced 13 mild adverse events. Five of the mild events were
`possibly related to the study drug (lower arm rash in one
`subject, and headache in four subjects) while the remaining
`events were unlikely to be related. At normal endogenous
`HA levels, both the pro-thrombin time and activated practi-
`
`cal thromboplastic time were within their respective refer-
`ence range for all subjects with the exception of subjects 9
`and 18.
`
`Kinetic Analysis of HA Elimination from Serum
`
`The study design was based on commonly used standard
`methods to obtain an optimal dosing regime for bioactive
`drugs [37]. However, the objective of the study was to
`evaluate the pharmacokinetic profile of increasing doses of
`1% HA injected as i.v. infusions over 120 min. Since the
`doses of i.v. administered HA in the present study were
`higher than those of previous reports (e.g., [38]) and were
`escalated over time, each dose was administered by a 2 h
`
`Table 4. Baseline Corrected Hyaluronan Mean Concentration (Cp), ng/mL
`
`1.5 mg/kg Mean [+/-SD]
`
`3.0 mg/kg Mean [+/-SD]
`
`6.0 mg/kg Mean [+/-SD]
`
`12.0 mg/kg Mean [+/-SD]
`
`N=24
`
`0
`
`[0]
`
`7761
`
`[1130]
`
`15280
`
`[2783]
`
`24052
`
`[3653]
`
`26284
`
`[3918]
`
`14419
`
`[3384]
`
`4633
`
`[3518]
`
`N=24
`
`0
`
`[0]
`
`17713
`
`[3021]
`
`34625
`
`[5621]
`
`52052
`
`[9039]
`
`63063
`
`[16099]
`
`52167
`
`[7646]
`
`36334
`
`[6390]
`
`N=24
`
`0
`
`[0]
`
`38838
`
`[6162]
`
`84083
`
`[13648]
`
`129204
`
`[16063]
`
`131549
`
`[17324]
`
`121122
`
`[13000]
`
`104426
`
`[16384]
`
`N=23
`
`0
`
`[0]
`
`77186
`
`[11297]
`
`154713
`
`[26503]
`
`254994
`
`[40017]
`
`313554
`
`[41271]
`
`325913
`
`[51980]
`
`264232
`
`[40440]
`
`21550
`
`Time (hr)
`
`0a
`
`0.5a
`
`1a
`
`1.5a
`
`2a
`
`4
`
`6
`
`Infusion Period
`
`10
`
`14
`
`18
`
`22
`
`26
`
`32
`
`38
`
`50
`
`74
`
`40
`
`[56]
`
`24
`
`[12]
`
`33
`
`[19]
`
`35
`
`[7]
`
`24
`
`[16]
`
`22
`
`[17]
`
`18
`
`[10]
`
`22
`
`[14]
`
`31
`
`[12]
`
`aThe infusion period was 0-4 hrs.
`
`12934
`
`[7139]
`
`1755
`
`[3188]
`
`61
`
`[103]
`
`23
`
`[14]
`
`19
`
`[10]
`
`7
`
`[4]
`
`23
`
`[14]
`
`29
`
`[10]
`
`15
`
`[10]
`
`73627
`
`[12514]
`
`50440
`
`[13547]
`
`26522
`
`[13128]
`
`10769
`
`[12496]
`
`1839
`
`[4367]
`
`28
`
`[55]
`
`16
`
`[11]
`
`16
`
`[11]
`
`22
`
`[16]
`
`[28998]
`
`195145
`
`[32153]
`
`153467
`
`[22857]
`
`146773
`
`[27505]
`
`94579
`
`[31429]
`
`54615
`
`[29332]
`
`21795
`
`[21939]
`
`419
`
`[1373]
`
`15
`
`[7]
`
`

`

`Pharmacodynamics of Escalating i.v. Infusions of Hyaluronan
`
`The Open Drug Metabolism Journal, 2009, Volume 3 49
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`102102102102102102102102102102102102102102102102102102102
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`Hyaluronan (ng/mL)
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`doses was 348 mg/hr/kg, which is greater than the value re-
`ported in an earlier study of a lower dose, bolus injection of
`HA in human subjects [38]. In contrast to the Vd and Vmax,
`the Km for the first (1.5 mg/kg) dose is significantly different
`from the last three doses. The first dose exhibits a smaller Km
`(6.4 mg/ml) than the other doses (the average of which is
`17.6 mg/ml) and the Km of the middle two doses (3.0 mg/kg
`and 6.0 mg/kg) is smaller than the largest, final dose (12
`mg/kg) (Table 5). The discrepancies amongst these Km val-
`ues may be due to the variability in data sets observed at
`lower serum HA concentrations and at the extended time
`periods of sampling (see Fig. 2). However, it is likely that
`these differences also reflect an altered capacity to eliminate
`increasing amounts of exogenous HA from serum.
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`HA Binding to PBMCs
`
`Time (hrs)
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`Fig. (2). Hyaluronan serum levels with dose-escalation. HA
`levels were determined following injection of escalating doses of
`HA [1.5 mg/kg (period 1), 3 mg/kg (period 2), 6 mg/kg (period 3),
`and 12 mg/kg (period 4)], administered as an i.v. infusion over 2 h.
`All values have been corrected for baseline, endogenous serum HA
`levels. Values represent the mean of n=24 for the 1.5, 3.0, and 6.0
`mg/kg doses and n=23 for the 12 mg/kg dose. Lines are obtained
`from least-squares fitting of the mean data for each set to the phar-
`macokinetic model (Fig. 1).
`
`infusion as this was judged to be safer than the bolus injec-
`tions used in previous studies. Serum HA concentrations
`below 40 ng/ml (baseline levels) were not included for ki-
`netic analyses and were subtracted from all other serum HA
`values. These corrected levels (Table 4) were plotted against
`time. The semi-log plot of these data (curve fitted to plotted
`data points) is shown in Fig. (2). There was a disproportion-
`ate increase in HA AUC with increasing dose (Table 5) indi-
`cating non-linear pharmacokinetics. By inspection of the log
`serum concentration vs. time profile between 2 h (end of
`infusion) and 75 h, the elimination of HA at all four doses
`was observed to be consistent with saturable or Michaelis-
`Menten kinetics as evidenced by the concave-down shape
`(Fig. 2). Indeed, the experimental data fit well with the
`pharmacokinetic model (Fig. 1) and the exercise yielded
`parameter values describing the zero-order endogenous for-
`mation, one-compartment distribution, and saturable elimi-
`nation of HA (Table 5). The apparent Vd for each dose was
`not significantly different from one another and the averaged
`Vd from all four doses was 38 ml/kg. This value is small but
`consistent with serum volume. The Vmax for each dose was
`also very close to one another. The average across the 4
`
`Table 5. Hyaluronan Pharmacokinetics
`
`PBMCs (including T and B lymphocytes, and mono-
`
`cytes) have been reported to bind to HA and to express HA
`receptors such as CD44, RHAMM and others [7, 39]. As a
`first step in assessing pharmacodynamics of exogenous HA
`infusions, we analyzed these cells that were obtained during
`each dosing period for their ability to bind FITC-HA (Table
`6). All cell types bound FITC-HA above background levels.
`However, monocytes (CD14+) bound considerably more HA
`than either B cells (CD19+) or T cells (CD4+ and CD8+). Of
`the cell types examined, T cells bound the least amount of
`FITC-HA, which was close to background levels (Table 6).
`Total FITC-HA binding to CD14+ PBMC was significantly
`greater in the 4th dosing period, compared to periods 1-3
`(Fig. 3). Further analysis of the dynamic changes in FITC-
`HA binding over time for each dosing period was conducted
`by comparing FITC-HA binding levels with HA serum lev-
`els. This analysis revealed that FITC-HA binding to circula-
`ting monocytes initially increased with rising serum HA lev-
`els during all infusion period. Nevertheless, the pharma-
`codynamics of binding vs. serum levels was different as
`serum levels began to fall in each dosing period (Fig. 4). In
`periods 1 and 2, FITC-HA binding to PBMC declined to
`baseline in rough coordination with return of serum HA lev-
`els to baseline. In period 3, FITC-HA binding levels re-
`mained above baseline while serum HA levels dropped. In-
`triguingly, by the 4th period, the rise and fall of serum HA
`levels were precisely synchronized with the rise and fall of
`FITC-HA binding to monocytes (Fig. 4). As well, the stan-
`dard deviations about the means were smallest during the
`fourth dosing period suggesting a more uniform binding in
`response of PBMC to increasing HA serum levels than was
`observed in previous dosing periods. These results suggest
`that exposure to either a high concentration of HA such as 12
`
`Dose
`
`Cmax
`
`AUC0-75 h,
`
`a
`ksyn
`
`a
`
`Vmax
`
`a
`
`Km
`
`Vmax/Km
`
`a
`Vd
`
`(mg/mL)
`
`(mg/hr/mL)
`
`(mg/hr/kg)
`
`(mg/hr/kg)
`
`(mg/mL)
`
`(mL/hr/kg)
`
`(mL/kg)
`
`1.5 mg/kg
`
`3.0 mg/kg
`
`6.0 mg/kg
`
`12.0 mg/kg
`
`26 ± 4
`
`63 ± 16
`
`131 ± 17
`
`314 ± 41
`
`103
`
`407
`
`1503
`