# Presentation: 527429

##  Pharmacokinetic and Pharmacodynamic Considerations in the Development  of Macromolecules

- Pamela D. Garzone, Ph.D.

## OUTLINE OF LECTURE TOPICS

- Macromolecules
- Interspecies Scaling
- Pharmacokinetic Characteristics
- Scientific Issues
- Pharmacodynamics
- Monoclonal Antibodies

**Notes:**
Good Evening! I would like to thank my hosts for the opportunity to be here again. I believe this is my 5th year and its been great. And in particular for this year, I have updated my slides to include more on monoclonal antibodies due to the number of approvals and number of monoclonal antibodies currently in development.  So this evening, here is an overview of my presentation.    

## REPRESENTATIVE MARKETED MACROMOLECULES

**Notes:**
At the time when I first began this lecture and was working on the chapter for the first edition of the Clin Pharm book that goes along with this course, these were the biologics that were marketed- also included in this list would be recombinant human insulin. Since that time, which seems like a long time ago, the macromolecule market has a new look.

## APPROVED MONOCLONAL ANTIBODIES

| Name | Approval | Indication |
| --- | --- | --- |
| Avastin
Bevacizumab  | Feb, 2004 | First line (with 5-FU) in metastatic colon CA |
| Erbitux
Cefuximab | Feb, 2004 | Alone or in combination in metastatic colon CA |
| Raptiva
Efalizumab | Oct, 2003 | Moderate to severe psoriasis |
| Xolair
Omalizumab | June, 2003 | Asthma |
| Humira
Adalimumab | Dec, 2002 | Prophylaxis of acute organ rejection |
| Campath
Alemtuzumab | May, 2001
 | Second line treatment of -cell CLL in patients  |

**Notes:**
You can see from this slide the FDA has approved a number of monoclonal antibodies. In 2004 alone, Avastin, Erbitux,Tarceva and the infamous Tysabri were approved. As you know, Tysabri since then was withdrawn from the market until a complete safety evaluation was done. There will be a delay before it comes back on the market but the companies are now permitted to dose patients enrolled in a clinical trial. 

## ASSAYS FOR MACROMOLECULES

- Immunoassays
- ELISA (Enzyme-Linked Immuno-sorbent Assay)
- RIA (Radioimmunoassay)
- IRMA (Immunoradiometric Assay)
- RRA (Radioreceptor Assay)

## INTERSPECIES SCALING OF MACROMOLECULES

- Factors to Consider
- Species specificity
- Glycosylation and sialation
- Binding proteins
- Size, shape and charge
- Relative abundance of tissue receptors

## ALLOMETRIC EQUATIONS FOR SOME MACROMOLECULES

**Notes:**
It was in 1991, that a paper was published by Mordenti et al out of 
Genentech suggesting that proteins can scale across species. I show two of those proteins here –GH and rt-PA.
The theoretical exponents for V1 and CL, as you learned from Dr. Dedrick’s lecture are 0.8 to 1.0 for V1 (in mL) or 0.6 to 0.8 for CL (in mL/hr). As you can see here, the exponents are within this range except for rH-FIX.
On a theoretical, when one does not have PK data, results can be scaled with the theoretical values, 0.67, 0.7 or 0.75 depending on mass units (BSA or mg/kg)  

## INITIAL COMPARTMENT VOLUME PREDICTED BY ALLOMETRIC SCALING COMPARED WITH OBSERVED V1

**Notes:**
Using those equations and substituting the human weight, predictions on 1 were made and after clinical trials, compared to observed values obtained in patients. As one might expect, rH-FIX was off almost 2 –fold while the others were reasonably close.

## ELIMINATION CLEARANCE PREDICTED BY ALLOMETRIC SCALING COMPARED WITH OBSERVED CL

**Notes:**
Again predictions on CL compared to observed in patients. Factor IX and IL-12 were off but the others were reasonably close. Since that time, there have been several publications that used approaches other than simple allometry shown here. For instance, Mahmood and Balian (2004) used three approaches: simple allometry, maximum life span potential and product of CL and brain weight as well as comparing results of 2 species with 3 species. They looked at 15 currently marketed products Drugs with exponents >1 required correction factors. Also more than 2 species proved more accurate. 

## ALLOMETRIC EQUATIONS for EGF Mab PK PARAMETERS

| Parameter 
(Y) | Coefficient 
(a) | Exponent
(b) | r |
| --- | --- | --- | --- |
| Vd (mL) | 219 | 0.84 | 0.92 |
| CL (mL/hr) | 4.07 | 0.85 | 0.94 |

**Notes:**
The authors conducted PK studies in mice, rats, rabbits and dogs after a single administration of 16 mg/kg, 8 mg/kg, 1.5 mg/kg and 0.2 mg/kg, respectively, of a murine Mab ior EGF/ r3. .  EGF concentrations were analyzed either by a radioreceptor assay (mice and rats) or a sandwich ELISA method (rabbit, dog and human). 

## COMPARISON BETWEEN the PREDICTED EGF PK PARAMETERS and OBSERVED PK PARAMETERS

| Parameter 
(Y) | Predicted PK Parameter
Estimate | Observed PK Parameter in Cancer Patients |
| --- | --- | --- |
| Vd (L/kg) | 0.01 | 0.04 |
| CL (mL/hr/kg) | 0.22 | 0.98 |

**Notes:**
Three female patients with non-small cell lung cancer were also studied.  They were participating in a Phase 1 trial and received a single IV infusion of 400 mg. The actual clearance in patients with cancer was four-fold greater than the predicted value.  The authors proposed that variation may suggest other drug clearance processes occur in patients with cancer that would not be present in healthy subjects or predicted by studying normal animals. Even with this disparate result, the authors used the scaling factor for clearance (0.85) to assist in the design of dose regimens for a clinical trial .

## PHARMACOKINETIC CHARACTERISTIC OF MACROMOLECULES

- Endogenous concentrations
- Absorption
- Distribution
- Metabolism
- Elimination

## THE PROBLEM OF ENDOGENOUS CONCENTRATIONS OF MACROMOLECULES

- Endogenous concentrations - What do you do with them?
- Two examples
- Growth Hormone
- Erythropoietin

## Growth Hormone

- Albertsson-Wikland K, et al. Am J Physiol 1989;257:E809-14.)

**Notes:**
A deconvolution method is employed to minimize the influence of circulating endogenous GH on PK parameter estimates derived from exogenously administered growth hormone.  In this method, the 24-hour secretory rate of GH is estimated by approximating endogenous plasma GH concentration data with cubic spline smoothing Analysis of plasma GH-concentration profile by a model-based deconvolution technique.  Panel A: observed concentrations (●) fitted by a spline approximation curve.  Panel B: plasma GH concentrations (●)and calculated GH secretory rate with 95% confidence limits (line and stippled area).  Panel C: GH secretion (thin line) and cumulative secretion with 95% confidence limits (line and stippled area). 

## ERYTHROPOIETIN

- Cheung et al CPT 1998; 64:412-423

**Notes:**
a cross-over study design can be employed such that subjects are randomized to placebo or treatment on one occasion and the alternate regimen on a second occasion, assuring an adequate washout period between the two occasions.  The endogenous concentrations determined in the same subjects after placebo administration can be subtracted from the matching sample collected after treatment administration.  This design accommodates the intrasubject variability and variations in endogenous concentrations due to pulsatile secretion, but assumes that the two separate study days are similar.
In this study, Epoetin alfa was administered as single subcutaneous doses of 300, 450, 600, 900, 1200, 1350, 1800, and 2400 IU/kg and in multiple subcutaneous dose regimens: 150 IU/kg 3 times a week for 4 weeks and 600 IU/kg once per week for 4 weeks in 2 open-label, randomized placebo-controlled studies in healthy volunteers. There was also a placebo group. In addition, 3 samples were collected prior to administration. Normal, circulating erythropoietin is about 10 mIU/mL. The authors subtracted the average of the 3 baseline values  before determining the PK parameters and  in order to do PK/PD modeling with reticulocyte counts.

## ABSORPTION OF MACROMOLECULES

- Flip-flop model
- Site of administration

## 	RELATIONSHIP BETWEEN MW AND LYHMPHATIC ABSORPTION OF WATER SOLUBLE COMPOUNDS

- Supersaxo A et al. Pharm Res 1990; 7:167-169

- Inulin

- Cytochrome C

- IFN-alpha-2b

**Notes:**
In 1990, Supersaxo, Hein and Steffen published a paper on the effect of molecular weight on the lymphatic absorption of water soluble compounds after SQ administration. The cumulative recovery of these compounds in lymph draining from the site of SQ administration in sheep was made. Inulin, with a MW of 5200, cytochrome C with a MW of 12,300. and IFN-alpha with a MW of 19,000.  The conclusions from this study were:
In the MW range studied, there was a linear relationship between the MW and the proportion of the dose absorbed lymphatically
Molecules with MW > 16,000 are absorbed mainly by the lymphatics that drain the injection site.

## COMPARISON OF ABSORPTION AND ELIMINATION RATE CONSTANTS

| Macromolecule | Route of 
Administration | Ka
(hr-1) |  Ke
(hr-1) |
| --- | --- | --- | --- |
| 
 GH | 
SC
IV | 
0.23  0.04  
 | 
0.43  0.05
2.58 |
| 
IFN--2b | 
SC
IV | 
0.24 | 
0.13
0.42 |
| 
Erythropoietin | 
SC
IV | 
0.0403  0.002 | 
0.206  0.004
0.077 |

**Notes:**
There are several important principles on this slide. The first is that in order to determine if ‘flip flop’ PK occurs, you must compare it to the parameter estimates obtained after IV administration. The second is the ‘flip-flop’ model wherein if ka is less than ke, absorption is rate limiting and the terminal of the curve reflects the absorption rate. I believe this was also addressed in one of the early lectures last year. Normally, the absorption rate constant, ka, is greater than the elimination rate constant, ke, elimination of the molecule from the body is the rate-limiting step and the terminal portion of the concentration-time curve is primarily determined by the elimination rate. so few published pharmacokinetic studies include IV administration to assess whether or not the macromolecule follows flip-flop pharmacokinetics.

## SITE OF INJECTION EFFECTS ON EPO ABSORPTION

- Jensen JD et al Eur J Clin Pharmacol 1994; 46:333-337

**Notes:**
In eleven healthy subjects, EPO was injected into the thigh on one occasion and on the second occasion, the abdomen. Samples were collected up until 144 hours and serum erythropoietin was measured with a RIA. The MRT was significantly longer in the thigh (32.7 hr) vs. the abdomen (26.2 hr) as was the absorption half-life, 14.9 in the thigh vs. 12.3 in the abdomen. The t1/2 was not significantly different, 4.4 vs 4.8 hr; the bioavailability was not significantly different. A twin peak configuration was noted was observed occurring around 24 hours, that was only observed in a couple of subjects after abdominal injection. The minor differences may be due to regional variations in lymph flow, regional blood flow due to activity.

## DISTRIBUTION OF MACROMOLECULES

- Volume of Distribution
- Binding Proteins

## DISTRIBUTION VOLUMES OF REPRESENTATIVE MACROMOLECULES

| Macromolecule | MW
(kDa) | V1
(mL/kg) | Vss
(mL/kg) |
| --- | --- | --- | --- |
| Inulin | 5.2 | 55 | 164 |
| Factor IX | 57 | 136* | 271* |
| IL-2 | 15.5 | 60 | 112 |
| IL-12 | 53 | 52 | 59 |
| G-CSF | 20 | 44 | 60 |
| rt-PA

* Calculated from literature | 65 | 59 | 106
 |

**Notes:**
Proteins and Mabs distribute initially into the plasma volume and then more slowly into the interstitial fluid space.  It can be seen from this slide that the initial distribution volume of interleukin-2 (IL-2) IL-12, G-CSF and rt-PA approximates that of plasma volume.  In contrast, the initial distribution volume of FIX is approximately twice that of plasma volume.  On the other hand, the volume of distribution at steady state (Vd(ss)) for IL-12, granulocyte colony stimulating factor (G-CSF), and recombinant tissue plasminogen activator (rt-PA) are considerably smaller than the Vd(ss) of inulin, a marker for extracellular fluid space (ECF).  When distribution volume estimates are much less than expected values for ECF, they could reflect the slow transport of large molecules across membranes and the fact that either assay sensitivity or sampling time have been inadequate to characterize the true elimination phase of the compound. 

## PHARMACOKINETICS  of MARKETED MONOCLONAL ANTIBODIES 

| Mabs | Molecular Weight
(kD) | T1/2a
(Days) | V1a
(L) | Vssa |
| --- | --- | --- | --- | --- |
| Avastin | 149 | 13-15 | 3 | 3.5-4.5    L |
| Erbitux | 152 | NDb | 2.7-3.4 |  2-3    L/m2 |
| Raptiva | 150 | 6-7.5c | NRd | 9       L e |
| Humira | 148 | 12-18 | 3 | 5      L |
| Campath | 150 | 1-14f | NRd | 7-28   L |

## EFFECTS & RELEVANCE OF MACROMOLECULE BINDING TO α2-MACROGLOBULIN

**Notes:**
Alpha2-macroglobulin, one of the major proteins in the serum, is highly conserved across species and can bind many molecules, such as cytokines, enzymes, lipopolysaccharide (LPS) and ions such as zinc and nickel (49).  Alpha2-macroglobulin is found in extravascular secretions, such as lymph.  It exists in two forms, a slow native form and a fast form, an alpha2-macroglobulin-protease complex that results in a conformational change that increases electrophoretic mobility.  This conformational change results in exposure of a hydrophobic region that can bind to cell surface receptors such as those on hepatocytes. Three different mechanisms for this binding have been identified (52).  The binding can be noncovalent and reversible.  An example of this type of binding is seen with growth factors such as tissue growth factor-β (TGF-).  Second, the binding to alpha2-macroglobulin can be covalent, and the third mechanism involves covalent linkages with proteinase reactions. Subsequent to the binding, the pharmacokinetics and pharmacodynamic properties of the macromolecule may be altered 

## HYPOTHETICAL MODEL of the BINDING EFFECTS of IGF-1

**Notes:**
Insulin-like growth factor-1 (IGF-1) is produced by many tissues in the body and it has approximately 50% structural homology with insulin.  In plasma, IGF-1 exists as “free” IGF-1 and “bound” IGF-1. To date, eight binding proteins (designated IGFBP-1 through 8) have been identified, with IGFBP-3 the most abundant.  The binding proteins vary in molecular weight, distribution, concentration in biological fluids, and binding affinity 
IGFBP-3           Exists in a 42 kDa “complete” form and a 31 kDa “fragment”   
IGF-1              Displaces IGF-2 from IGFBP-3   
                      Drives reaction towards forming a ternary complex with ALS; Formed complex is retained within the 		    ntravascular space.   
		    Suppresses GH secretion   
IGF-2             Induces IGFBP-2  
GH                Increases synthesis of IGFBP-3 and ALS

## METABOLIC EFFECTS OF MACROMOLECULES

- Effects on P450s

## EFFECTS OF MACROMOLECULES ON P450 CYP ENZYMES

**Notes:**
With the exception of IL-2, these cytokines depress the activity of CYP enzymes.  Data on cytokine-mediated depression of drug metabolizing ability has been obtained primarily in rodents under conditions of inflammation or infection  The reduction in drug biotransformation capacity parallels a decrease in total CYP content and enzyme activity, and is due primarily to a down-regulation of CYP gene transcription but modulation of RNA and enzyme inhibition may also be involved 
In vitro study results have been consistent with those obtained in vivo.  For example, in primary rat hepatocyte cultures IL-1, tumor necrosis factor (TNF) and interleukin-6 (IL-6) concentrations ranging from 0.5 to 10.0 ng/mL) suppressed the expression of CYP2C11 mRNA 
The clinical significance of the aforementioned findings is unknown  a pharmacokinetic interaction was not demonstrated between IFN-α-2b and ribavirin or an additive effect of the combination therapy on safety assessments.  In another study, administration of IFN-α prior to the administration of cyclophosphamide significantly impaired the metabolism of cyclophosphamide and 4‐hydroxy-cyclophosphamide. In contrast, the administration of IFN-α after cyclophosphamide resulted in higher 4-hydroxycyclophosphamide concentrations and produced a significant decrease in leukocyte count 
Doxorubicin, given before IL-2, prevented IL-2-induced lymphocyte rebounds but not did not qualitatively alter non-major histocompatibility complex-restricted cytotoxicity. there are few reports that evaluate the extent and clinical significance of corresponding PK or PD changes.

## EXCRETION OF MACROMOLECULES

- Contributions of kidney and liver
- CHO vs E. Coli produced
- Receptor mediated clearance

## RELATIONSHIP BETWEEN MOLECULAR WEIGHT AND ELIMINATION CLEARANCE

## LIVER CELL SURFACE RECEPTORS FOR CLEARANCE OF CARBOHYDRATES & MONOSACCHARIDES

**Notes:**
This slide depicts the cell surface receptors that catabolize proteins. The most famous of these, The asialoglycoprotein receptor or the ASHWELL receptor was first identified 
 Gal – galactose
NAc – N-acetyl
GalNAc - N-acetylgalactosamine
Man – Mannose
GLcNAC – N-glycans N-acetylglucosamine
Fuc - fucose

## DIFFERENCES BETWEEN rhEPO AND NESP (NOVEL ERYTHROPOIESIS-STIMULATING PROTEIN)

- rhEPO
- 165 normal amino acid sequence
- Up to 40% carbohydrate
- 3 N-linked sugar chains
- Up to 14 sialic acids
- 30.4 Kd
- Plasma T1/2= 4-8 hrs

- NESP
- 5 amino acid exchanges
- Up to 52% carbohydrate
- 5 N-linked sugar chains
- Up to 22 sialic acids
- 38.5 Kd
- Plasma T1/2= 24 hrs

**Notes:**
rhEPO has a relatively short half-life, requiring 2-3 times weekly administration. Aransep was recently approved and you can see in this slide that its half-life is much longer. What is different about the two molecules. NESP has 2 additional N-gllycans compared to EPO. It is the composition of these N-glycans that are important in determining biologic activity and its rate of degradation.the terminal end of monosaccharides of the N-glycans is commonly sialic acid (N-acetylneuraminic acid) attached to galactose – NESP is considered a hyperglycosylated product  

## METABOLIC FATE OF EPO

- Hepatocytic galactose receptor mediated uptake

- Renal Filtration

- EPO receptor mediated uptake

*Extracellular deglycosylation and proteolytic cleavage*

*Intracellular degradation*

*Intracellular degradation*

*Tubular reabsorption and degradation*

**Notes:**
There remains lack of knowledge of the main site and mechanism of removal of EPO from the circulation.This slide shows possible sites of catabolism. Heptocytes can take desialylated glycoproteins via the galactose receptor. The kidney may not be a primary site of removal but may contribute although several studies investigating EPO pharmacokinetics in normal and in patients with chronic renal failure have not shown differences between the two groups. The most likely catabolic site is by EPO-receptor mediated uptake and degradation in erythropoietic tissues. So that the greater the activity and myeloid red blood cell precursors, the lower the plasma EPO level  

## SERUM CONCENTRATION-TIME PROFILES FOR CHO VS. E. Coli PRODUCED GM-CSF 

_**Type**_        _**Cmax**_       _**t1/2**__****_     _**t1/2**__****_
- CHO          138            20         68
- E. Coli         57              8         75

- Mortensen HD et al. Eur J Haematol 1993; 50:32-36

## SERUM CONCENTRATION-TIME PROFILES FOR NON-GLYCOSYLATED VS. GLYCOSYLATED G-CSF

- Watts et al. Br J Haematol 1997; 98:474-479

## RELATIONSHIP BETWEEN G-CSF CLEARANCE AND ABSOLUTE NEUTROPHIL COUNT

- Ericson SG et al. Exper Hematol 1997; 25:1313-1325

## MONOCLONAL ANTIBODY PRODUCTION

|  |
| --- |

**Notes:**
Monoclonal antibodies, by definition, are produced by a single clone of hybridoma cells, i.e., a single species of antibody molecule. The production of antibodies appears to be simple (Figure 32.2).  However, their commercialization is challenging.  The need for specificity makes the market small; thus, the costs of doing clinical trials for small markets are unattractive to most companies.  Quality control of the production and manufacture of monoclonal antibodies is another issue since a high degree of purification and low degree of contamination is necessary before approval.  Finally, a major limitation is the stability of the mammalian cells expressing the immunoglobulin  

## HUMAN IgG

**Notes:**
The Ig molecule consists of the Fc region and the Fab region allowing for multivalent, high avidity and specificity.  It should be noted that immunoglobulin sequences are conserved across species such that considerable homology between mouse and human variable regions exists ( 

## IgG and SINGLE-CHAIN Fv

## CONCEPT OF ANTIBODIES

**Notes:**
First murine monoclonal antibody to be approved in 1986 was OrthoClone OKT3. Total murine antibodies provoke a strong human anti-murine antibody (or HAMA) response which restricts their usefulness for repeated administration.
Chimeric antibodies –the whole of the variable regions of a rodent antibody are expressed along with human constant regions. Reduces the HAMA response
Humanized antibodies-

## PROPOSED HUMAN PLASMA CLEARANCE of DIFFERENT ANTIBODY MOLECULES 

| Antibody Molecule | Molecular Weight (kD) | Relative Plasma Clearance (Cl) |
| --- | --- | --- |
| Native intact human IgG | 150 |          ≈ 21 days |
| Fully human/humanized | 150 |  |
| Chimeric human-mouse IgG | 150 |  |
| Whole mouse IgG | 150 |  |
| F (ab)2 | 110 |  |
| Fab’ | 50 |  |
| Single chain FV (scFV) | 25 | ≈ 1 day |

**Notes:**
The most characteristic features of monoclonal antibodies are their low blood clearance and prolonged elimination half-life.  It has been demonstrated for both intact Mabs and fragments that clearance is inversely related to molecular size (Table 32.2) (11, 12).  Detailed investigations have been undertaken to explore the specific Ig structures that may be affect clearance and half-life.  In particular, the Fc receptor, FcRn, has been shown to play an important role in determining Ig half-life, and specific sequences in the CH2 and CH3 regions of IgG regulate clearance rate through their interaction with FcRn 

## DESIGN OF ANTIBODIES

- Molecules that can be attached:
- Enzymes
- Toxins
- Viruses
- Cationic tails
- Biosensors

## CHARACTERISTICS THAT AFFECT THE PHARMACOKINETICS OF MACROMOLECULES

- Physical characteristics
- Post-translational modification
- Binding
- Route of administration
- Duration of administration
- Frequency of administration

## PATIENT CHARACTERISTICS THAT AFFECT PHARMACOKINETICS OF MACROMOLECULES

- Age
- Gender
- Disease
- Concurrent drugs

## EFFECTS OF GENDER ON GROWTH HORMONE PK/PD

- Daily rhGH dose/kg required to normalize IGF-1 response in GH deficient women is higher than in men
- Estrogen replacement also significantly increases rhGH dose requirement

## PHARMACODYNAMICS OF MACROMOLECULES

- Important considerations
- Regimen dependency
- Endpoints
- Models

## REGIMEN DEPENDENCY OF IL-12 PHARMACOKINETICS AND IFN-γ STIMULATION

- Motzer RJ et al. Clin Cancer Res 1998;4:1183-1191

## PHARMACODYNAMIC ENDPOINTS

- Easy - replacement proteins
- rFIX
- Difficult- casade of events
- IGF-1

## RELATIONSHIP BETWEEN rFIX CONCENTRATION AND ACTIVITY

- Schaub et al. Seminars in Hematology 1998; 35:28-32

## PK-PD MODEL OF rhGH WITH MEASURED VS. PREDICTED [IGF-1] AFTER SINGLE AND DAILY SC rhGH INJECTIONS

- Sun YN et al. JPET 1999; 289:1523-1532

## PHARMACODYNAMIC ENDPOINTS

- Omalizumab:  Free IgE levels
- 			      Clinical outcomes
- Basiliximab:   Soluble IL-2 receptor
-                     CD25+ T lymphocytes ≦1%

**Notes:**
Omalizumab= Xolair
Basiliximab =Simulect. Indicated for renal transplantation in combination with immunosuppressive agents.

Targeted therapies lend themselves well to using cell based assays - fluorogenic assay kits that enable researchers to easily measure enzyme activity in living cells, enhancing their ability to detect small changes in enzyme activity, cell migration, cell adhesion, etc. 

## Summary

- Use scientific judgement and good sense in the interpretation of PK/PD results with macromolecules
- Application of PK principles that have been developed work with macromolecules
- Difficult to select the most appropriate pharmacodynamic endpoint

## Acknowledgements

- Genetic Institute
- PK/PD Sciences
- Dr. Joyce Mordenti
- Dr. Art Atkinson