Molecular Imaging

1
Biodistribution of Protein Drugs
2
Origin of Peptide Pharmaceuticals

• Therapy using protein hormones dates back to the
  1920s
• Due to the Genomics Revolution cir.
  1980-199015-17 other proteins became serious
  candidates for therapy14
• Specificity

3
Cetuximab (Erbitux) bound to EGFR
Trastuzumab (Herceptin) bound to HER-2
pertuzumab
SR Hubbard Cell 2005
4
Pharmacokinetics Biodistribution

• Biodistribution is a term favored by researchers
• Pharmacokinetics is a term favored by clinicians
• Distribution,
• Metabolism,
• Elimination

5
Monitoring Biodistribution

• Radioactivity (99Tc, 123/131I, 161Tb, 88/90Y,
  111In)
• Immunohistochemistry
• Magnetic Resonance Imaging
• Fluorescence
• Radiation toxicity can occur months or years
  after administration
• The action of pharmaceuticals is drastically
  reduced by unfavorable pharmacokinetics
• Many peptide imaging molecules are directed at
  specific structures

6
Factors Influencing Biodistribution

• Specific interactions
• Nonspecific interactions
• Tissue Perfusion

7
Tissue Perfusion
Organ O2 Delivery ml/min/100 g tissue Blood Flow Rate ml/min/100 g O2 Consumption ml/min/100 g O2 Consumption/O2 Delivery ()
Brain 10.8 54 3.7 34
Heart 16.8 87 11.0 65
Skel. Musc. 0.5 2.7 0.18 34
Liver 11.6 58 2.2 18
Kidney 84 420 6.8 8
GM Coritsidis, MD State U of NY, Stony Brook
University Hospital and Medical Center Department
of Medicine Division of Nephrology
8
Blood Brain barrier

• 0.5 kDa molecular mass cutoff

http//www.satori-5.co.uk/word_articles/mcs/engagi
ng_with_mcs.html
9
Circulating Proteases
10
Reticuloendothelial System
11
Immunogenicity can be reduced by humanization

• Utilizing human sequences for all CH and the CL
  results in a chimeric antibody
• Humanization Utilizing a completely human
  antibody with only the CDR of a mouse antibody
  results

12
The Liver
Racanelli V and Rehermann B, Hepatology 2006
13

• The only way to lower the liver uptake of
  radiolabled peptides is to co-administer the same
  peptide that lacks the radiometal to cause
  competition.
• Beatty BG, O'Conner-Tressel M, Do T, Paxton RJ,
  Beatty JD Cancer Res. 1990 Feb 150(3
  Suppl)846s-851s

14
The Kidney

• 1-5 total bodyweight24
• 25 total Cardiac Output24
• Filtration, Reabsorption Secretion

15
Bohrer, et al in Journal of Chemical
Investigation (1978)

• The kidney is more efficient at eliminating
  positively charged solutes than negatively
  charged solutes.
• When the glomerular filtration barrier is
  disrupted, this difference is abolished

http//www.uhmc.sunysb.edu/internalmed/nephro/webp
ages/Part_A.htm
16
Albumin distribution gives insight to renal
processing of proteins

• Less than 100 mg/ dL Albumin is in the urine 20
• albumin is 220-320mg/dL in ultrafiltrate20
• Albumin binds to the apical membrane of proximal
  tubule cells with Kd app 100-300 nM (97-20mg/L)
  20

http//www.uhmc.sunysb.edu/internalmed/nephro/webp
ages/Part_A.htm
17
Albumin Destruction in the Kidney
M Abbate G Remuzzi Am J of Kidney Diseases
(2001)
18
A Closer Look at Megalin and Cubulin

• Megalin (517 kDa)
• Necessary for peptide scavenging in the kidney
  22
• Cubilin (460 kDa glycoprotein)
• 0.63 ?M Kd for albumin 19
• In cubulin deficient dogs the albumin excretion
  rate increases by a factor of ten or more 20
• Can be purified using albumin columns 19
• 27 ligand bindin domains!26

• Christensen, et al Pediat Nephrol 2003

19
Megalin and Cubulin are nonspecific

• In concious rats, lysozyme, cytochrome c, and
  ß2-macroglobulin compete for uptake in proximal
  tubules 25
• Cubulin binds to light chains 26, megalin does
  so with a higher affinity 28
• Tamm-Horsfall protein may be involved in protein
  re-absorption and destruction 27

20
Increasing the negative charge density of an
octreotide reduces renal accumulation

• RH
• RCOOH

21
Isoelectric Point and Pharmacokinetics
H Kobayashi et al 1999 Cancer Research
22
Fab and F(ab)2
MT micromet/MedimmuneTM MT-103
pim.medicine.dal.ca/images/atb-2.jpg
23
Isoelectric Point and Pharmacokinetics
Biodistribution of an sa-glyco-Fab (black), an
ma-glyco-Fab (checkered), an mc-glyco-Fab
(dotted), and a non-glyco-Fab (striped) at 15
(A), 45 (B), and 180 (C) minutes after injection
H Kobayashi et al 1999 Cancer Research
24
Isoelectric Point and Pharmacokinetics
H Kobayashi et al 1999 Cancer Research
25
Isoelectric Point and Pharmacokinetics

• The decreased negative charge probably causes an
  unfavorable interaction with the filtration
  barrier in the glomerulus.

26
Effects of Isoelectric Point and
Co-administration of Lysine
The more negative Fab did not accumulate in the
kidney as well The administration of Lysine can
allow for easier elimination
H Kobayashi et al 1999 Cancer Research
27
Co-administration of Basic Amino Acids
28
Co-administration of Basic Amino Acids

• Cationic amino acids diminish kidney uptake of
  Fab and F(ab)2 fragments 30
• Lysine ethyl ester was more potent than lysine
  30
• D-Lysine and L-lysine are equally effective ip or
  orally
• D-glucosamine was also effective

Corroborated by PJ Hammond et al Br J Cancer
1993 MV Pim Eur J Nucl Med 1994 TM Behr Cancer
Res 1995
29
PEGylation
FM Veronese G Pasut, Drug Discovery Today 2005
30
Pharmacokinetics of PEGylated IFN a-2a

• 1 out of 10 animals tested produced an anti
  PEG2-IFN antibody
• 10 out of 10 animals tested produced and anti IFN
  a-2a
• PEGylation drastically prolonged serum activity

P Bailon et al, Bioconjugate Chem 2001
31
Biodistribution and Pharmacokinetics of PEGylated
proteins

• Pegfilgrastim - selectively PEGylated at the
  N-terminal 31
• Cysteine, Lysine, and N-terminal PEGylating
  moieties available 31
• PEGylation may extend circulation time to a full
  week

32
(No Transcript)
33
(No Transcript)
34
N-acetylation and Modification of Basic Residues

• This method risks enhancing the immunogenicity of
  humanised peptides

JP Tarburton J Biol Response Mod 1990
35
Summary

• By far, the major point of difficulty for protein
  pharmaceuticals is the kidney.
• Renal accumulation is almost certainly a result
  of megalin and cubulin activity.
• In addition to size/effective radius, the
  isoelectric point of a protein influences its
  circulation time, with more positive proteins
  filtered easier.
• Accumulation in the proximal convoluted tubule
  seems to be hindered by co-administration of
  lysine or other aminated molecules
• The liver, spleen, and lymphatic tissues are apt
  to metabolize circulating proteins, even those
  that are not immunogenic.
• PEGylation, and pI adjustments are valid means of
  altering filtration at the glomerulus.

36
references

• 1. Kaptain S, Tan LK, Chen B (2001). Her-2/neu
  and breast cancer. Diagn Mol Pathol 10139-152,
  2. Ravdin PM, Chameness GC. The c-erb-2
  proto-oncogene as a prognostic and predictive
  marker in breast cancer a paradigm for the
  development of other macromolecular markers a
  review. Gene 15919-27, 1995
• 3. Bernstein JL, Lopez-Carrillo L, Wang L (1999).
  The epidemiology of Her-2/neu and P53 in breast
  cancer. Salud Publica Mex 41(S2)S114-S123
• 4. Elledge RM, Clark GM, Chamness GC, Osborne CK
  (1994). Tumor biologic factors and breast cancer
  prognosis among white, Hispanic, and black woment
  in the United States. J Natl Cancer Inst 86
  705-712,
• 5. Al-Abbadi MA, Washington TA, Saleh HA,
  Tekyi-MEnsah SE, Lucas DR. Differential
  expression of HER-2/NEU receptor of invasive
  mammary carcinoma between Caucasian and African
  American patients in the Detroit metropolitan
  area. Breast Cancer Research and Treatment
• 6. Stark AT, Claud S, Kapke A, Lu M, Linden M,
  Griggs J (2006). Race Modifies the Association
  between Breast Carcinoma Pathologic Prognostic
  Indicators and Positive Status for HER-2/neu.
  Cancer 104 (10) 2189-2196
• 7. Sidoni A, Ferri I, Cavaliere A, Bellezza G,
  Scheibel M, Bucciarelli E. Detection of
  Her-2/neu (c-erbB-2) overexpression and
  amplification in breast carcinomas with ambiguous
  immunohistochemical results. A further
  contribution to defining the role of fluorescent
  in situ hybridization. Anticancer Res. 26 (3B)
  2333-2337
• 8. Casalini P, Iorio MV, Galmozzi E, Menard S
  (2004). Role of HER Receptors Family in
  Development and Differentiation. J Cell Phys
  200343-350
• 9. Sawaki M, et al (2006). High Prevalence of
  HER-2/neu and p53 Overexpression Inflammatory
  Breast Cancer. Breast Cancer 13 (2) 172-179
• 10. Li CI, Malone KE, Daling JR (2002).
  Differences in Breast Cancer Hormone Receptor
  Status and Histology by Race and Ethnicity among
  Women 50 Years of Age and Older. Canc Ep Bio
  Prev 11601-607
• 11. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan
  J (2006). An Allosteric Mechanism for Activation
  of the Kinase Domain of Epidermal Growth Factor
  Receptor. Cell 125 1137-1149
• 12. Hubbard SR. EGFR Attacks on Multiple
  Fronts. Cancer Cell 7 287-288
• 13. Jurado P, et al (2002). Production of
  functional single-chain Fv antibodies in the
  cytoplasm of Escherichia coli. J Mol Biol 320
  1-10
• 14. Nispen JW, Pinder RM (1987) Neuropeptides and
  their processing Targets for Drug Design. Ann
  Reports Med Chem 22 51-62
• 15. Van Noordwijk (1988). Quality assurance of
  products manufactured by recombinant DNA
  technology Introduction and elements of a
  philosophy. Drug Res. 38(2)7943-947
• 16. Hess PN (1987). Biotechnology in bulk drug
  production objectives and strategies of process
  development. Drug Res. 37(2)101210-1215
• 17. Bachmayer H (1988). Quality control of
  protein drugs. Drug Res 38 (1)4590-591
• 18. Kobayashi H, et al (1999). The
  pharmacokinetic Characteristics of Glycolated
  Humanized Anti-Tac Fabs are Determined by Their
  Isoelectric Points. Cancer Research 59 422-430
• 19. Abbate, M., and Remuzzi, G. (2001) Am J of
  Kidney Diseases 38, 1, 196-204 Novel Mechanisms
  Implicated in Tubular Albumin Reabsorption and
  Handling Am J of Kid D July 38 (1) 196-204
• 20. Gekle, M. 2005 Renal Tubule Albumin
  Transport. An Rev Physiol 67, 573-594

37
Biodistribution of Protein Drugs
38
The Danger of Her-2 Overexpression

• The HER-2 is locked in the dimerization-competent
  conformation, making it the preferred
  homodimerization partner for all other HERs.

39
Limitations
SGMIB pI 8.1-8.5 SIPC pI 7.4-7.8 SIPMB pI
5.8-6.5