Cambridge MedChem Consulting

Distribution and Plasma Protein Binding

The distribution of a drug is often measured as a volume of distribution (Vdss), and is a measure of the fluid volume that would be required to contain the amount of drug present in the body at the same concentration as that measured in the plasma. It is important to note that the measured volumes are not actual physical volumes but are apparent volumes based on the dilution of drug in plasma. However the liquid volumes in various compartments can put the observed volumes of distribution into context.

Typical liquid volumes for a 70kg man are (% and L)

total water: 60%, 42 L
intracellular volume: 40%, 28L
extracellular volume: 20%, 14L
plasma volume: 4%, 3L
blood volume: 8%, 5.5L

Examples of compounds with different volume of distribution

PPB

Drug VD Comments Properties
Warfarin 8L Reflects a high degree of plasma protein binding. PPB=99%, T1/2=37-89h
Theophylline 30L Represents distribution in total body water. PPB=40%, T1/2=8h
Chloroquine 15000L Highly lipophilic molecules which partitions into body fat PPB=55%, T1/2=1-2 months
NXY-059 8L Highly-charged hydrophilic molecule. PPB=30%, T1/2=2-4h

The distribution of drug from plasma to target tissues can be effected by a number of factors, such as high molecular wight, but perhaps the most important is Plasma Protein Binding (PPB). Compounds that are extensively bound to plasma proteins will have a low Volume of distribution (Vdss), can have long plasma half-lives (T1/2), and have low clearance (Cl) by both liver (hepatic) and kidney (Renal) routes. High plasma protein binding may also have an impact on efficacy since it is usually the free fraction of drug that is responsible for the pharmacological action.

Measuring Plasma Protein Binding

A quick way to get an idea if plasma protein binding may be an issue is to add serum to the in vitro screen, if the apparent affinity drops it is often evidence that the ligands are binding to plasma proteins, you can also add the purified components (e.g. Human serum albumin) to get an idea of which proteins might be involved.

Equilibrium dialysis is the most widely accepted method for assessing plasma protein binding as non specific binding effects are minimised compared with other methods such as ultrafiltration, but is a relatively slow process, from 4-24 hours at 37 C. For very highly bound compounds it can be difficult to measure the concentration of unbound drug, in these cases it may be better to use 10% plasma in buffer. Very poorly soluble compounds may also give anomalous results as will compounds that are unstable in plasma.

Ultrafiltration is suitable for fast screening and requires very little compound. Compounds are incubated in plasma for 1 hour, then centrifuged at 2700 rpm 10 mins. The supernatant is then analyzed for drug concentration. This method can be susceptible to non-specific binding.

A publication from the Univ of Washington DOI describes an inexpensive Microdialysis Device for Measuring Drug–Protein Binding (DIYM).

The device is based on the standard equilibrium dialysis method to measure the fraction of low molecular weight compound bound to proteins. It is constructed from a standard polypropylene 96-well plate, dialysis tubing, and low viscosity epoxy resin. The device can be readily prepared for a small fraction of the cost of a commercial, multi-chamber, micro-dialysis device.

The results obtained agree favourably with literature values.

Compound DIYM (%) Lit (%)
Dextromethorphan 66.8 65
Diclofenac 98.0 99.5
Mefloquine 98.9 >98
Methotrexate 54.0 50.4
Paclitaxel 94.2 95
Progesterone 97.0 98
Propranolol 82.5 82
Testosterone 93.3 98

Internal standards such as Warfarin, Propanalol, Digoxin or Diclofenac are used. Plasma from different species can also be used.

The results are usually expressed as fraction unbound (fu), where [D] is the free drug concentration and [DP] is the concentration of drug protein complex.

fu_equation
or as % Protein Binding

percentPB

Until the drug is greater than 90% bound there is not usually any issues, once you get to >99% bound then plasma protein binding is likely to have a significant impact.

Strategies to reduce plasma protein binding

I’ve been compiling a database of measured plasma protein binding data for small molecules taken from the literature, as the plot below shows, it is clear that the majority of small molecules bind to plasma proteins, with many being found to be >90% bound.

percentBindingplot

Different classes of compounds are more likely to have problems with PPB, acids tend to display the highest affinity for plasma proteins as shown in the graph below (taken from the talk by Rupert Austin at the RSC MedChem School). The graph shows a plot of log(%bound/%free) versus LogD. In general the more lipophilic compounds display greater plasma protein binding, in the case of acids compounds with LogD > 0 are almost all >99% bound. Neutral and basic molecules usually require LogD greater than > 4 before plasma protein binding becomes an issue. Several in silico models have been described DOI in which lipophilicity and molecular weight have critical and independent affects on plasma protein binding.

The major plasma proteins that can interact with drugs are:

In general, acidic and neutral drugs primarily bind to albumin, and basic drugs bind to the acidic alpha-1 acid glycoprotein or Lipoproteins

Albumin

(MWt 66 KDa), (HSA) is the most abundant protein in human blood plasma at 3.5 to 5.0 g/dl. It is produced in the liver. Albumin comprises about half of the blood serum protein, (30 to 50 g/L). There are almost 100 crystal structures of albumin, 58 with ligands bound, in the Protein Database PDB. An example is 2vdb in which S-Naproxen is bound. Mutations in the albumin protein have been shown to have an effect on drug binding DOI90466-V). Human serum albumin has multiple potential binding sites and this is nicely illustrated in the X-ray structure of HSA complexed with difunisal (green) shown below. DOI.

has_diflunisal

Alpha-1-acid glycoprotein

(MWt 44 KDa), (AAG, AGP, Orosomucoid, ORM ) has a normal plasma concentration between 0.04-0.1 g/dl (1-3% plasma protein). It is negatively charged at physiological pH and interacts mainly with basic drugs, including beta-adrenergic-receptor blockers, antidepressants, neuroleptics and local anaesthetics. There is a crystal structure in the PDB 3BX6 the branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation pattern. The crystal structure of the A variant of human alpha1-acid glycoprotein and amitriptyline complex 3APV has been recently de[posited in the PDB and highlights pi-stacking interactions are important in ligand binding.

alpha_amytrip

Table of Major Plasma Proteins

Plasma protein Mol Wt(kDa) Conc in Plasma (g/dl) Drugs Binding to protein Function
Albumins 69 3.5-5.0 Acidic maintains colloid osmotic pressure and transport insoluble molecules
alpha1-acidic glycoprotein 44 0.04-0.1 Basic acute phase reactive protein
Lipoprotein 200-3400 Varies Basic Fatty acid transport
Globulins 140 2.0-2.5 Participate in immune system
Steroid Binding globulin 53 0.003-0.007 Steroids
Fibrinogen 400 0.2-0,4 Blood coagulation

Examples of specific blood proteins

Prealbumin, Alpha 1 antitrypsin , Alpha 1 acid glycoprotein, Alpha 1 fetoprotein, alpha2-macroglobulin, Gamma globulins, Beta 2 microglobulin, Haptoglobin, Ceruloplasmin, Complement component 3, Complement component 4, Lipoproteins, C-reactive protein (CRP), Lipoproteins (chylomicrons, VLDL, LDL, HDL), Serum amyloid P component (SAP), Transferrin, Transthyretin (TTR), Prothrombin, MBL or MBP,

Binding of some drugs to plasma proteins

Albumin

Ceftriaxone (A), Clindamycin (A), Clofibrate (A), Dexamethasone (N), Diazepam (N), Diazoxide (A), Dicloxacillin (N), Digitoxin (N), Etoposide (N), Ibuprofen (A), Indomethacin (A), Nafcillin (A), Naproxen (A), Oxacillin (A), Phenylbutazone (A), Phenytoin (A), Probenecid (A), Salicylic add (A), Sulphisoxaole (A), Teniposide (N), Thiopental (A), Tolbutamide (A), Valproic acid (A), Warfarin (A).

Albumin and AAG

Alprenolol (B), Carbamazepine (N), Disopyramide (B), Erythromycin (B), Lidocaine (B), Meperidine (B), Methadone (B), Verapamil (B).

Albumin and Lipoproteins

Cyclosporine (N), Probucol (N).

All

Amitriptylline (B), Bupivacaine (B), Chlorpromazine (B), Diltiazem (B), lmipramine (B), Nortriptyline (B), Perazine (B), Propanolol (B), Quinidine (B).

A=Acid; B=Base ; N=Neutral.

Implications of Plasma Protein Binding

The efficacy of drugs can be compromised by high plasma protein binding since only the unbound fraction is available for binding to the molecular target.

Clearance of a drug can be affected by plasma protein binding, in general it is only the unbound drug that is subject to hepatic clearance. Similarly renal clearance is reduced for compounds with high plasma protein binding. The volume of distribution at steady state will also be influenced by protein binding and consequently the terminal half-life.

Drugs can compete for the same binding site and thus there is the potential for drug-drug interactions, this would be a particular concern for highly protein bound drugs that have a low therapeutic index such as warfarin.

Whilst the plasma proteins serve similar functions and are at similar concentrations across species, small differences in the protein sequence can have an impact on the extent of protein binding, thus in vitro evaluation in multiple species is advisable.

Updated 18 November 2017