A Literature Review to Investigate if Blood Compatibility Tests are a Necessary Procedure in the Pharmaceutical Industry to Predict Adverse Toxicology Sophie Taylor

Safety Assessment is a major department within AstraZeneca The main aim of my dissertation was to investigate whether Blood Compatibility Tests (BCT’s) are a necessary procedure which have the potential to predict adverse toxicology when an animal is dosed in vivo I investigated all the BCT studies that had been conducted in Safety Assessment Alderley Park in the past 5 years If a BCT showed that intravenous (IV) dosing of a compound could cause adverse effects and the compound was still carried forward to a study, I then assessed the corresponding MTD’s or One Month Toxicity Study

IV route of administration is frequently used in man and animals for many classes of drugs In the process of developing an IV formulation its haemolytic potential must be considered Within SAUK the BCT is preferably carried out before any in vivo procedures (eg surgery) are performed The results of a BCT are dependant on the structure/class of a compound and also the constitution of the formulation that is being tested

A BCT test is split into 3 main areas for experimentation; haemolysis, erythrocyte clumping and plasma precipitation Haemolysis is the disruption of erythrocytes causing them to ‘break’ open releasing their contents into the surrounding fluid Erythrocyte Clumping is when red blood cells clump together within the plasma Plasma Precipitation is when constituents within the plasma precipitate out giving an effect similar to clumping

I investigated BCT studies conducted in house in the last 5 years I also used various search engines and databases to gain any additional data to support the findings in the studies Inclusion criteria's were applied for both the BCTs and Literature, These are as follows: BCT= A positive haemolysis result, positive erythrocyte clumping, positive plasma precipitation, being advised not to dose, being advised to dose with caution, being advised to dose at lower levels Literature= haemolysis, plasma precipitation, erythrocyte clumping, BCT’s, Information of pH’s of formulations, information on solubility and methods of solubilising compounds, regulatory guidelines in relation to IV dosing and animal welfare

To further support the review, analysis of a variety of IV toxicology studies within AZ was carried out. These were identified when the BCT that supported the study showed adverse effects

Out of the 32 BCT reports that were examined only 11 demonstrated the inclusion criteria Furthermore only 7 reports were available that had follow on MTD’s, DRF’s or One Month Studies to review The individual criteria for each study can be viewed on the following tables:

Table to show the findings of the BCT studies that produced Clumping, Haemolysis or Plasma Precipitaion in the last 5 years BCTClumpingHaemolysisPlasma PrecipitationAdvice Given 1 (human blood)No clumping observedNo haemolysis observed mg/ml= mg/ml= mg/ml=0 Can dose at 0.45mg/ml but dose with caution 2 (human and dog blood) Human-12.5µmol/ml=C 6.25µmol/ml=M (male) N (female) 3.75µmol/ml= N (both sexes) Dog-12.5µmol/ml= C (male) M (female) 6.25 and 3.75µmol/ml =N (both sexes) Human- 3.75µmol/ml=0 Dog-3.75 µmol/ml=0 Human-3.75 µmol/ml= +++ (male) ++ (female) 3.75 µmol/ml (in serum)= 0 (both sexes) Dog-3.75 µmol/ml= ++ (male) + (female) 3.75 µmol/ml (in serum)= 0 (both sexes) Dose at 3.75µmol/ml with caution at a slow infusion rate. But note the clear sex difference. Male blood more sensitive than female and human blood more than dog blood 3 (rat and dog blood)50mg/ml= M 25mg/ml=N Control (1) pH = C Control (2) pH 4.57= N 1:1 at 50mg/ml= precipitate occurred therefore no reading could be taken. 1:10 at 50mg/ml (rats)= % Rat -Control (1)-1:10=10-13% Dog-Control (1)-1:10= 0 (male) white cloudy precipitation (female) Control (1)-1:1 rat/dog=40-50% Control (2)- 1:10 or 1:1=0 rat/dog Plasma-50mg/ml= +++ (1:1 rat/dog) Control (1) and (2)=0 (1:1 rat/dog) Serum-50mg/ml=+++, 1:4 dilution=++, 1:8 dilution=0 Control (1) and (2)=0 Formulation not compatible for I.V administration in rat or dog at 50mg/ml or control (1) due to Clumping, haemolysis and Precipitaion Control (2) is ok to dose 4 (rat and dog blood)No clumping observedNo haemolysis observedDog-25mg/ml=+++ (plasma) 12.5mg/ml=0 (plasma) Rat- 25/12.5mgml=++/+ (plasma) 6.25mg/ml=0 (plasma) Dog- 25mg/ml=0 (serum) Rat-25mg/ml=+ (serum) 6.25mg/ml=0 (serum) 12.5mg/ml + Control acceptable for I.V administration to dogs 6.25mg/ml + Control acceptable for I.V administration to rats

BCTClumpingHaemolysisPlasma PrecipitationAdvice Given 5 (rat blood) 4 Compound BCT (A- D) Compound D- method 1pH adjusted with Methanesulfonic Acid, the other method 2 pH adjusted with Hydrochloric Acid (HCl) Compounds A-C=N D- Method 1+2=C Compounds A-C- 1:1/1:10=0 Compound D(methods 1+2)-1:1=1.5-6% Compound D(methods 1+2) and control-1:10=0 Compounds A-C-1:1=0 Compound D (both methods)-1:1=+-++ Compounds A-C are ok to dose, compound D dose cautiously 6 (rat blood) 2 Compounds Both compounds 1:1=N Both compounds 1:1=45- 65% Both compounds 1:10=3- 10% Control-1:1=50% 1:10=5% Both compounds 1:1=0Dose cautiously due to haemolysis 8 (rat blood) 4 Compound BCT Compounds A-D Compounds A-C and their respective controls-1:1=N Compound D-1:1=C Control for compound D-1:1=N No haemolysis observed in all 4 compounds Compounds A and B- 1:1=0 Compound C-1:1 (plasma)=+++ 10mg/ml(serum) 1:1=+ 5mg/ml(serum)1:1=0 Compound D-1:1 (plasma) 20mg/ml=+++ 5,10,20mg/ml (serum)- 1:1= +,++,+++ respectively 2.5mg/ml(serum)-1:1=0 Compounds A and B ok to dose, Compound C ok to dose Compound D was to be used cautiously starting with low concentrations and close monitoring of animals before proceeding to higher concentrations

Key: Clumping- C-Complete Clumping M- Medium Clumping N-No Clumping Precipitation- + -Weak Positive ++-Medium Grade +++-Strong Positive

Only one BCT out of the seven that were investigated showed in vivo adverse findings Following feedback from the pathologists report and the clinical pathology data a potential haemolytic effect was observed All other toxic effects were considered to be ‘normal’ for the associated class of compound

After looking at the BCT data it is difficult to associate pathology findings with the BCT This gives variability to a human trial situation - Animal studies have a higher dose level and faster infusion rate than clinical studies - Different compound solubility levels - Less toxic? Only one BCT predicted that there would be adverse toxicology if further studies were carried out

BCTs are a useful indicator to show what dose levels in vivo studies should start at Clinical pathologists can also use the data obtained in a BCT to give advice on how to proceed with dosing A BCT also indicates the pH and osmolality of the formulation which is another useful indicator in showing how the formulation will react upon contact with the blood-again showing concern for animal welfare This also provides useful information for formulation trials, scale up and batch checks of different batches of compound The recommendation is that we will continue to perform BCT’s due to animal welfare benefits

Method- the way that a BCT is performed can be looked at: -Test is performed in a static test tube - Has an effect on a variety of variables which can potentially invalidate BCT results- doesn’t truly reflect an in vivo situation Improvements- -Make the test more dynamic - Perform the test at 37°C (body temperature)