Chapter 5: Biological and Solubility Evaluation of the Substituted Ajoenes
5.3 Animal study ( in vivo )
The study was conducted by Dr Catherine Kaschula from the UCT IIDMM (Institute of Infectious Diseases and Molecular Medicine) to assess the in vivo activity of the PMB-amide 29, which was chosen to be a suitable candidate based on its polar nature. Based on the solubility data, the latter was considered as having a good enough solubility for delivery into the mouse subject, since one of the objectives was to enhance the aqueous solubility of the ajoene molecules. There are reports in the literature that suggest that compounds with phenols may be toxic to subjects as they form reactive metabolites. Similarly, phenolic acetates (27) could be hydrolysed to the phenol, and the PMB-ester 28 could also be hydrolysed (to its acid form), which would also probably be toxic. All these factors suggested that the other three analogues were not as suitable candidates as amide 29.52
The study comprised of 20 nude mice, which each received a subcutaneous injection of 2.50 x 105 A375 cells (in 100 L of PBS) into their hind right quarter. The mice were split into two groups of 10 mice assigned to the treatment or the control group and the tumours were allowed to grow for 21 days.
The test mice each received PMB-amide (8mg/kg) from the day of inoculation every day for the duration of the study by intraperitoneal injection, in which the drug was solubilised easily into a solution of 2.5% chondroitin, 10% DMSO and PEG400. The tumours were measured twice weekly and recorded as a tumour volume (volume calculated by: (length2 x height)/2).53 Upon completion of the study on day 21, the mice were sacrificed (halothane inhalation), the tumours were removed and accurately weighed to determine if there had been any size reduction during the course of the study (see Figure 29).
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67 Figure 29: A steady increase in weight of the mice from the treatment and control groups was observed over the duration of the study which indicated that the drug was not cytotoxic to the mice.
The results revealed that treatment with the PMB-amide had no significant effect on the tumour growth (see Figure 30) when compared with their control counterparts. From the collected data, it was apparent that there was no tumour volume reduction observed in the treatment group; rather the tumours steadily grew over time (see Figure 30 (i)). A similar result was observed for mice that were treated with the advanced lead (bis-PMB) 5 that had also returned excellent in vitro activity.
(i) (ii)
Figure 1: Figure 30: (i) Growth of tumours - Treated vs Untreated. Tumour size were measured by calipers (volume calculated by: (length2 x height)/2) 53; (ii) Tumour size as measured by weight of tumour obtained from biopsy.
A375 Nude mice treated with PMB-amide
28 Nov 4 Dec 7 Dec 11 Dec 14 Dec 19 Dec 8
12 16 20 24 28
32 mouse 1
mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 mouse 8 mouse 9 mouse 10
Weight / g
A375 Nude mice - control
28 Nov 4 Dec 7 Dec 11 Dec 14 Dec 19 Dec 17
21 25
29 mouse 1
mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7
mouse 8
mouse 9 mouse 10
Weight / g
A375 tumours in nude mice, PMB-amide treatment
6 days 9 days 13 days 16 days 21 days 0
100 200 300 400 500 600 700
800 control
PMB-amide
Tumour volume / mm3
A375 nude mouse xenografts, tumour mass on day 21.
PMB-amide treatment
control PMB-amide
0 500 1000 1500 2000
Tumour mass / mg
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68 The PMB-amide was ineffective at reducing tumour growth in the nude mice, yet it still had good in vitro activity. Upon initial design of this compound it was thought that its precursor bis-PMB 5 was ineffective due to its poor-bioavailability as a result of poor solubility in the intralipid solution used for drug delivery.
The PMB-amide had a twofold increase in inherent solubility into the delivery solution compared to the bis-PMB 5 and as such it was administered in a solubilised form. Thus, the poor-bioavailability due to solubility was ruled out as an explanation for the failure.The two studies involving both bis-PMB and the PMB-amide have shown that administering of the drug via intraperitoneal injection is not the best method and as such oral or intravenous administration should be considered. However, owing to time constraints the other two methods could not be attempted. Other experiments were carried out in an attempt to explain the ineffectiveness of the two compounds in reduction of tumours in nude mice and these were carried out at the Department of Pharmacology, UCT. The bis-PMB 5 was incubated with mouse whole blood in vitro at 37°C and the amount of compound present in the sample monitored over three hours by removing an aliquot and analysing it by liquid chromatography-mass spectrometry (LC/MS), which gave a peak area as a reflection of ajoene derivative concentration. Concurrently, red blood cells (RBC) and blood plasma were separated and the same experiment conducted, and the results analysed, which are summarised in Figure 31.54
Figure 31: Metabolic stability of bis-PMB in blood.
The results from Figure 31 indicate that the bis-PMB incubated in both the whole blood and the red blood cells had strong affinity towards them and as such could not be detected in the two samples by LC/MS beyond 5 minutes of incubation, whereas in plasma alone it had a half-life of two hours. This data suggests that the bis-PMB possibly binds to the red blood cells and is thus not available for
Stability of bPMB in whole blood, plasma and RBC (37 C)
0 25 50 75 100 125 150
0 2.5×105 5.0×105 7.5×105 1.0×106
Whole blood, t1/2 < 5min Plasma, t1/2 = 120min RBC, t1/2 <5min
time / min
Average peak area
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69 delivery into the tumours when it is administered via intraperitoneal injection. However, further investigation needs to be carried out to support this hypothesis and to also evaluate exactly which receptors are likely responsible for the interaction observed. A study conducted by Apitz-Castro showed that ajoene (with the same vinyl disulfide/sulfoxide pharmacophore) has affinity towards the fibrinogen receptor, which is found in human platelets and prevents platelet aggregation. Thus, it is likely that the bis-PMB might bind to similar receptors reversibly, but this is still to be proven.55