2.4 Performance-in-Service
2.4.2 Performance-in-Service: in vivo (Preclinical)
is stabilized by PEG-lipid. As a consequence, the hydrogen ion gradient rapidly equalizes, DoxH1 comes out in seconds (large red arrow), as does any remaining embedded Dox in the bilayer. From dextran permeability measurements and other calculations,43the size of these nanopores appears to be B10 nm in diameter, more than large enough to allow the very rapid transport seen for CF, dithionite and doxorubicin.
2.4.2.2 MRI Contrast Data
The timing of liposome injection in relation to application of hyperthermia using manganese as a released contrast agent was tested in a rat fibrosarcoma model.103When trapped inside the liposome, the Mn11did not affect the MR signal, because it was sequestered from the surrounding water; when it was released, the MR signal increased profoundly as a result of the interaction of Mn11 with the water surrounding the liposome. When Mn-LTSL was injected into an animal with a preheated tumor, release was predominantly in the peripheral tumor vasculature. Overall average tumor drug concentrations were doubled compared with LTSL injection in a tumor that was heated after drug administration; the time to progression (5initial tumor volume) was longer (34 daysvs. 18.5 days, respectively). These results showed that attempts to preload a tumor in the first hourvia EPR, even with thermally triggered drug-releasing liposomes, were inferior to a vascular release mechanism. One reason is that when LTSL is first injected (as a bolus injection) and then the tumor heated, it takes 15–20 min. to reach thermal steady state with a passive thermal conduction heating method. Over this time the plasma concentration of Dox-LTSL does fall somewhat before the transition temperature in the tumor is reached. This is even more reason to heat first, and inject the LTSL while maintaining the required 421C hyperthermic temperature of the tumor.
2.4.2.3 Effect of Drug Release on Tumor Vasculature
If drug was being released into the blood vessels of the tumor, what effects could it be having, perhaps on the vasculature itself? In studies that measured the red blood cell velocity using fluorescent red cells as ‘‘tracers’’,104we found that tumor blood flow can actually be shut down by Dox-LTSLþHT in FaDu tumors.
The average red blood cells (RBC) velocity was reduced from 0.428 mm/s to 0.003 mm/s and the microvascular density was reduced from 3.93 mm/mm2 to 0.86 mm/mm2at 24 h after just a 1 h treatment. In addition, blood flow stasis and severe hemorrhage occurred immediately after treatment, and there was no blood flow in micro-vessels in five out of six tumors at 6 h and 24 h after the treatment.
To determine if the treatment had the same effects on tumor blood flow in other tumors, Chenet al.34treated 4T07 tumors with Dox-LTSL plus HT, and concluded that tumor microvascular permeability to drug was more critical than the sensitivity of tumor cells to doxorubicin in determining the anti- vascular efficacy of Dox-LTSLþHT treatment.34
2.4.2.4 Other Tumors
Expanding the preclinical testing to a series of other tumors the efficacy of the commercial formulation (ThermoDoxs) was re-examined in FaDu and compared in HCT116, PC3, SKOV-3 and 4T07 cancer cell lines.102 Dox-LTSLþHT resulted in the best antitumor effect in each of the five Materials Science and Engineering of the Low Temperature Sensitive Liposome 61
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tumor types. Interestingly, these variations in efficacy were most correlated to in vitrocell doubling time.
2.4.2.5 Triggered, Intravascular Release to Improve Drug Penetration into Tumors
The most compelling and dramatic evidence for not only release in the bloodstream but also deeper penetration into a tumor than has ever been achieved and measured before, is presented in a new paper by Manzooret al.17 The traditional goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors and, as mentioned earlier, the EPR effect can permit passive accumulation into tumor interstitium in some subcutaneous animal tumors. However, only suboptimal delivery is achieved, especially for 100 nm liposomes, because of heterogeneities of vascular permeability and the density of the interstitial stroma, which limits nanoparticle extravasation and penetration. Further, slow drug release from non-thermally sensitive or environment insensitive liposomes limits bioavail- ability of the encapsulated drug. We have demonstrated, quite categorically, that the LTSLs release doxorubicin inside the tumor vasculature, but only when the tumor is heated to 421C.17
As shown in Figure 2.18, real-time confocal imaging of doxorubicin delivery to the FaDu xenograft in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases the amount of free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional PEGylated liposomes (Figure 2.18).
Figure 2.18 Tumor uptake of doxorubicin as a function of time when free doxo- rubicin (Free DoxþHT) or doxorubicin loaded LTSL (Dox-LTSLþHT) were injected in the warmed tumor (421C). Time sequence images of blood vessels (green) and doxorubin (red) for pre-injection, and at time points 1, 5, 10 and 20 min after injection. Scale bar¼100mm.
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Intravenous injection of free Dox, even with applied HT, results in the appearance of drug in interstitial tissue within 1 minute that is quickly reab- sorbed into the vasculature within five minutes, with few cells taking up any drug. Heating the tumors with concurrent administration of the Dox-LTSL results in continuous drug delivery to tissue, with uptake of doxorubicin by cells far from vessels that continues to increase though the 20 minutes of obser- vation. Importantly, drug is delivered without liposome extravasation. This proves that the release mechanism occurs by intravascular release. Although not shown here, when LTSL is injected without heating the tumor, there is very little if any liposome extravasation and even less doxorubicin in the interstitial tissue than for free drug administration.
The histologic assessment of drug concentration-penetration from vessels in flank FaDu tumors is shown in Figure 2.19.17Regarding the actual penetration of drug into the tumor tissue, the Dox-LTSL plus HT regimen achieves much greater concentrations of drug at the endothelial cells and far superior distances from blood vessels into the tumor. Drug levels are expressed as median fluo- rescence intensity at distances out to 100mm from the nearest blood vessel for heated tumors. As is clear, Dox-LTSL delivers much more total drug at all distances from vessels compared to Doxiltand free doxorubicin, including 3.5 times more than free drug at the endothelial cells. Dox-LTSL actually shows maximum delivery at 20mm (several cell diameters) from blood vessels into the tissue. At the distance at which Dox-LTSL levels start to fall, Doxilt drug levels are less than half that of Dox-LTSL and have already fallen to approximately one-third of their maximal concentration close to blood vessels.
Maximum measureable drug penetration from tumor vasculaturevs.treatment group shows drug delivered with Dox-LTSL penetrates twice as far as Doxilt liposomes (78mmvs. 34mm). These huge improvements in drug bioavailability establish the LTSL plus mild hyperthermia as a new paradigm in drug delivery:
rapidly triggered drug release in the tumor bloodstream and deep penetration of drug into the tumor tissue.17The average intervascular distance in human
Figure 2.19 Histological assessment of drug concentration-penetration from vessels in flank tumors for Dox-LTSLþHT, DoxilþHT and free DoxHT.
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esophageal and cervix cancer has been measured and estimated to be in the range of 90–160mm, respectively.105Thus, Dox-LTSL can, on average, deliver doxorubicin to nearly every tumor cell, since the penetration distance is over 70mm and drug would be delivered from both sides of a tumor core.105 Of course, these are average values and heterogeneity in delivery is not taken into account. The important point is whether the drug reaches all tumor cells in a concentration adequate to kill them. That is not yet known.