Impact of the Enhanced Permeability and Retention (epr) Effect and Cathepsins Levels on the Activity of Polymer-drug Conjugates

Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i) the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR) effect) and (ii) the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B). Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates). An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer), although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.


Introduction
Polymer-drug conjugates (PDCs) are nano-sized drug delivery systems, in which one or more chemotherapeutic agent is covalently linked to a water-soluble polymer (Figure 1a) [1,2].The main rationale for PDCs stems from their ability to passively accumulate into the tumour tissue by means of the enhanced permeability and retention (EPR) effect (Figure 1b) [3].This is a unique feature of tumour vasculature, which makes the tumour vasculature hyper-permeable to macromolecules compared to normal vasculature.As a result, PDCs have been shown to: (a) selectively target tumour tissues; (b) be less toxic and display an extended half-life compared to the parent free drug (e.g., maximum tolerated dose (MTD) for N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (320 mg/m 2 ) is four-fold higher than for the parent compound doxorubicin (60 mg/m 2 )) [4].Nineteen PDCs have undergone/are undergoing clinical evaluation (Table 1 and Figure 2).Clinical trials have shown evidence of tumour responses to PDC to various degrees, and some conjugates (e.g., poly-L-glutamic acid (PGA)-paclitaxel) have progressed to Phase III trials [5,6].In spite of several advantages of PDCs over the relevant parent drugs, such as a more favourable toxicity profile, superior quality of life and a significant survival rate [7], to date, no such conjugate has entered the market place, which suggests that system design and/or patient selection are still suboptimal.
When looking at possible reasons for the slow progression of the development of this technology, various factors need to be considered.First, PDCs are intrinsically more complex systems than low molecular weight drugs, for instance they have an inherent degree of variability due to the polydispersity of the carrier.In addition, and of greater significance, the anti-tumour activity of PDCs relies on two factors: (a) passive tumour accumulation via the EPR effect; (b) drug release following a biological stimulus (e.g., enzyme or pH) (Figure 1c).Failure to identify the correct patient/tumour type population, which displays a sufficient level of EPR effect and a sufficient level of enzymes, would negatively bias the results obtained with this technology.
Many studies have reported evidence for the EPR effect in a variety of solid tumours [8][9][10][11] but the factors that affect the magnitude of such an effect are still unclear.Recent studies have considered different in vivo tumour models and these have reported that both the size and tumour type can affect the magnitude of the EPR effect [12,13].A discussion group constituted by world experts of the EPR effect has looked at the heterogeneity of such a phenomenon and has identified its key influencing factors, mainly: (a) the nature of the vascular bed and stroma, including the presence or absence of lymphatics; (b) tumour size, type and location; and (c) patient characteristics such as age, gender, body composition, and treatment [10,13,14].With regards to expression of the various activating enzymes, again the situation is heterogeneous and unclear.It is well known that enzymes that have been exploited for activation of PDCs, such as cathepsin B, are expressed in tumour tissues [2,13,[15][16][17].However, it has been recently noted that various factors, such as hormone levels, can affect the abundance of such enzymes.For instance, cathepsin B levels are higher in pre-menopausal women than in post-menopausal women as the former have higher oestrogen levels than the latter [5,18,19].In this paper, we retrospectively analyse data obtained from the literature concerning clinical trials carried out on PDCs in order to determine whether there is a connection between the clinical responses of various tumour types and the levels of enzyme expression/magnitude of the EPR effect in such tumour types.First, we analysed clinical data within the literature to identify the tumour types for which marked tumour responses were observed.Then, we documented the content of cathepsin B that has been reported for the various tumour types.Finally, we determined the extent of the EPR effect, which has been, reported for these various tumour types.

Experimental Section
The data presented in this paper were collected via systematic literature searches using search engines such as PubMed, Science Direct and Web of Science.No date restrictions were applied to the searches.

Terminology
This paper reports data collected from various sources that are derived from both clinical and pre-clinical studies.Different studies used different terminologies when referring to the various cancer types, with the terminologies ranging from technical/very specific (e.g., glioblastoma) to more general (e.g., brain cancer).In addition, in some cases, the effects of the tumours on adjacent organs are reported (e.g., gastroesophageal cancer).To ensure that a consistent terminology is used within this paper, clinical tumour types and preclinical models were grouped under general umbrella terms summarised in Table 2.

Clinical Status of the PDCs
The data on the current clinical status of the PDCs, reported in Table 1, were gathered from articles obtained using the key word "polymer-drug conjugate*".The clinical data related to each individual conjugate, which are reported in the supplementary information, were gathered using the name of the individual conjugate.
The percentage tumour response reported for each conjugate, per tumour type, was determined considering all of the clinical responses observed (i.e., number of (partial response (PR) + complete response (CR) + stable disease (SD) + minor responses (MR)), as well as the total number of patients evaluated per tumour type using the following formula: Response to conjugate A in tumour X (%) = (Total clinical responses observed for conjugate A in tumour X/ Number of patients evaluated for conjugate A in tumour X) × 100 The results obtained for each conjugate for each tumour type (reported in supplementary information, Tables S1 to S19) were then further processed in order to obtain an overall response to all PDCs per tumour type.
The overall percentage tumour response for each tumour type was determined using the following formula: Overall response in tumour X (%) = Σ[(Response to conjugate A in tumour X (%) × number of patients with tumour X in which conjugate A was tested) + (Response to conjugate B in tumour X (%) × number of patients with tumour X in which conjugate B was tested) + (Response to conjugate n in tumour X (%) × number of patients with tumour X in which conjugate n was tested)]/Total number of patients with tumour X

Cathepsin Level
The data on cathepsin levels in various tumour types were collected via literature searches using the following key words and their combinations: "cathepsin level*", "cathepsin content*", "tumour/tumor types".

EPR Effect
The data related to the EPR effect in different tumour types were retrieved via literature searches using the following key words (and their combinations): "enhanced permeability and retention effect", "EPR effect", "tumour/tumor*", "drug accumulation", "biodistribution" and the names of the individual conjugates.In addition, original research articles quoted in reviews related to the EPR effect, which were not identified from the general search, were also considered.
Priority was given to articles related to PDCs, however, as the EPR effect is a phenomenon that applies to any macromolecular system, clinical and preclinical data relating to other such systems (e.g., liposomes and micelles) were also included.

Results and Discussion
Many clinical studies have been carried out on PDCs (e.g., OPAXIO) [26].The main clinical outcomes have been summarized in extensive reviews [55][56][57], but, as yet, a detailed analysis of which tumour types are proving more responsive to this type of drug delivery technology is still missing.A number of studies have already attempted to identify correlations between tumour types and enzyme content or the magnitude of the EPR effect, but this approach has been largely carried out in preclinical models and systematic assessments of these factors and clinical activities are missing, which is surprising since the latter are two key pre-requisites for the activity of PDCs.
In an attempt to provide a more comprehensive study that links clinical outcomes, enzyme content, and the EPR effect, we initially documented all the PDCs that have been explored in clinical trials, alongside the triggers that have been used to promote drug release in each (Table 1).Of the 19 conjugates that have been tested in patients, the vast majority ( 17) rely, at least partially, on the presence of enzymes for drug release (Table 1).Of the various enzymes exploited as triggers, cathepsins, a family of lysosomal proteases, are the most widely targeted (eight of the conjugates reported in Table 1 rely on cathepsins action for drug release).This is not surprising, as cathepsins have been reported to be linked with cancer progression.In particular, cathepsin B has been connected with tumour invasion [58].

Effect of Tumour Type on Clinical Response
To assess the clinical responses observed in different tumour types, we analysed clinical data for PDCs retrieved from our literature searches.Although the purpose of phase I, II and III clinical trials and their patient selection criterias are different, the objective responses (i.e., partial response (PR), complete response (CR), stable disease (SD) and minor responses (MR)) are considered as the end point in all such studies [59].Therefore for each conjugate, evidence of a clinical response (PR, CR SD and MR) in the various tumour types has been summarised in detail in Tables S1-S19 in the supplementary information.A representative data set with clinical responses for the top three highest responsive tumour types for each conjugate has been provided in Table 3.For example, in the case of HPMA copolymer-doxorubicin (PK1; FCE28068), the top three highest responsive tumour types are lung, breast, colon and/or rectum, with tumour responsive rate of 57%, 53%, and 4%, respectively calculated from both phase II clinical trials and I. Likewise, the top three highest responsive tumour types based on the calculation from all the available clinical phase trials have been summarised for the other eighteen PDCs.
Oesophagus, stomach and intestine Oesophagus, stomach and intestine  Finally, summary graphics assembling the percentage responses per tumour type for all conjugates were compiled (Figure 3 and Table 4).Figure 3 and Table 4 shows the percentage responses observed for cathepsin-activated conjugates.Breast, ovary and lung cancer were the cancer types in which the highest response rates were observed (>30%).However, the responses observed for the various conjugates in each tumour type were variable, for example, the HPMA copolymer-oxaliplatin conjugate (AP5346/ProLindac) showed no responses in breast cancer while the HPMA copolymer-doxorubicin conjugate (PK1) showed 53% response in that tumour.This variability might be due to the fact that the latter relies solely on cathepsins for drug release, and hence has a more specific activation than the former, for which both cathepsins and acid hydrolysis play a role in the release of oxaliplatin.This finding however can also be affected to some extent by the nature of the therapeutic agent.Notes: * Averages were normalised to take into account of the number of patients evaluated for each conjugated, see experimental section; "-" indicates that the conjugate was not evaluated against the particular tumour type.
Figure 4 and Table 5 show the result for all conjugates, independent of their activation mechanism.The results are similar to that observed for cathepsin-activated PDCs, with breast, ovary and lung cancer still achieving >30% responses.However, urinary cancer and cancers of the oesophagus, stomach and intestines showed a high response as well (48% and 34%, respectively).

Cathepsin Levels in Different Tumour Types
Having assessed which tumour types showed the highest responses to PDCs, we investigated which tumour types had been reported to express the highest levels of cathepsins.
Cathepsins are lysosomal cysteine proteases involved in the bulk degradation of intracellular and endocytosed proteins.Cathepsin B is a large lysosmal protease that is generally over expressed in tumour tissues and plays a very important role in the release and activation of PDCs.Several studies have indicated that its co-existing isoenzymes such as cathepsin D, L, H, S and X are also overexpressed in tumour tissues and are thought to play a role in proteolysis [60][61][62][63].Some studies have been carried out to elucidate precisely which isoenzyme contributes to drug release.For example, extensive studies carried out in the 80s have identified cathepsin B as the primary enzyme responsible for doxorubicin release from the HPMA copolymer-Dox conjugate, when a Gly-Phe-Leu-Gly (GFLG) linker was used.However, the release mechanism for other conjugates has not been investigated so extensively.Also, it is known that various isoenzymes can contribute to drug release from peptidyl linkers, for example, both cathepsin L and cathepsin B were found to contribute to the release of daunorubicin when GPLG and GPPL linkers were used [64].Similarly, both cathepsin B and cathepsin H contributed to the release of 5-FU, albeit to a different extent [65].The level of expression of these cathepsins in tumour tissues (especially cathepsin B) is very crucial for the selective release of the drug from the PDC within the tumour environment (Table 1).Also, several studies have indicated that cathepsin expression in tumours varies greatly [63] which is affected by various parameters including gender, age and hormone levels [14,18].Therefore, to determine if there is a correlation between the level of cathepsin expression in a tumour type, and the clinical response to cathepsin-activated PDCs, data on cathepsin levels is presented for clinical as well as for in vivo and in vitro samples of different tumour types to obtain reliable results (Table 6).The information on tumour grades and other parameters that are related to the cathepsin's expression were limited in the original studies, hence these details have not been considered in our present study.Notes: * Averages were normalised to take into account of the number of patients evaluated for each conjugated, see experimental section; "-" indicates that the conjugate was not evaluated against the particular tumour type; a The clinical responses for this conjugate are considered under the tumour type "others" as the responses per specific tumour type has not been mentioned in the original references [36][37][38][39].NC: Not considered.Notes: * The cathepsin levels are expressed as high and low (rather than a specific value) for those entries in which definite quantitative values were not available in the original study; NA, Not applicable; "n" = number of human/animal subjects.
Two tumours, lung and breast, which have shown the highest percentage response clinically to PDCs activated by cathepsins, also showed a generally high content of this class of enzyme (in some studies, as high as 3835 ng/mg for lung cancer).This correlation is as expected, as the presence of a trigger is essential for the activation of a prodrug, but has not been widely reported from analysis of experimental data.It should also be noted that the content of cathepsins reported in various studies concerning the same tumour type were highly variable.This finding suggests that heterogeneity might be also present in the tumour samples of the patients that have undergone treatment with PDCs and this could potentially explain why only a fraction of the patients enrolled in the clinical studies responded to the treatment [92,93].Differences in the content of cathepsins are particularly marked for breast cancer.For example, the content of cathepsin B was reported to be "high" in the study from Bremer et al. [86] but "low" from that of Hulkower et al. [87].It is also key to highlight that, as mentioned previously, cathepsins level have been found to be affected by oestrogen levels and therefore an additional element of variability is added depending on whether the sample is taken from a post-menopausal or pre-menopausal woman [5,18].

Magnitude of the EPR Effect in Different Tumour Types
A second, but at least equally important, factor for activity of PDCs is the extent of the EPR effect in a tumour.To establish if there was a correlation between the magnitude of the EPR effect and the tumour's response to PDCs, data on drug accumulation of different drug delivery technologies such as PDCs and liposomes in clinical tumour samples as well as in preclinical samples were collected as summarised in Table 7.Although this comparison holds a limitation with respect to the heterogeneity of systems (different nanoconstructs and different phases of clinical research) and heterogeneous tumour types, pooling the results from such heterogeneous data was necessary due to the limited clinical studies on EPR effect with PDCs.Different methodologies have been used in different studies to evaluate the extent of passive accumulation in the tumour both qualitatively and quantitatively.For example, in some studies analogues of conjugates containing radioisotopes were used with imaging techniques where the results were expressed qualitatively (i.e., expressed as whether drug accumulation was or was not observed) and quantitatively (expressed as % ID/Kg of drug uptake in tumour).Similarly with the staining technique using Evan's blue dye the results were expressed both qualitatively and quantitatively (expressed as % dose/g/tumour).A few studies involved the comparison between the free drug accumulation and the nano particle accumulation in which case the results were expressed in terms of degree of their accumulation.Some studies have indicated that the extent of the EPR effect is also dependent on the tumour sizes [3,13,94] but due to the lack of information on the tumour sizes in most of the studies, this factor was not considered in our evaluation.
EPR-mediated accumulation was generally observed in all the tumour types where the highest percentage responses were reported (namely breast, lung and ovary).Studies on liposomal drug accumulation have shown poor EPR effects in liver and pancreatic tumours [95,96].Colorectal cancer was another tumour type where poor EPR effect has been reported in some studies ( [11,20] and Table 7).This observation is probably mainly due to the fact that large tumours in liver, pancreas and prostate possess hypovascular properties with low drug accumulation potential.However, it should be remembered that the smaller tumours possess high vascular density and can exhibit profound EPR effect [97].
This observation also correlates with a recent study on validation of the tumour models for EPR activity, where high drug accumulation has been reported for both large and small lung tumours, and a low drug accumulation has been reported for the large breast tumours [13].Disparities observed across the various studies are might be due to the heterogeneity within and between the tumour types (size and histological differences) and the diversity of the nanopharmaceutical characteristics [14].Notes: * The quantitative data on accumulation in tumour is not available in the original research paper; # related free drug uptake is not stated in the original study; "n" = number of human/animal subjects.
Two decades since the first PDC entered the clinical evaluation [2], one might wonder about the margin of advantage of PDCs over their corresponding parent drugs in terms of overall survival and the patient's quality of life.Such an advantage of a drug is evaluated only at phase III of the clinical trial, thus since only 2 PDCs have undergone phase III clinical trials (Opaxio and NKTR-102) and the clinical data is available to-date only for Opaxio, any conclusion with regards to the overall survival and patient's quality of life is derived from Opaxio's performance.Opaxio, in comparison with its conventional paclitaxel-based treatment, showed some safety-related advantages such as alopecia was rare, nausea and vomiting were uncommon and hypersensitivity reactions were rarely observed.A significant survival benefit has also been observed for women receiving Opaxio vs. paclitaxel [7].These promising results have provided the confidence that PDCs can give therapeutic benefits in the field of oncology.The use of PDCs in combination with the application of diagnostic tools to measure protease expression and EPR effect would be a rational approach for the further development and clinical use of PDCs.

Conclusions
In this study, we have reported analysis of data from the literature whereby we have documented and examined the expression of enzymes (cathepsins) within various tumour types, the magnitude of the EPR effect in the various tumour types, and the clinical responses observed for PDCs.We have determined that the highest percentages of clinical responses to PDCs were observed for lung cancer, which was also found to express high levels of the cathepsin enzymes.Breast and ovarian cancer also showed high clinical responses to PDCs, which correlated well with high levels of cathepsins observed in these tumour types, and with reports indicating the presence of the EPR effect.This finding is in line with other studies [13,14] and in agreement with that concluded by others (e.g., [13]).Moreover, our study also suggests that careful patient selection, in the form of pre-screening for enzyme content and the EPR effect, would be a rational approach for the further development and clinical application of PDCs.This could ultimately result in a more consistent efficacy of this drug delivery system in the clinical setting.

Figure 2 .Figure 2 .
Figure 2. Chemical structures of the PDCs discussed in this article.(a) PDCs for which the release of the drug is triggered via an enzymatic mechanism; and (b) PDCs for which the release of the drug is triggered by a non-enzymatic mechanism.Note: The chemical structure of NKTR-105 is not available.(a)

Figure 3 .
Figure 3. Graphical representation of the tumuor response rates for PDCs (release mediated only by cathepsin).

Figure 4 .
Figure 4. Graphical representation of the tumuor response rates for PDCs (release mediated both by cathepsin and pH).

Table 1 .
Current clinical status of polymer-drug conjugates (PDCs) and types of release triggers.

Table 2 .
Summary of the terminology used in this article to identify various tumour types/tumour models.

Table 3 .
Clinical data for the top three tumour types for the nineteen PDCs that are released by both cathepsins and change in pH.

Multi-arm-polyethylene glycol-docetaxel; NKTR-105 [51]
Indicates total number of patients considered for the clinical evaluation; b Clinical responses includes the total of SD, Stable disease; PR, Partial response; MR, Minor response; CR, Complete response; OS, Overall survival; NR, No response; c Tumour response rate (%) is the added responses (SD + PR + MR + CR + OS) per tumour type.NA,Not available, NC, Not calculated.*Indicatesnumber of patients evaluated for the tumour responses.The term "Solid tumours" has been mentioned wherever the clinical responses per tumour type have not been reported in the respective study.1Solidtumours includes (number of patients is denoted in the brackets for 4Solid tumours includes-Ovary; Breast; Adrenal; Oesophagus, stomach and intestine; Lymphoma; Lung; Cervix; Head/Neck and brain; Urinary and Breast.

Table 4 .
Tumour response rates for PDCs (release mediated only by cathepsin).

Table 5 .
Tumour response rates for PDCs (release mediated both by cathepsin and pH).

Table 6 .
Cathepsin levels found in clinical and preclinical sample of different tumour types (clinical studies highlighted in grey).

Table 7 .
Extent of passive accumulation of macromolecular system in different tumour types in both clinical and pre-clinical samples, clinical studies highlighted in grey.