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Science, Technology and Medicine open access publisher.Publish, read and share novel research. Colon Cancer: Current Treatments and Preclinical Models for the Discovery and Development of New TherapiesSamuel Constant1, Song Huang1, Ludovic Wiszniewski1 and Christophe Mas1[1] OncoTheis, 14 Chemin des aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland1. The Intestinal Stem Cell Signature Identifies Colorectal Cancer Stem Cells and Predicts Disease Relapse. An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth. Taniguchi H, Yamamoto H, Hirata T, Miyamoto N, Oki M, Nosho K, Adachi Y, Endo T, Imai K, Shinomura Y.
These highlights do not include all the information needed to use AXIRON safely and effectively.
Starting AXIRON dose is 60 mg of testosterone (1 pump actuation of 30 mg of testosterone to each axilla), applied once daily, at the same time each morning. Dose adjustment: The dose of testosterone may be decreased from 60 mg (2 pump actuations) to 30 mg (1 pump actuation) or increased from 60 mg to 90 mg (3 pump actuations) or from 90 mg to 120 mg (4 pump actuations) based on the serum testosterone concentration from a single blood draw 2 – 8 hours after applying AXIRON and at least 14 days after starting treatment or following dose adjustment. Patients should wash hands immediately with soap and water after applying AXIRON and cover the application site with clothing after the solution has dried.
The application site and dose of AXIRON are not interchangeable with other topical testosterone products. Edema with or without congestive heart failure, may be a complication in patients with preexisting cardiac, renal, or hepatic disease (5.7). Use of testosterone with Adreno-corticotropic Hormone (ACTH) or corticosteroids may result in increased fluid retention.
There are insufficient long-term safety data in geriatric patients using AXIRON to assess the potential risks of cardiovascular disease and prostate cancer (8.5). AXIRON is an androgen indicated for replacement therapy in males for conditions associated with a deficiency or absence of endogenous testosterone. Primary hypogonadism (congenital or acquired): testicular failure due to conditions such as cryptorchidism, bilateral torsion, orchitis, vanishing testis syndrome, orchiectomy, Klinefelter's syndrome, chemotherapy, or toxic damage from alcohol or heavy metals. Hypogonadotropic hypogonadism (congenital or acquired): idiopathic gonadotropin or luteinizing hormone-releasing hormone (LHRH) deficiency or pituitary-hypothalamic injury from tumors, trauma, or radiation. The recommended starting dose of AXIRON (testosterone) topical solution is 60 mg of testosterone (2 pump actuations) applied once daily.
AXIRON is applied to the axilla, preferably at the same time each morning, to clean, dry, intact skin.
Keeping the applicator upright, patients should place it up into the axilla and wipe steadily down and up into the axilla. After use, the applicator should be rinsed under room temperature, running water and then patted dry with a tissue.
When deodorants or antiperspirants are used as part of a regular program for personal hygiene, they should not interfere with the efficacy of AXIRON in treating hypogonadism. Patients should be advised to avoid swimming or washing the application site until two hours following application of AXIRON [see Clinical Pharmacology (12.3)]. To reduce the likelihood of interpersonal transfer of testosterone, the application site should always be washed prior to any skin-to-skin contact regardless of the length of time since application.
Apply once to the left and once to the right axilla, wait for the product to dry, and then apply once again to the left OR right axilla.
Apply once to the left and once to the right axilla, wait for the product to dry, and then apply once again to the left AND once to the right axilla.
Hands should be washed thoroughly with soap and water after AXIRON has been applied [see Warnings and Precautions (5.2)].
Children and women should avoid contact with the unclothed or unwashed application sites on the skin of men using AXIRON.
Patients should wash their hands immediately with soap and water after application of AXIRON. Prior to any situation in which direct skin-to-skin contact is anticipated, patients should wash the application site thoroughly with soap and water to remove any testosterone residue. In the event that unwashed or unclothed skin to which AXIRON has been applied comes in direct contact with the skin of another person, the general area of contact on the other person should be washed with soap and water as soon as possible.
While interpersonal testosterone transfer can occur with a T-shirt on, it has been shown that transfer can be substantially reduced by wearing a T-shirt and the majority of residual testosterone is removed from the skin surface by washing with soap and water. AXIRON is contraindicated in men with carcinoma of the breast or known or suspected carcinoma of the prostate [see Warnings and Precaution (5.1)].
AXIRON is contraindicated in women who are, or who may become pregnant, or who are breastfeeding.
Monitor patients with benign prostatic hyperplasia (BPH) for worsening of signs and symptoms of BPH. Cases of secondary exposure to testosterone in children and women have been reported with topical testosterone products applied to the abdomen or upper arms, including cases of secondary exposure resulting in virilization of children. Inappropriate changes in genital size or development of pubic hair or libido in children, or changes in body hair distribution, significant increase in acne, or other signs of virilization in adult women should be brought to the attention of a physician and the possibility of secondary exposure to testosterone should also be brought to the attention of a physician.
Increases in hematocrit, reflective of increases in red blood cell mass, may require lowering or discontinuation of testosterone.
At large doses of exogenous androgens, including AXIRON, spermatogenesis may be suppressed through feedback inhibition of pituitary follicle-stimulating hormone (FSH) which could possibly lead to adverse effects on semen parameters including sperm count. Prolonged use of high doses of orally active 17-alpha-alkyl androgens (methyltestosterone) has been associated with serious hepatic adverse effects (peliosis hepatitis, hepatic neoplasms, cholestatic hepatitis, and jaundice).
Gynecomastia may develop and may persist in patients being treated with androgens, including AXIRON, for hypogonadism.
The treatment of hypogonadal men with testosterone may potentiate sleep apnea in some patients, especially those with risk factors such as obesity and chronic lung disease.
Changes in serum lipid profile may require dose adjustment or discontinuation of testosterone therapy. Androgens, including AXIRON, should be used with caution in cancer patients at risk of hypercalcemia (and associated hypercalciuria).
Androgens, including AXIRON, may decrease concentrations of thyroxin-binding globulins, resulting in decreased total T4 serum concentration and increased resin uptake of T3 and T4. Alcohol based products, including AXIRON, are flammable; therefore, patients should be advised to avoid smoking, fire or flame until the AXIRON dose applied has dried.
Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in practice.
Other less common adverse reactions reported by at least 2 patients in the 120 day trial included: application site edema, application site warmth, increased hemoglobin, increased blood pressure, increased blood testosterone, increased blood glucose, acne, nasopharyngitis, anger and anxiety.
Following the 120 day study, seventy-one (71) patients entered a two-month extension study with AXIRON. No serious adverse reactions to AXIRON were reported during either the 120 day trial, or the extension to 180 days. Changes in insulin sensitivity or glycemic control may occur in patients treated with androgens.
The concurrent use of testosterone with ACTH or corticosteroids may result in increased fluid retention and should be monitored cautiously, particularly in patients with cardiac, renal or hepatic disease.
Pregnancy Category X [see Contraindications (4)] — AXIRON is contraindicated during pregnancy or in women who may become pregnant. Although it is not known how much testosterone transfers into human milk, AXIRON is contraindicated in nursing women because of the potential for serious adverse reactions in nursing infants. There have not been sufficient numbers of geriatric patients involved in controlled clinical studies utilizing AXIRON to determine whether efficacy in those over 65 years of age differs from younger patients. AXIRON contains testosterone, a Schedule III controlled substance as defined by the Anabolic Steroids Control Act.
Although drug dependence is not documented in individuals using therapeutic doses of anabolic steroids for approved indications, dependence is observed in some individuals abusing high doses of anabolic steroids. AXIRON (testosterone) topical solution is a clear, colorless, single phase solution containing 30 mg of testosterone in 1.5 mL of AXIRON solution for topical administration through the axilla.
Endogenous androgens, including testosterone and dihydrotestosterone (DHT), are responsible for the normal growth and development of the male sex organs and for maintenance of secondary sex characteristics.
On the skin, the ethanol and isopropyl alcohol evaporate leaving testosterone and octisalate.
When AXIRON treatment is discontinued after achieving steady-state, serum testosterone concentrations returned to their pretreatment concentrations by 7 – 10 days after the last application.
Distribution — Circulating testosterone is primarily bound in the serum to sex hormone-binding globulin (SHBG) and albumin.
Metabolism —Testosterone is metabolized to various 17-keto steroids through two different pathways. DHT concentration increased in parallel with testosterone concentration during AXIRON treatment.
Excretion — There is considerable variation in the half-life of testosterone as reported in the literature, ranging from 10 to 100 minutes. Potential for testosterone transfer: The potential for testosterone transfer from males dosed with AXIRON to healthy females was evaluated in a clinical study conducted with a 2% testosterone formulation. In a clinical study conducted with a 2% testosterone formulation to evaluate the effect of washing on the residual amount of testosterone at the axilla, 10 healthy male subjects received 60 mg (2 pump actuations) of testosterone to each axilla (the maximum testosterone dose of 120 mg).
AXIRON was evaluated in a multicenter, open label, 120-day trial that enrolled 155 hypogonadal men at 26 clinical research centers. During the initial AXIRON treatment period (Days 1-15) 143 patients were treated with 60 mg of testosterone daily.
On day 120, 75% of responding patients finished the study on the starting dose of 60 mg of testosterone, while 2% had been titrated to 30 mg, 17% had been titrated to 90 mg and 6% had been titrated to the 120 mg dose.
Table 3 summarizes the proportion of subjects having average testosterone concentrations within the normal range on Days 60 and 120. Of the 135 patients who completed the 120 day treatment, 123 patients did so with no deviation from the protocol. Figure 2 summarizes the pharmacokinetic profiles of total testosterone in patients completing 120 days of AXIRON treatment administered as 60 mg of testosterone for the initial 15 days followed by possible titration according to follow-up testosterone measurements. AXIRON (testosterone) topical solution is available as a metered-dose pump containing 110 mL of solution.
Used AXIRON bottles and applicators should be discarded in household trash in a manner that prevents accidental exposure of children or pets.
Cases of secondary exposure to testosterone in children and women have been reported with topical testosterone products applied to the abdomen, shoulders or upper arms, including cases of secondary exposure resulting in virilization of children, with signs and symptoms including enlargement of the penis or clitoris, premature development of pubic hair, increased erections, aggressive behavior and advanced bone age. Children and women should avoid contact with the unwashed skin of the axilla or unclothed application sites of men where AXIRON has been applied.
Prior to any situation in which direct skin-to-skin contact of the axilla is anticipated, patients should wash the axilla to which AXIRON has been applied thoroughly with soap and water to remove any testosterone residue. Changes in urinary habits such as increased urination at night, trouble starting your urine stream, passing urine many times during the day, having an urge that you have to go to the bathroom right away, having urine accident, being unable to pass urine and having a weak urine flow. Breathing disturbances, including those associated with sleep, or excessive daytime sleepiness.
With testosterone doses greater than 60 mg, which require two applications of AXIRON to the same axilla, the product should be allowed to dry after the first application before the second is applied. Avoid swimming or washing the application site until two hours following application of AXIRON [see Dosage and Administration (2) and Clinical Pharmacology (12.3)]. Signs of puberty that are not expected (for example, pubic hair) have happened in young children who were accidentally exposed to testosterone through skin to skin contact with men using topical testosterone products like AXIRON.
Women and children should avoid contact with the unwashed or unclothed area where AXIRON has been applied. If you expect another person to have direct skin-to-skin contact with your armpits, first wash the application area well with soap and water.
Your healthcare provider will test your blood before you start and while you are taking AXIRON.
AXIRON is a controlled substance (CIII) because it contains testosterone that can be a target for people who abuse prescription medicines. Women who are pregnant or who may become pregnant should avoid contact with the area of skin where AXIRON has been applied.
Talk to your healthcare provider before taking this medicine if you have any of the above conditions. Tell your healthcare provider about all of the medicines you take, including prescription and non-prescription medicines, vitamins, and herbal supplements.
Apply 3 applications: one to the left and one to the right armpit, wait for the product to dry, and then apply again one to the left OR right armpit. Apply 4 applications: one to the left and one to the right armpit, wait for the product to dry, and then apply again one to the left AND one to the right armpit. Before applying AXIRON, make sure that your armpit is clean, dry and that there is no broken skin. After you have finished applying AXIRON, rinse the applicator cup with room temperature running water, and then pat it dry with a tissue. Clean up any spilled solution from surfaces such as the sink or floor to make sure others do not come into contact with it. If you already have enlargement of your prostate gland your signs and symptoms can get worse while using AXIRON. Call your healthcare provider right away if you have any of the serious side effects listed above. Other side effects include more erections than are normal for you or erections that last a long time. Tell your healthcare provider if you have any side effect that bothers you or that does not go away.
When it is time to throw away the bottle, safely throw away all parts of the AXIRON dispenser including bottle applicator cup and cap. Medicines are sometimes prescribed for purposes other than those listed in a Medication Guide. IntroductionMore than 10 years after the first sequencing of the human genome and despite major advances in scientific and technological expertise into drug research and development processes (R&D), the fact remains that we are facing a dearth of new drugs. Wash the application site thoroughly with soap and water prior to any situation where skin-to-skin contact of the application site with another person is anticipated.
More frequent monitoring of International Normalized Ratio (INR) and prothrombin time is recommended (7.2).
These men usually have low serum testosterone concentrations and gonadotropins (FSH, LH) above the normal range. These men have low testosterone serum concentrations but have gonadotropins in the normal or low range.


The AXIRON dose can be adjusted based on the serum testosterone concentration from a single blood draw 2 – 8 hours after applying AXIRON and at least 14 days after starting treatment or following dose adjustment.
Do not apply AXIRON to other parts of the body including to the scrotum, penis, abdomen, shoulders or upper arms.
When using AXIRON for the first time, patients should be instructed to prime the pump by depressing the pump three (3) times, discard any product dispensed directly into a basin, sink, or toilet and then wash the liquid away thoroughly. If patients use an antiperspirant or deodorant (stick or roll-on) then it should be applied prior to the application of AXIRON to avoid contamination of the stick or roll-on product. Signs and symptoms have included enlargement of the penis or clitoris, development of pubic hair, increased erections and libido, aggressive behavior, and advanced bone age. Testosterone therapy should be promptly discontinued at least until the cause of virilization has been identified.
Edema, with or without congestive heart failure, may be a serious complication in patients with pre-existing cardiac, renal, or hepatic disease [see Adverse Reactions (6)].
Free thyroid hormone concentration remain unchanged, however there is no clinical evidence of thyroid dysfunction. These data reflect the experience primarily with a testosterone dose of 60 mg, which was taken by all patients at the start of the study, and was the maintenance dose for 97 patients. Other less common adverse reactions reported in fewer than 1% of patients in the 120 day trial included: asthenia, affect lability, erythema (general), folliculitis, anxiety, increased lacrimation, breast tenderness, hypertension, neoplasm prostate and elevated red blood cell count. Two patients (3%) had adverse reactions that led to discontinuation of treatment during the period from Day 120 to Day 180.
In diabetic patients, the metabolic effects of androgens may decrease blood glucose and, therefore, insulin requirement.
More frequent monitoring of INR and prothrombin time is recommended in patients taking anticoagulants, especially at the initiation and termination of androgen therapy.
Of the 155 patients enrolled in the pivotal clinical study utilizing AXIRON, 21 were over 65 years of age. Primary hypogonadism is caused by defects of the gonads, such as Klinefelter's Syndrome or Leydig cell aplasia, whereas secondary hypogonadism is the failure of the hypothalamus (or pituitary) to produce sufficient gonadotropins (FSH, LH). In general, steady-state serum concentrations are achieved by approximately 14 days of daily dosing. Approximately 40% of testosterone in plasma is bound to SHBG, 2% remains unbound (free) and the rest is bound to albumin and other proteins.
About 90% of a dose of testosterone given intramuscularly is excreted in the urine as glucuronic and sulfuric acid conjugates of testosterone and its metabolites; about 6% of a dose is excreted in the feces, mostly in the unconjugated form.
10 males were treated with 60 mg (2 pump actuations) of testosterone in each axilla (the maximum testosterone dose of 120 mg). Following 5 minutes of drying time, the left axilla was wiped with alcohol towelettes which were assayed for testosterone content.
A control group of 6 subjects only applied 30 mg (1 pump actuation) of testosterone to a single axilla. The application sites of each group were washed with soap and water 2 hours or 6 hours after the application of AXIRON. On Day 45 of the trial, patients were maintained at the same dose, or were titrated up or down, based on their 24 hour average serum testosterone concentration measured on Day 15. By day 120, average serum testosterone concentration was within normal range for 67% of those who titrated down on the 30 mg dose, 89% of those on the 60 mg dose, 86% of those who titrated up to 90 mg and 70% of those who titrated up to the 120 mg dose.
Inappropriate changes in genital size or premature development of pubic hair or libido in children, or changes in hair distribution, increase in acne, or other signs of testosterone effects in adult women should be brought to the attention of a physician and the possibility of secondary exposure to AXIRON also should be brought to the attention of a physician. If patients use a stick or roll-on antiperspirant or deodorant, then it should be applied prior to application of AXIRON to avoid contamination of the stick or roll-on product.
This can happen if other people come into contact with the area where the AXIRON was applied. If a woman or child makes contact with the application area, the contact area on the woman or child should be washed well with soap and water right away. Ask your healthcare provider or pharmacist for a list of all of your medicines if you are not sure. Do not apply AXIRON to any other parts of your body such as your stomach area (abdomen), penis, scrotum, shoulders or upper arms. If you use antiperspirant or deodorant, then it should be applied at least 2 minutes before you apply AXIRON.
Carefully replace the applicator cup and cap back onto the bottle and make sure you store the bottle safely. Your healthcare provider should check you for prostate cancer or any other prostate problems before you start and while you use AXIRON. Overall success rate of clinical trials for phases I-III from 2003 to 2010 corresponding to 4275 drugs and 7300 indications (a), success rate for phase II and III divided according to therapeutic areas (b) and overall success rate within specific oncologic areas (c). Estimated leading cancer sites mortality in US and in European Union (EU-27) for the year 2011 expressed as percent of total cancer deaths. This table gives an overview of the main colorectal cancer therapies being currently evaluated in clinical trials. Sequential steps leading to the establishment of a CRC primary Patient-Derived Tumor Xenograft collection.
Indeed, the number of drugs approved by the US Food and Drug Administration (FDA) has roughly fallen to 50% over the last ten years [1]. After applying the solution, the application site should be allowed to dry completely prior to dressing. It would be appropriate to reevaluate patients 3 to 6 months after initiation of treatment, and then in accordance with prostate cancer screening practices. In most cases, these signs and symptoms regressed with removal of the exposure to testosterone. It would be appropriate to re-evaluate the hematocrit 3 to 6 months after starting testosterone treatment, and then annually.
Long-term therapy with intramuscular testosterone enanthate has produced multiple hepatic adenomas.
These reactions were: one patient with application site irritation (considered possibly related to AXIRON application) and one patient with dry skin and erythema, but not at the application site (considered not related to AXIRON administration) and application site erythema (considered possibly related to AXIRON administration). Additionally, there were insufficient long-term safety data in these patients utilizing AXIRON to assess a potential incremental risk of cardiovascular disease and prostate cancer. Treatment of overdosage would consist of discontinuation of AXIRON together with appropriate symptomatic and supportive care. Testosterone USP is a white to practically white crystalline powder chemically described as 17-beta hydroxyandrost-4-en-3-one.
Testosterone and DHT are necessary for the normal development of secondary sex characteristics. At 2 hours after the application of AXIRON to the males, the females rubbed their outer forearms for 15 minutes on the axilla of the males. Blood samples were collected for 72 hours from all subjects following AXIRON administration. A control group of 6 female subjects applied 30 mg (1 pump actuation) of testosterone to a single axilla and did not wash the application site. There is suggestive evidence that injection of testosterone into some strains of female mice increases their susceptibility to hepatoma.
On Day 90 of the trial, patients were maintained at the same dose, or were titrated up or down, based on their 24 hour average serum testosterone concentration measured on Day 60. Table 4 below summarizes the testosterone concentration data in the patients who completed 120 days. AXIRON should be promptly discontinued at least until the cause of virilization is identified.
This information does not take the place of talking with your healthcare provider about your medical condition or treatment. Keep a list of them and show it to your healthcare provider and pharmacist when you get a new medicine. You can ask your pharmacist or healthcare provider for information about AXIRON that is written for health professionals. Column diagrams highlight the mortality rate within the population specifically affected by colon cancer. Briefly, a CRC tumor fragment coming from surgical waste is directly xenografted in an immunodeficient mouse (Passage 0). Unfortunately for pharmaceutical companies, at present this attrition in drug discovery combined with the expiration of major product patents logically lead to the development of generics. Avoid fire, flames or smoking until the solution has dried since alcohol based products, including AXIRON, are flammable. After priming, patients should completely depress the pump one time (one pump actuation) to dispense 30 mg of testosterone. The process is then repeated with application of 30 mg of testosterone (1 pump actuation) to the other axilla to achieve a total of 60 mg of testosterone applied.
If a pregnant woman is exposed to AXIRON, she should be apprised of the potential hazard to the fetus.
In a few cases, however, enlarged genitalia did not fully return to age-appropriate normal size, and bone age remained modestly greater than chronological age. If hematocrit becomes elevated, stop therapy until hematocrit decreases to an acceptable level.
If this drug is used during pregnancy, or if the patient becomes pregnant while taking this drug, the patient should be apprised of the potential hazard to a fetus. Male hypogonadism results from insufficient secretion of testosterone and is characterized by low serum testosterone concentrations.
The right axilla was then wiped with alcohol towelettes which were assayed for testosterone content.
Although a decrease of up to 33% of testosterone exposure (AUC[0-72]) was observed when antiperspirants or deodorants are used 2 minutes prior to AXIRON application, underarm deodorant or antiperspirant spray or stick products may be used 2 minutes prior to AXIRON application as part of normal, consistent, and daily routine.
Testosterone is also known to increase the number of tumors and decrease the degree of differentiation of chemically induced carcinomas of the liver in rats. After successful engraftment, new fragments are taken from the mouse hosted human tumor and xenografted again in multiple immunodeficient mice (Passage 1).
Facing both a major medical need and an obvious economical challenge, there is an urgent need to make significant improvements in the research output.Analyses of the clinical trials landscape reveal that a large number of promising drug leads fail in late stages, mainly in phase II, with an overall failure rate of 67% (Fig. To dispense the solution, position the nozzle over the applicator cup and carefully depress the pump fully once. For patients prescribed the 90 mg dose of testosterone, the procedure is the same, but three applications are required. The risk of transfer was increased in some of these cases by not adhering to precautions for the appropriate use of the topical testosterone product. Serum concentrations of testosterone were monitored in the female subjects for 72 hours after the transfer procedure. A decrease of up to 35% of testosterone exposure (AUC[0-72]) was observed when applications sites were washed 2 hours and 6 hours after AXIRON application.
Testosterone was negative in the in vitro Ames and in the in vivo mouse micronucleus assays. A collection of fragments from the resulting tumors can then be cryopreserved in a tissue bank for subsequent experiments or directly re-engrafted in mice for expansion (P2, P3, etc…). To dose 120 mg of testosterone, four applications are required alternating left and right for each application as shown in Table 1.
Study results show a 13% and 17% increase in testosterone exposure (AUC[0-24]) and maximum testosterone concentration (Cmax), respectively, compared to baseline in these females.
Patients should be advised to avoid swimming or washing the application site until 2 hours following application of AXIRON. The administration of exogenous testosterone has been reported to suppress spermatogenesis in the rat, dog and non-human primates, which was reversible on cessation of the treatment. All studies agree on the reasons by pinpointing either insufficient efficacy (~55%) or safety issues (~20%) as major causes of human trials failure [2, 3]. When repeat application to the same axilla is required, the axilla should be allowed to dry completely before more AXIRON is applied. In a prior clinical study conducted with a 1% testosterone formulation under similar study conditions, direct skin-to-skin transfer showed a 131% and 297% increase in testosterone exposure (AUC[0-72]) and maximum testosterone concentration (Cmax), respectively, compared to when men had covered the application area with a T-shirt. Remarkably, the therapeutic area showing the largest number of failures is oncology, with only 29% of success rate in Phase II and 34% in Phase III (Fig.1b). Dosing that requires greater than one pump actuation must be applied in increments of 30 mg as is shown in Table 1.
Within oncology indications, the status of colorectal cancer (CRC) is the most dramatic with an overall drug approval of only 3% (Fig.1c) over the last 10 years!
More surprisingly, more than half of the drugs currently approved to treat CRC work through the general inhibition of DNA synthesis and cellular division, instead of targeting molecular processes specifically involved in CRC progression (Table 1).
This strategy may save years of efforts and millions of dollars, giving that the average usual time for developing a new drug is ten years and with a total cost amount to billions of dollars.But in contrast, because a new drug has to show a benefit compared to an already approved treatment, the number of patients involved in a pivotal trials is increasing more and more in order to reach significance, and a similar trend is noted for the duration of the trial, that is directly linked to safety.
That was the time of the inevitable high-throughput screening, which combined with the “all-Omics” supposed to reduce costs and blew up success rates [4]. As we have seen, this approach, maybe too reductionist in the sense that it does not allow getting an idea of the full biological properties (ADME, toxicity,etc…) of a compound at an early stage, has favored the quantity instead of quality and has not kept its promises [1].Today, efforts have to be made to clearly address the early clinical discovery steps, with the goal to better qualify “leads” to increase the signal-to-noise ratio of drugs entering into clinical trials. This point of view is supported by the important failure rate subsisting in Phases III (Fig1b), suggesting an overestimation of the efficacy of candidate molecules during preclinical tests. One of the important reasons may be the use of irrelevant models or models not predictive enough. Therefore, the development of relevant and predictive models is key to increase the quality of preclinical researches and to increase the success rate of new drugs.
Consequently, the foundations of the drug discovery process have to be reconsidered by giving definitively more emphasis to the quality of preclinical validations and by encouraging the design of new pertinent models, including human 3D (three dimensional) in vitro cell models and tissue explants. This article is intended to give an overview of the current knowledge about CRC and the different models commonly used to study CRC, in order to identify the most suitable bio-systems for optimal development of new CRC therapies. The first part will describe the pathology and its molecular basis, and the various drugs that are currently in clinical use or under development.
Then, in the second part we will review and discuss the use of cancer cell line collections, genetically engineered mouse models (GEM), primary human tumors xenografts (PDX) and ex vivo organotypic cultures (EVOC) to identify and validate anticancer colon therapeutics.
Colorectal CancerColorectal cancer is one of the major health concerns in the Western world. CRC is the second most frequently diagnosed cancer in men and women, right after lung cancer. It represents the second leading cause of cancer-related deaths, both in the United States and in Europe, with a significant rate of 9% and 13% of total cancer deaths, respectively (Fig.2).


The vast majority (~75%) of colon cancers are sporadic adenocarcinomas, arising from mutations in the epithelial cells lining the wall of the intestine that is in continuous renewal. CRC often begins as an adenomatous polyp, a benign growth on the interior surface of the organ.
Molecular mechanismsLoss of APC function is the initial molecular event that leads to adenoma formation. Indeed, germline mutations in the gene APC have been identified as the cause of familial adenomatous polyposis (FAP), an inheritable intestinal cancer syndrome [5], and APC is mutated in more than 80% of all sporadic cancers [6]. APC belongs to the WNT signaling pathway (Figure 3) where it interacts with other proteins like AXINS and GSK3? to make a complex that down-regulates the cellular levels of ?-CATENIN (see [7] for review).
Activating mutations in ?-CATENIN gene have also been observed in more than 10% of CRC [8].
Through several cytoplasmic components, the signal is transduced to ?-CATENIN, which enters the nucleus and forms a complex with LEF and TCF4 to activate transcription of WNT target genes. Mutations in APC, Axin and ?-CATENIN genes lead to constitutive activation of WNT signaling and ultimately to cancer development. Clinical managementIt is commonly accepted that CRC results from complex interactions between inherited and environmental factors, with a large contribution of dietary and life style factors as suggested by wide geographical risk variations. However, the primary risk factor of CRC is age, as 90% of the cases are diagnosed over the age of 50 years [9]. Surgical removal remains the most efficient treatment for early stage colorectal cancer, and may be curative for cancers that have not spread. Patients whose cancer is detected at an early, localized stage present a 5-year survival around 90% [9]. For these reasons, US and European Union have implemented preventive screening programs that have contributed to slightly reduce morbidity and mortality [10].Unfortunately, as in many other forms of cancer, colon cancer does not display too many symptoms, develops slowly over a period of several years, and only manifests itself when the disease begins to extend. Adjuvant chemotherapy in combination with surgery or radiation is then the usual treatment.
However, 5 of the 9 anti-CRC drugs approved by the FDA today are basic cytotoxic chemotherapeutics that attack cancer cells at a very fundamental level (i.e.
These figures underline the urgent need to expand the standard therapy options by turning to more focused therapeutic strategies. In recent years, combination of basic chemotherapies with targeted therapies, in the form of humanized monoclonal antibodies directed against the vascular endothelial growth factor VEGF (Bevacizumab) to prevent the growth of blood vessels to the tumor, or directed against the EGF receptor (Cetuximab, Panitumumab) to block mitogenic factors that promote cancer growth, have been introduced as possible therapeutic protocol and used routinely to treat standard CRCs, as well as metastatic CRCs (Table 1). During the preparation of this manuscript (August 2012), another recombinant protein active against angiogenesis, Aflibercept, has been approved by the FDA for the treatment of metastatic CRC in second-line therapy (Table 1). Designing new therapiesA classical approach of drug design in oncology is to identify modulators of specific signal transduction pathways that are important for tumor growth, survival, invasion, and metastasis. These results can be achieved by modulating the pathway at different levels, from the membrane receptor to the final nuclear transcription factors (Figure 3). It is now well documented that a number of critical pathways regulating stem cell maintenance and normal developmental processes (e.g. HEDGEHOG-GLI, NOTCH, TGF) are also involved in the self-renewal and differentiation of cancer stem cells whose tumors are initiated [20].
To date, the only compound designed to specifically disrupt ?-CATENIN is developed for the treatment of Familial Adenomatous Polyposis (FAP), an inherited form of colon cancer.
This new RNAi-based therapeutic known as CEQ508 consists of a modified E.coli bacterium that is able to express and deliver a shRNA to the epithelial cells of the gastrointestinal mucosa after ingestion by the patient [23].
KLF4 (Kruppel-like factor 4) is a tumor suppressor factor which is typically deficient in a variety of cancers, including colorectal cancer. In addition to controlling the cell cycle regulator cyclin D1, KLF4 has also been shown to inhibit the expression of ?-CATENIN [24]. Therefore, the modulation of KLF4 expression may represent a novel therapeutic approach for ?-CATENIN-driven malignancies. Acquired tumor resistance and targeted therapies In the recent years, a cohort of oncogenes, including BRAF, KRAS, NRAS, PI3K, PTEN and SMAD4, have been found mutated in CRC with significant frequencies ranging from 6% (NRAS) to 40% (KRAS) [26]. These observations pinpoint one of the most challenging aspects of anticancer therapy that is intrinsic or acquired drug resistance. Indeed, several studies have shown that these mutations are associated with the lack of response to Cetuximab and Panitumumab (anti-EGFR therapies) observed in a subset of chemorefractory metastatic CRCs, suggesting that the corresponding deregulated signaling pathways are responsible for the occurrence of resistance of the tumor to the clinical treatment [27-28]. As a result, downstream key components (mostly protein kinases) of these constitutively activated growth-related signaling cascades have become targets for drug development.
Small molecules inhibitors of BRAF (ARQ 736), MEK (Selumetinib, PD-0325901), PI3K (PX-866, BEZ235, BKM120), and MET (Tivantinib) that were able to reverse resistance to EGFR inhibitor therapy in pre-clinical studies [29-31] are currently in CRC Phase II clinical studies (Table 2). This new class of drugs appears therefore as a promising third-line therapeutic strategy for colon cancer patients, especially after recurrence of tumor resistance.
However, a recent publication reporting the apparition of resistance to PI3K and AKT inhibitors mediated by ?-CATENIN overactivation, may temper this enthusiasm. Depending on the tumor status, from pro-apoptotic tumor suppressor, PI3K or AKT inhibitors could become metastatic inducers [32].
Similar side effect induction mechanisms have also been reported in CRC for the BRAF(V600E) inhibitor Vemurafenib that triggers paradoxical EGFR activation [33].
New anti-angiogenesis therapiesAs previously mentioned, until recently the humanized monoclonal antibody Bevacizumab against VEGF was the only anti-angiogenesis agent approved by FDA. It is now completed by Aflibercept, a recombinant protein consisting of the key domains of VEGF receptors 1 and 2. The compound captures and blocks all isoforms of VEGF-A and VEGF-B growth factors, as well as placental growth factors [34]. Due to improvement in the understanding of the critical role of angiogenesis in the maintenance of CRC tumors and the spread of their metastasis, anti-angiogenesis has become an area of active investigation [35]. However, the recent failure in Phase III first-line studies of two promising compounds (Sunitunib in 2009 and Cediranib in 2010) has cast serious doubt on that strategy.
Therefore, the approval of Aflibercept provides timely support to the further development of anti-angiogenics as treatment for metastatic CRC.
Today, 4 additional therapeutic agents that target VEGF, Ramucirumab [36], Icrucumab [37], Regorafenib [38] and Vatalanib [39-40] are under clinical evaluation (Table 2).
This battery of anti-angiogenics is supplemented by AMG386, a recombinant peptide-antibody fusion protein (peptibody) which targets another signaling pathway involved in tumoral angiogenesis, the angiopoietin axis [41].
AMG386, which inhibits the interaction between the ligands ANGIOPOIETIN-1 and ANGIOPOIETIN-2 with their TIE2 receptor, is currently in Phase II. Finally, a phase III trial was also recently initiated (May 2012) to evaluate TAS-102, a combination agent composed of the cytotoxic pyrimidine analog TFT and a thymidine phosphorylase inhibitor (TPI) with antineoplastic activity (Table 2).
TAS-102 mechanism of action is based on the inhibition of the thymidine phosphorylase (TYMP) also known as the platelet-derived endothelial cell growth factor, a potent angiogenic factor [42].
In this context, it is important to point out that differences in the efficiency to block angiogenesis and tumor progression have been observed between preclinical models and clinical trials, when comparing antibodies with small molecules [35]. Other cellular mechanisms under targetModifications in the epigenetic landscape are commonly associated with cancer, but on the contrary to genetic mutations, these changes are potentially reversible and therefore druggable. Most of the epigenetic drugs discovered to date modulate DNA methylation or histone acetylation. Four epigenetic drugs have already been approved by FDA for use in clinic against various cancers. Unconventional approachesOncolytic viral therapy represents an appealing alternative therapeutic strategy for the treatment of CRC, both as single agent or in combination with existing clinical regimens.
Oncolytic viruses, like the vaccinia virus (a virus previously used for worldwide vaccination against smallpox), have the property to selectively infect and destroy tumor cells with limited or no toxicity to normal tissues. These viruses efficiently replicate in tumor tissue, cause tumor lyses and stimulate antitumor immune response. During the last decade, numerous mutants have been engineered to improve their tumor specificity and antitumor efficacy, and to allow tracking of viral delivering by non-invasive imaging [44]. No less than five oncolytic virotherapies are currently evaluated in clinical trials for metastatic CRC indication, including ColoAd1, derived from an adenovirus [45], NV1020, derived from an Herpes simplex virus [46], Reolysin, a reovirus [47], and JX-594 [48] and GL-ONC1 [49] both derived from vaccinia viruses, reflecting the many hopes carried by this emerging treatment modality. However, it is noteworthy to mention that there are still some difficulties to viral infection. Solid tumors have a complex microenvironment that includes disorganized surrounding stroma, poor vascular network as well as high interstitial fluid pressure. All these parameters will limit viral delivery since viral penetration directly depends on cellular packing density and adhesion between cancer cells [50]. Moreover, hypoxia reduces viral replication, and therefore oncolytic efficiency, without affecting tumoral cells viability [51].
3D cell cultures or spheroids in vitro, or patient primary-derived xenografts that retain tumoral architecture complexity in vivo, will be critical for future clinical success.This inventory of new drugs for the treatment of colorectal cancer highlights the diversity of approaches being considered to combat the disease.
Whether based on small molecules, humanized antibodies or modified viruses, their success in further clinical assessment is largely related to the quality of their preclinical evaluation. This is why both the choice of appropriate existing model systems and the development of more clinically relevant and predictive pre-clinical models appear critical in overcoming the high attrition rates of compounds entering clinical trials.Current research is also focusing on the development of biomarkers that will be useful for the early detection of CRC, as well as for fine-tuning drug regimen and following efficacy during trials and treatments.
To date, only a few markers have been recommended for practical use in clinic [52] but large-scale genomics technology combined with advanced statistical analyses should generate soon new biomarker panels for CRC diagnosis [53].
Then, it will be interesting to see how these biomarkers could be implemented in preclinical stages to improve drug selection. 3.
Colon cancer cell linesIt is worth mentioning that most of our understanding of the molecular mechanisms involved in CRC come from studies done on mouse or human cell lines that represent only a highly selected fraction of the original tumor and that may have acquired in vitro additional genetic abnormalities. Clearly, the scientific community has taken into account these limitations, as shown by the growing interest for more complex models (e.g.
However, although imperfect, colon cancer cell lines still represent a unique resource that can be extremely valuable in term of genetic manipulation and high-throughput screening, with cell viability, cell proliferation or promoter specific reporter activity being the usual endpoints followed. Several initiatives have been launched to maximize their utility in large scale drug discovery programs. In an attempt to identify new active molecules, over 100,000 chemical compounds were pharmacologically tested in this cell line set. But disappointingly, most of the selected positive candidates were typical cytotoxics, affecting cancer cells via general fundamental cellular processes, like cell cycle regulation.
These cell lines are under further characterization by sequencing for mutations in known human oncogenes. Interestingly, this resource can be screened on demand for any chemical or biological agent. The Cancer Genome ProjectThe emergence of tumor acquired resistance to pharmacological inhibitors linked to mutations in driver oncogenes has recently revived the interest for cancer cell lines. Indeed, an extensive characterization of cell lines at the genomic and genetic levels will allow determining a genetic profile predictive of drug sensitivity. Such a signature will help to stratify patient population and identify efficient therapeutics combination, as long as cell lines reflect real tumor biology. Using current high throughput techniques this program intends to provide information on mutations, copy number variations, single nucleotide polymorphisms (SNPs) and microsatellite instability of usual cancer cell lines.
Biomimetic cell culture modelsThe derivation of a cancer cell line from the primary tumor is not an obvious process, and for many cancers, few if any cell line can be obtained. A success rate of less than 10% has been reported for the establishment of human colon cancer cell lines grew immediately in vitro from fresh tumors [56]. Elasticity of the surrounding microenvironment has been pointed out as a critical parameter of in vitro cell growth.
Indeed, culture plastic dishes are much more rigid than the epithelial wall of the intestine (10000 kPa vs 40 kPa). More importantly, depending on the stiffness of the substrate, cells can be differentially sensitive to drugs in term of spreading and apoptosis-induction, notably because of the expression and presentation of surface receptors [57].
Therefore, the choice of an appropriate biomimetic substrate that will preserve the in vivo phenotype appears decisive not only for cell survival but also for clinical relevance. Colon cancer stem cell modelsCancer stem cells (CSCs) are a discrete self-renewing tumor cell subpopulation that can differentiate into multiple lineages, drive tumor growth and metastasis. Moreover, CSCs are thought to be responsible for tumor recurrence after chemotherapy and radiotherapy. One of the characteristic of the CSCs is their ability to form spherical cell colonies when they are cultured in chemically defined serum-free medium at a relative low density [58]. This model, also called colonospheres, constitutes a unique in vitro system to elaborate therapeutic strategies that specifically target colon CSCs, like oncolytic adenoviruses developed to target specific CSCs antigens (e.g.
Multicellular Spheroid modelsEarly stage development of novel anti cancer treatment requires in vitro methods able to deliver fast, reliable and predictive results. To select the most active molecule lead in a library, pharmaceutical industry has turned its attention to High Throughput Screening (HTS) tests which mimic human tissues. Furthermore, 3-Dimensional (3D) test system has been widely accepted as being more informative and relevant than classical 2D cell systems. Combination of HTS and 3D models such as the multicellular tumor spheroid model has been pointed out having the potential to increase predictability of clinical efficacy from in vitro validation therefore contributing savings in both development cost and time [59].
Advantages of spheroids compared to classical 2D cell line culture have been reported [60]. Indeed, proteomic analysis of multicellular spheroids versus monolayers cultures identifies differential protein expression relevant to tumor cell proliferation, survival, and chemoresistance. Consequently, spheroids strategy has been used for the screening of new anticancer agents, like compounds that modulate apoptosis pathways [61].Standardized spherical microtissue production in a 96 or 384-well hanging-drop multiwell plate format on robotic platform has been successfully achieved by 3D Biomatrix and Insphero AG. Formation of standardized spheroids rely on the use of A431.H9, a human epithelial carcinoma cells, [62] or the colon cancer cell line HCT116 [63]. Interestingly, loss of cancer drug activity in HCT-116 cells during spheroid formation in a 3D spheroid cell culture system has been reported [64].
Spheroid cell models also enable the study of colon cancer chemoresistance and metastasis [65]. Chemically induced animal modelsColon cancer can be induced in mouse by specific carcinogens like 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM).
Exposure of the mouse intestine to these chemicals triggers rapid and reproducible tumor induction which recapitulates the adenoma-carcinoma sequence that occurs in human sporadic CRCs, with the notable exception however of the invasive and metastatic stage.
Interestingly, differences in genetic mutations that arise in chemically induced colon tumor models are largely carcinogen specific.
K-Ras mutations are predominant in the DMH model, while AOM treated mice exhibit tumors with activating mutations in the ?-catenin gene [66].



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Comments

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    22.10.2015

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    22.10.2015