HAMLET: Functional Properties and Therapeutic Potential

James Ho CS; Anna Rydström; Maria Trulsson; Johannes Bålfors; Petter Storm; Manoj Puthia; Aftab Nadeem; Catharina Svanborg

Disclosures

Future Oncol. 2012;8(10):1301-1313. 

In This Article

In vivo Effects of HAMLET

Most current therapies lack tumor specificity and toxicity for healthy tissues is therefore a major concern. A variety of approaches are being explored to identify new drugs that selectively kill tumor cells. Significant progress is being made and new, targeted therapies include inhibitors of growth factors and their receptors, blockers of angiogenesis and proapoptotic drugs.[4–6,8] A high degree of selectivity for tumor cells in vitro and in vivo has identified HAMLET as an interesting candidate drug with tumor specificity in animal models and clinical studies.[13–15,75]

HAMLET Limits the Progression of Human Glioblastoma Xenografts

Malignant tumors of the brain may arise from cells in the brain tissue per se or from metastatic spread of tumor cells from peripheral organs. In both cases, therapeutic options are limited. Due to infiltrating growth, glioblastomas are not amenable to selective surgical removal and are resistant to irradiation and most, if not all, chemotherapy. Experimental therapies such as gene therapy and antisense, engineered and defective viruses may be efficient in brain tumor models, but those few that have made it to clinical trials have not been promising.[76] For example, regional infusion of a transferrin-diphtheria toxin complex has been shown to decrease tumor volume,[77] but complications included necrosis, brain edema and destruction of brain capillary endothelial cells. There remains, therefore, a great unmet need for novel therapeutic approaches that selectively eliminate the tumor cells without damaging functional brain tissue.

Xenotransplantation of human glioblastoma cells into nude rats has been used extensively to screen novel therapeutic agents.[78] The human tumor is cultured in vitro and approximately 5–10 µl of phosphate-buffered saline containing five biopsy spheroids is injected into the striatum. Rats are monitored daily and sacrificed when they develop symptoms such as passivity, clumsiness and paresis, and the tumor mass is quantified by MRI scans.

To administer HAMLET, the authors chose to use convection-enhanced delivery, where the region of the tumor was infused with HAMLET or α-lactalbumin through a cannula connected to an osmotic mini pump.[13] A 24-h infusion of HAMLET was sufficient to dramatically delay tumor development (Figure 4A) and the onset of pressure symptoms. By immunohistochemistry, the protein complex was shown to trigger apoptosis in tumor tissue and HAMLET penetrated throughout the hemisphere that was injected. However, HAMLET infusions did not appear to harm the normal brain or cause neurological symptoms. It appears from these studies that regional therapy of HAMLET may be proposed as a possible novel approach to control the progression of malignant and invasive brain tumors and possibly of metastases of tumors from distant sites. Use of convection-enhanced delivery into the CNS is essential for future studies, as HAMLET is inactivated in human serum.

Figure 4.

Therapeutic effects of HAMLET.
(A) Delayed brain tumor development after convection-enhanced delivery of HAMLET into the brain of nude rats bearing human glioblastoma xenografts, compared with α-lactalbumin controls. (B) Human skin papillomas showing a reduction in lesion size after topical HAMLET administration. (C) Reduction in bladder cancer size after 1 week of HAMLET instillation into the bladder.
(A) Reproduced with permission from [13].
(B) Reproduced with permission from [14].
(C) Reproduced with permission from [15].

Effects of HAMLET on Human Skin Papillomas: A Placebo-controlled Study

The authors' group selected skin papillomas for the first human study of HAMLET's effect as a topical therapeutic.[14] Human papillomavirus (HPV)-transformed keratinocytes proliferate and form warts, and most of the skin lesions remain benign. Cutaneous papillomas are caused by one or more of approximately 130 different HPV types.[79] Current treatments include cryotherapy, curettage, cautery, topical virucidal agents,[80] lasers,[81,82] antimitotic agents[83] and immunoactivators.[84–86] Immunosuppressed patients run an increased risk of developing papillomas and often carry multiple HPV types.[87]

The authors tested topical treatment of human skin papillomas with HAMLET in a placebo-controlled, double-blind study. HAMLET (0.7 mM in 0.9% NaCl) or placebo (0.9% NaCl) was applied topically once a day for 3 weeks and the volume change was recorded. After the first trial, both the placebo and the treatment group were offered a 3-week course of HAMLET in an open arm of the study. The lesion volume was reduced by ≥75% in the HAMLET group compared with 15% in the placebo group (p < 0.001). Complete resolution of all lesions had occurred in 90% of all HAMLET-treated patients after 5 months, and the time to resolution was shorter in the group receiving HAMLET from the start compared with the placebo group (Figure 4B). No adverse reactions were reported, and there was no difference in treatment outcome between immunocompetent and immunosuppressed patients. It was concluded that topical HAMLET treatment has a beneficial and lasting effect on skin papillomas.

Intravesical Instillation of HAMLET in Patients With Bladder Cancer

Bladder cancers are common, differing in severity and accessibility to therapy. Surgery alone or in combination with cytostatic drugs is used successfully, but therapy-resistant tumors still cause significant morbidity and mortality.[88] After removal by transurethral resection of superficial papillary tumors the short-term prognosis is excellent. Intravesical instillation of the Bacille Calmette–Guerin vaccine results in a recurrence-free interval of at least 2 years in approximately 70% of the treated patients.[89] Still, the recurrence rate is high and due to the risk of dedifferentiation, patients require life-long follow-up. Invasive tumors are removed by radical cystectomy and some patients receive adjuvant systemic chemotherapy but may still have a poor prognosis.[90] Furthermore, Bacille Calmette–Guerin treatment has significant side effects and, especially in immunosuppressed patients, systemic antituberculous therapy may be required.

The authors' group therefore studied whether intravesical HAMLET instillations may be used to kill cancer cells in vivo.[15] In patients with superficial, exophytic bladder cancer or cancer in situ, an image of the tumor was obtained through the cystoscope at the time of diagnosis. After five daily intravesical instillations of HAMLET during the week before scheduled surgery, the tumor was again photographed and biopsies were obtained to examine tissue integrity and to compare the apoptotic response of the tumor and surrounding healthy tissues. By cystoscopy, a reduction in tumor size was detected (Figure 4C) and in biopsy specimens, apoptotic cells were seen in the remaining tumor. The patients with cancer in situ showed a reduction in the number of tumor-positive biopsies. By contrast, there was no TUNEL response in healthy tissue biopsies adjacent to the tumor and no cell exfoliation after intravesical NaCl instillations in the patients. In addition, each HAMLET instillation triggered the exfoliation of large numbers of tumor cells.

The results show that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo, and that local administration of HAMLET may cause a rapid reduction in tumor mass.

processing....