The mode of action of the HIV protease inhibitor lopinavir against HPV

Human papillomavirus (HPV) related cervical cancer is still the most common gynaecological malignancy in developing countries and, as yet, there is no alternative to surgery for the treatment of HPV-associated pre-malignant lesions. HPV 'hijacks' the host-cell ubiquitin-proteasome system to degrade the p53 and Rb tumour suppressor proteins which in turn, leads to the development of cancer. Previous studies have shown that the HIV protease inhibitor lopinavir selectively inhibits the chymotryptic-like activity of the 26S proteasome which stabilises p53 and induces the apoptosis of HPV positive cervical carcinoma cells. Based on this it was hypothesised that lopinavir treatment of HPV positive cervical carcinoma cells would produce changes in the levels of a wide range of cellular proteins that are dis-regulated by HPV-related activation of the proteasome. In order to address this, antibody microarray screening was carried out on lopinavir treated and control untreated HPV positive SiHa cervical carcinoma cells. This showed lopinavir induced alterations in 51 proteins including the cellular antiviral defence protein RNase L. Lopinavir induced both a dose and time dependent increase in RNase L which was subsequently confirmed by western blotting. Transient siRNA silencing of RNase L expression reduced the lopinavir-dependent toxicity in SiHa cells, suggesting an important role for this protein in the toxicity of lopinavir in HPV infected cells. SiHa cells were much more sensitive to lopinavir than CaSKi cervical carcinoma cells which had much higher levels of the E6 protein and did not up regulate RNase L. Furthermore, lopinavir treated HPV16 E6/E7 immortalised keratinocytes were also shown to up regulate RNase L protein expression and these cells were much more sensitive to lopinavir induced apoptosis than mortal control keratinocytes. In addition, transient expression of RNase L in RNase L-deficient C33A cells and the same cells stably transfected with HPV16 E6 (C33AE6) demonstrated that E6 protected these cells from RNaseL-induced cell death. Surprisingly, analysis of RNase L protein levels in these cells demonstrated that E6 did not induce the degradation of the RNase L protein. Instead it was found that E6 stabilised the interaction between RNase L and its endogenous inhibitor protein, ABCE1, and that lopinavir de-stabilised this interaction. Given that C33A tumour cells, E6/E7 immortalised keratinocytes and hTert immortalised keratinocytes are all sensitive to lopinavir, this implies that this compound does not specifically target HPV immortalised cells but rather targets immortalised cells in general, regardless of how this was achieved. The optimum concentration of lopinavir for all these effects was 25 μM, which is 15-fold higher than is observed in cervico-vaginal secretions following oral dosing with the drug Kaletra. In conclusion these results have confirmed the potential of lopinavir to treat HPV related pre-cancerous cervical lesions and provided at least part of the mode-of-action. Indeed they strongly support the use of lopinavir as a low-cost, self-applied topical alternative to surgery for this disease which will be of particular benefit in low-resource countries. Finally, the ability of lopinavir to induce apoptosis of non-HPV related immortalised cells merits further investigation since this indicates this drug may be useful for the treatment of other non HPV related pre-malignant conditions.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A Single-Arm, Proof-Of-Concept Trial of Lopimune (Lopinavir/Ritonavir) as a Treatment for HPV-Related Pre-Invasive Cervical Disease

BACKGROUND: Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings. METHODS: Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC -ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration. RESULTS: A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 (52.2%, 95%CI: 30.6-73.2%). Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22 (63.6%, 95%CI: 40.6-82.8%) had no dysplasia and 4/22 (18.2%, 95%CI: 9.9-65.1%) were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions. CONCLUSIONS: These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy. TRIAL REGISTRATION: ISRCTN Registry 48776874

Participant characteristics at study baseline.

<p>Participant characteristics at study baseline.</p

Changes in cytology and HPV results between baseline and 3-month visits following Lopimune treatment.

<p>Changes in cytology and HPV results between baseline and 3-month visits following Lopimune treatment.</p

Patient screening, testing and management flow chart used for the study.

<p>Patient screening, testing and management flow chart used for the study.</p