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"These data indicated that lopinavir could potentially be used topically as an anti-HPV therapeutic"
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Download the PDF here
from 2011 published paper in Antiviral Therapy of in vitro study
"Prior to a clinical trial to test this new indication, we set out to further characterise how lopinavir works against HPV.........In conclusion, our data support the potential use of lopinavir as a topical, self-administered treatment for HPV-related cervical dysplasia. If successful, this treatment would be particularly useful in low-resource countries which lack cytology screening and surgical services, and where HIV infection is often a significant additional complication. With current pricing strategies, anti-HPV vaccines are not an option in such settings, and as previously discussed, they will most likely have reduced efficacy in HIV-positive women. Furthermore, in view of the observation that infection with HPV can enhance the sexual transmission of HIV in both men and women [29,30], this clearly indicates that a simple, non-surgical treatment for HPV-related precancerous lesions may not only reduce the burden of cervical cancer but may also reduce the chances of HIV transmission."
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End in sight for cervical cancer? Lopinavir (Kaletra) - attached below is in vitro study paper published in Antiviral Therapy in 2011
A Manchester university team believe they have developed a radical new therapy for women at high risk of the disease
video on formulation:
http://www.telegraph.co.uk/women/womens-health/10642431/End-in-sight-for-cervical-cancer.html
The study, published in the journal Antiviral Therapy, builds on the team's previous work in 2006 that first identified lopinavir as a potential therapeutic for HPV-related cervical cancer following laboratory tests on cell cultures.
"Since publishing our earlier work, we have now found that lopinavir selectively kills HPV-infected, non-cancerous cells, while leaving healthy cells relatively unaffected," said Dr Ian Hampson, from Manchester's School of Cancer and Enabling Sciences.
"This is a very significant finding as these cells are not cancer cells but are the closest thing to being like the cells found in a pre-cancerous HPV infection of the cervix. In addition we were also able to show that lopinavir kills these HPV-infected cells by re-activating a well-known antiviral system that is suppressed by HPV."
In many developing countries, HPV-related cervical cancer is still one of the most common women's cancers accounting for approximately 290,000 deaths per year worldwide. The same virus also causes a significant proportion of cancers of the mouth and throat in both men and women and this disease is showing an alarming increase in developed countries, such as the UK, where it is now more than twice as common as cervical cancer.
Dr Hampson said: "Our results suggest that for this drug to work against HPV it would be necessary to treat virus-infected cells of the cervix with roughly 10-15 times the concentration that is normally found in HIV-infected patients taking lopinavir as tablets. This implies that, for this treatment to work, it would need to be locally applied as a cream or pessary."
By Cherrill Hicks
6:22PM GMT 16 Feb 2014
Ian Hampson is finding it hard to contain his excitement. A molecular virologist with a passionate interest in understanding the relationship between viruses and cancer, he thinks that he and his wife, Lynne, may - he stresses the word may - have found a revolutionary self-help therapy for women with pre-cancerous cell changes in the cervix.
Almost by chance, the Hampsons discovered in their lab at St Mary's Hospital, Manchester, that the drug lopinavir, licensed for the treatment of HIV, attacks the strain of human papillomavirus (HPV) that causes virtually all cases of cervical cancer. An as-yet unpublished clinical trial has found that lopinavir capsules, when inserted into the vagina, appear to kill off abnormal cells in the cervix. There appear to be no side effects.
Cervical cancer is still the main killer of women in the developing world, causing more than 280,000 deaths worldwide annually, so the idea that women might use a simple vaginal pessary to protect themselves against it is an exciting prospect.
But the results of Dr Hampson's trial are also promising for women in developed countries. Up to 40,000 women in England alone are found to have abnormalities of the cervix every year, following smear tests or biopsies. At present the only treatment for high grade, pre-cancerous changes is invasive - and far from risk-free - surgery.
"The study looked at 40 women with both high and low-grade pre-cancerous disease of the cervix and the antiviral drug, normally used orally to treat HIV, was self-applied directly to the cervix as a pessary [A therapeutic pessary is a medical device similar to the outer ring of a diaphragm. The pessary can be placed temporarily or permanently, and must be fitted by a medical person.]
The results, due to be presented at two international scientific conferences later this month and next, showed a high proportion of women diagnosed with HPV positive high-grade disease returned to normal following a short course of the new treatment.
The findings build on previous peer-reviewed laboratory based research carried out by Drs Hampson and will be submitted to a journal soon. They have been described by an independent leading specialist in gynaecological cancer as very impressive.
The 40 women, who were all HPV positive with either high-grade, borderline or low grade disease, were treated with one capsule of the antiviral drug twice a day for 2 weeks. Repeat cervical smears showed a marked improvement within one month of the treatment although after three months, there was a definite response. Out of 23 women initially diagnosed with high-grade disease, 19 (82.6%) had returned to normal and two now had low-grade disease giving an overall positive response in 91.2%.of those treated. Furthermore the 17 women initially diagnosed with borderline or low-grade disease also showed similar improvement."
Sensational news, then, but it has taken Dr Hampson 12 years and a battle against numerous obstacles - including UK red tape, lack of interest and funding - to reach this point. "Worldwide, one woman dies every two minutes of cervical cancer," he says. "The results of the early trial are staggering, but I might have to wait another six months to get this study published, and another year to get a bigger study off the ground. I want to get this treatment on the road."
The story begins in 2002, when Lynne, a lecturer in viral oncology at the University of Manchester, was studying HPV16 - the main cancer-causing strain of HPV - and analysing how human proteins interacted with virus proteins.
"It's well known that viruses like HPV take over human cells by hijacking their 'waste disposal systems' to make cells throw away certain proteins inappropriately," Dr Hampson says. "Human cells use enzymes called proteases to get rid of waste proteins in different ways. Lynne's results suggested that HPV was enhancing the function of a specific type of protease.
"The findings made me speculate that there might be a drug that could selectively block this particular function of the virus. A class of drugs used to treat HIV, known as protease inhibitors, primarily targets the HIV protease enzyme; but previous work has shown they could also block the same type of human protease, which our results had shown was important for HPV. It was an epiphany."
At his wife's suggestion, they got hold of as many protease inhibitors as they could lay their hands on (using a national Aids repository in the US, which supplies small quantities of drugs for research). "We literally smashed up tablets and stuck them into cultures both of HPV and cervical cancer cells," recalls Dr Hampson. Several seemed to have activity against HPV and cancer, but lopinavir was the most potent. "Lopinavir was remarkably toxic. We could see the cells dying in the lab."
By 2011, he says, they had shown that lopinavir was "multifunctional", meaning it inhibited both viral proteins and the proteins in cancer cells. Still nothing had been proven clinically, and as Dr Hampson soon discovered, setting up a patient trial proved difficult.
Lopinavir is made by the US company Abbott Laboratories as part of a combination drug for HIV called Kaletra. The good news was that, as one of the most widely prescribed drugs worldwide and already licensed for both children and adults with HIV, it would not need extensive safety testing. The bad news was that it was only licensed for oral delivery. This formulation would not, at the strength needed, get enough of the drug delivered to the cervix.
"Our work showed we needed a 10-fold higher concentration of the drug at the site of infection - the cervix - than you can get by taking the drug orally," says Dr Hampson. "The obvious solution was to reformulate the drug, so it could be applied locally as a cream or pessary, and organise a clinical trial."
But the cost of reformulating the drug meant there was little interest from drug companies, cancer charities or research agencies. Then one of Dr Hampson's PhD students pointed out that one oral form of the drug available was a soft gelatin capsule, which might "melt very nicely" in the vagina, and could be trialled as a pessary.
There was another obstacle, though. In Europe the makers of the capsule had phased it out in favour of a hard tablet. A generic lopinavir capsule called Lopimune, made by an Indian company, was only licensed for Africa.
"I wanted to bring some Lopimune into the UK for a trial," says Dr Hampson, "but it would have involved a lot of red tape - and would have cost a fortune. I felt as if I was banging my head against a brick wall."
The same PhD student, Dr Orora Maranga, pointed out how useful such a self-help treatment - if it worked - would be to women in Africa, where there are few screening programmes to pick up pre-cancerous conditions, or surgical facilities to treat them.
So it was that, near the end of 2011, the Hampsons finally won approval from Kenyatta National Hospital in Nairobi (where Dr Maranga was then a senior registrar) for a preliminary trial of lopinavir in women with pre-cancerous cells in the cervix. In return they would set up a cervical-cancer screening programme - needed to identify women suitable for the trial - and facilities for surgery, if it was needed.
Funding such a trial was difficult, and Dr Hampson is enormously grateful to those who supplied money or equipment. Donors included St Mary's, Central Lancashire Healthcare Trust, a company called Hologic, a UK philanthropist, Ken Cholerton, and a small charity on the Isle of Wight, the Caring Cancer Trust.
Finally, last year, in a trial overseen by Dr Maranga, more than 800 Kenyan women were screened for cervical cancer. Of those, 21 per cent were found to be HPV positive, of whom 40 had pre-cancerous cells in the cervix. Twenty-three of this group had high-grade disease - where there is a higher risk of cancer developing and for which the only treatment is surgery. A further 17 had low-grade or borderline disease.
All 40 women were asked to insert a lopinavir capsule as a pessary, twice a day, for two weeks. At three months, each participant underwent a biopsy of the cervix, together with a smear test. In 19 of the 23 women with high-grade disease, the abnormal cells had disappeared. Two women now had low-grade changes, while two still had high-grade disease, for which they needed surgery.
Dr Hampson emphasises that this was an early trial, the type normally used to look at how well a new drug is tolerated. And he points out that there were no side effects. "We had a 90 per cent treatment response in the high-grade group - we couldn't believe it," he says. Although high-grade disease can sometimes disappear spontaneously after a year or two, this is rare after three months. There was also, he reports, "very high clearance" among women with low-grade and borderline cell changes.
Dr Hampson is now planning a larger controlled trial involving 500-1,000 women based in three sites in Africa - Nairobi, Kampala and Johannesburg. If he can raise the funds, that is. If the results can be replicated, the advantages for women everywhere are obvious. This potential treatment, less invasive than surgery, would also avoid the risk of premature labour in pregnancy that is associated with the operation.
"Research has found that one surgical excision of abnormal cervical cells increases the risk of premature delivery by 14 per cent," says Prof Pierre Martin-Hirsch, a gynaecology oncologist at Lancashire Teaching Hospitals, who finds the results of this first trial "impressive".
By contrast, Dr Hampson points out that in cases where the disease is persistent, "treatment with lopinavir can be repeated as often as needed, because it appears to be safe."
Lopinavir could also be an option for women with low-grade pre-cancers who are currently offered a "watch and wait" approach - "a horrible time of anxiety".
"While a vaccine is now being offered against HPV in some parts of the world," he points out, "it will take 20 to 30 years before we have immunity."
Cancer Research UK says it cannot comment on a trial that has not yet been peer-reviewed or published. "This is raising the hopes of patients," warns a spokesman. "A peer review might find flaws in the trial. It may never get published."
What frustrates Dr Hampson is the fact that, even if further trials were successful, it could be years before this pessary becomes available. There is now talk of of a "window trial" - one in which lopinavir could be offered to women with high-grade disease while they are waiting for surgery. "The drug has been shown to be safe, and the women have nothing to lose and everything to gain. If it works they will not need an operation.
"We are looking at this possibility, but clinicians in the UK are understandably cautious about using treatments off-label. And who can blame them? In our litigation culture, it doesn't pay to take risks."
Which is why Dr Hampson supports Lord Saatchi's Medical Innovation Bill, designed to encourage doctors to innovate without fear of prosecution, and which is the subject of a government-backed consultation.
"At present there is a great reluctance to deviate from the accepted best clinical practice. Had we been able to obtain the particular drug formulation in the UK, it would have been far easier to convince a clinician to try the treatment - if the fear of litigation was reduced."
The University of Manchester has filed a patent for treatment with lopinavir for HPV-related disease
Saatchi's Medical Innovation Bill
Lord Saatchi's Bill, now open to public consultation, would allow doctors to prescribe ''off label'' drugs. These are drugs that have been tried and tested for one specific use (in this case lopinavir used to combat HIV). lopinavir is known to be safe, but doctors cannot easily prescribe it for other, non-standard uses. The Bill aims to speed up the development of new and innovative treatments for cancer and other diseases.
For details of Lord Saatchi's bill, see saatchibill.tumblr.com
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Antiviral Therapy 2011
Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells
Gavin Batman1, Anthony W Oliver1, Ingeborg Zehbe2, Christina Richard3, Lynne Hampson1,, Ian N Hampson1,*,
1Gynaecological Oncology Laboratories, School of Cancer & Enabling Sciences, University of Manchester, St Mary's Hospital, Manchester, UK
2Probe Development & Biomarker Exploration, Regional Research Institute, Thunder Bay, ON, Canada
3Regional Cancer Care, Thunder Bay, Thunder Bay Regional Health Sciences Centre, Thunder Bay, ON, Canada
Abstract
Background: We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism.
Methods: SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used.
Results: Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression.
Conclusions: These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.
Introduction
Human papillomavirus (HPV) has been unequivocally implicated as a causative agent in the development of cervical cancer [1,2] with >490,000 women worldwide diagnosed every year and >273,000 deaths per annum. Although there are over 100 subtypes of HPV, 70% of cervical cancer cases are attributed to infection with just two high-risk types, 16 and 18 [3,4].
There are currently large differences in the incidence of this disease between developed and developing countries, mainly due to the implementation of effective screening strategies in the former [5]. For example, in the UK, cervical cancer now rates as the 11th most common women's malignancy, whereas in Africa it remains the most prevalent. Lack of screening is not the only contributory factor here, as many African nations also carry the additional burden of HIV infection, which is known to predispose to HPV-related cervical neoplasia [6]. Whilst surgery remains the current treatment of choice for HPV-related pre-cancerous lesions, this is not generally advocated for low-grade disease for which a 'wait and see' approach is usually adopted. Unfortunately for HIV-positive women, surgery is far less effective and recurrence rates of cervical cancer are much higher in these women than those with a negative HIV status [7]. Surgical intervention for cervical cancer in HIV-positive women can also lead to a massive post-operative increase in vaginal fluid HIV titre, thereby greatly increasing the chances of sexual transmission of the virus [8,9].
The production of an HPV vaccine has prompted many developed countries to implement anti-HPV vaccination programmes which aim to target females prior to their first sexual exposure [10]. However, this will still leave large numbers of already infected women at risk, in addition to those infected with high-risk types of HPV not covered by the current vaccines. In developing areas like Africa the cost of such a vaccination programme, even with subsidies, is likely to be prohibitively expensive. Furthermore, there is also evidence to indicate that vaccination will be less effective in HIV-positive women [11,12].
For the foreseeable future it is clear that development of a non-surgical, preferably self-applied, treatment for HPV-related cervical dysplasia would be extremely valuable. This would have particular impact and benefit in low-resource settings where high levels of HIV infection can aggravate this condition.
We previously investigated the ability of HIV protease inhibitors to combat HPV infection and demonstrated that the antiretroviral drug lopinavir could stabilise the p53 protein and induce apoptosis of HPV-positive cervical carcinoma cells in vitro, albeit at higher doses than those achieved by oral administration [13]. These data indicated that lopinavir could potentially be used topically as an anti-HPV therapeutic. Prior to a clinical trial to test this new indication, we set out to further characterise how lopinavir works against HPV. In this regard we have used Fourier Transformation Infra Red spectroscopy to analyse the metabolic changes occurring in lopinavir-treated cervical carcinoma cells [14] and direct Raman spectroscopic imaging to identify the site of action of the drug in cells [15]. We now provide evidence to further support our initial observations and present data on the specific molecular mechanisms by which lopinavir works against HPV.
Discussion
Our data are the first to show that the HIV protease inhibitor lopinavir induces an increase in the levels of the RNASEL protein in HPV-positive cervical carcinoma cells and in stable HPV16 E6/E7 transfected human keratinocytes. Lopinavir was also shown to have enhanced toxicity against E6/E7 immortalised PHFKs when compared to normal PHFKs and our results are consistent with RNASEL contributing to this selective toxicity.
In agreement with the results shown in Figure 1, other groups have shown lopinavir can induce selective inhibition of the proteasome and this ability is thought to play a significant role in the anti-HIV effects of this, and other related compounds [19-22]. In addition to HIV, many viruses including HPV, have been shown to subvert/hijack the activity of the host proteasome in order to eliminate proteins that would be detrimental to viral persistence [23]. In HPV-infected cells the E6 and E7 oncoproteins are largely responsible for this activity, whereby they signal the destruction of selected cellular proteins (for example, p53 and Rb) in order to execute the viral life cycle [24].
Based on these observations, we opted to use antibody array screening of lopinavir-treated SiHa cells to analyse changes in protein expression following this treatment. This identified a whole range of cellular proteins that were up-regulated (Table 1), including Tp53 which is consistent with our previously reported findings [13]. Indeed, as we have discussed, lopinavir induced changes in the levels of several other apoptosis-related proteins and it is also significant that the majority of the proteins listed in Table 1 are known to be regulated by proteasomal degradation. On this point, an interesting finding was the observed lopinavir-induced down-regulation of GFAP which is known to be capable of inhibiting the proteasome [25]. Since p53 is a target of high-risk HPV E6, we searched the Virus Molecular INTeraction (VirusMINT) database [26] for any other known E6 and E7 targeted proteins which are affected by lopinavir treatment (Table 1). At this time, only Tp53 is a confirmed target of high-risk HPV.
Given that SiHa cells are a fully malignant cell type, we opted to investigate the toxicity of lopinavir against E6/E7 immortalised and normal control PHFKs. This showed that at a concentration of 25 μM, lopinavir up-regulated RNASEL expression in the E6/E7 cells which were much more susceptible to lopinavir-induced cell death than control PHFKs. At higher concentrations of drug, this selectivity was lost and these data indicate that lopinavir has a good therapeutic index for E6/E7 PHFKs at concentrations up to 25 μM. Since these cells are not transformed it is most likely that this is due to the ability of lopinavir to activate apoptotic antiviral systems which are suppressed by E6/E7.
Curiously, it is known that HIV patients receiving oral lopinavir as part of HAART do not show enhanced clearance of HPV-related lesions [27]. On this point, it is noteworthy that the concentration of lopinavir found in the vaginal fluid of HIV patients taking oral Kaletra is <2 μM [28], which has been discussed in our previous study [13].
In order to address the issue of whether the observed HPV-specific toxicity of lopinavir is related to its ability to block RNASEL degradation by E6/E7, siRNA RNASEL gene silencing experiments were carried out in lopinavir-treated SiHa cells. These experiments showed that, at the optimum therapeutic dose range of 25 μM, lopinavir had much reduced toxicity in RNASEL siRNA transfected SiHa cells when compared to the same cells transfected with AllStar control siRNA. These results support the hypothesis that the selective toxicity of lopinavir in HPV E6/E7 expressing cells is at least in part mediated by up-regulated expression of the RNASEL protein.
It is also notable that our results support the conclusion that HPV can compromise the antiviral activity of RNASEL. The observation that CaSki cells are insensitive to lopinavir [13] and do not up-regulate RNASEL is significant since CaSki cells express much higher levels of E6 than do SiHa (Figure 3B). Indeed, investigation of the effects of E6 on RNASEL toxicity is the subject of our continued work.
Thus, the logical conclusion from these observations is that inactivation of the RNASEL response by HPV infection of the cervix could lower host antiviral defences and increase the likelihood of infection with other viruses. This is highly significant when considered with the previously discussed findings that genital HPV infections can predispose to increased risk of HIV transmission in both men and women [29,30].
In conclusion, our data support the potential use of lopinavir as a topical, self-administered treatment for HPV-related cervical dysplasia. If successful, this treatment would be particularly useful in low-resource countries which lack cytology screening and surgical services, and where HIV infection is often a significant additional complication. With current pricing strategies, anti-HPV vaccines are not an option in such settings, and as previously discussed, they will most likely have reduced efficacy in HIV-positive women. Furthermore, in view of the observation that infection with HPV can enhance the sexual transmission of HIV in both men and women [29,30], this clearly indicates that a simple, non-surgical treatment for HPV-related precancerous lesions may not only reduce the burden of cervical cancer but may also reduce the chances of HIV transmission.
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