The creators of Liposomal Cisplatin: Lipoplatin™
Regulon, Inc (Regulon) is a biopharmaceutical company committed to the discovery, development, testing, and commercialization of low toxicity anti-cancer pharmaceuticals based on a unique patented encapsulation technology.
Our objective is to develop a broad range of drugs to be used in cancer therapy. We believe we have achieved a breakthrough in the development of a unique liposome encapsulation technology.Regulon engages in the discovery and development of nanopharmaceutics in oncology based on liposome encapsulation platform technology. Its products include Lipoplatin, a liposomally encapsulated cisplatin for various cancer indications, including non-small cell lung cancer and pancreatic cancer.
The company was founded in 1997 and is based in Athens, Greece & California with operations in Europe and Asia.
Regulon's unique liposome encapsulation technology applicable to drugs, small molecules, peptides, proteins and viruses, significantly reduces the side effects of chemotherapy known to exacerbate the quality of life (QOL) of cancer patients. The firm has successfully applied this technology to encapsulate two members of the platinum family of anticancer drugs: cisplatin and oxaliplatin.
One approach that has gained much attention in molecular biology is the development of liposome formulations that can be used for the encapsulation of drugs and other molecules for delivery to an organism.
Regulon has developed a unique liposome encapsulation technology applicable to drugs, small molecules, peptides, proteins, and viruses.
Lipoplatin nanoparticles, loaded with cisplatin are uptaken by tumors and metastases (10 to 200-fold higher than normal tissue) by leaking through the compromised endothelium of tumor vasculature sprouted during neoangiogenesis, a process known as extravasation, and by the avidity of tumors for nutrients with Lipoplatin disguised as a nutrient with its lipid shell.
Moreover, Lipoplatin nanoparticles fuse with the cell membrane or are rapidly uptaken by cancer cells thus emptying their toxic payload inside tumor cells.
In order to overcome inefficient drug delivery, one of the major obstacles associated with efficient cancer therapy, Regulon, Inc. has developed a unique liposome encapsulation technology applicable to drugs, small molecules, peptides, proteins, and viruses.
Drug encapsulation takes advantage of chemical structural characteristics of various drugs especially those used for chemotherapy on cancer patients. Lipoplatin, the liposomally encapsulated form of cisplatin, brings a major breakthrough in molecular medicine reducing significantly the toxicity of cisplatin and enhancing its tumor targeting after intravenous injection to animal models implanted with human cancers. The encapsulation efficiency reaches extremely high yields unlike any other similar technology.
Passive delivery to tumors is achieved secretly from immune cells and normal tissues by encapsulation of the cytotoxic drugs into a natural lipid capsule protected with a PEG polymer; the 110-nm liposome nanoparticles exploit the compromised endothelium of tumor vasculature for their preferential extravasation to tumors and metastases. Lipids, composing the nanoparticle shell, are natural products, one of the four classes of biomacromolecules, compatible with the lipids of the cell membrane, unlike synthetic polymers used in other nanotechnology capsules with dubious cumulative toxicities.
Figure 1. The scheme shows the PEGylated liposome that is the carrier of the toxic drug cisplatin with its long-circulating properties in body fluids after intravenous administration.
Regulon’s nanoparticles carrying therapeutic drugs have long circulating properties in body fluids (Figure 1); this longevity in circulation is required for passively identifying the tumors and metastases in the body for their preferential targeting and extravasation in tumors. This preferential targeting of cancer tissue takes advantage of the imperfections in the vasculature sprouted by tumors to accelerate their growth during neoangiogenesis; the arteries, veins and micro-vessels in normal tissue have endothelial walls more compact compared to the “leaky” vasculature of tumors; as a result, tumors uptake 10- to 200-times more Lipoplatin nanoparticles than normal tissue.(Figure 2)
Figure 2. The scheme shows a blood vessel in tumor tissue. Lipoplatin nanoparticles of 100nm in diameter are depicted as spheres with the yellow toxic payload of cisplatin inside them. In normal tissue, blood vessels are impenetrable by small nanoparticles. On the contrary, tumor blood vessels have imperfections (tiny holes) in their walls (called endothelium); tumor blood vessels are established during the process of neo-angiogenesis (meaning sprouting of new blood vessels by a tumor cell mass during its growth phase). Lipoplatin nanoparticles take advantage of these tiny holes to pass through and extravasate inside the tumor reaching a concentration that can be 10- to 200-fold higher compared to the adjacent normal tissue.
This was demonstrated in human studies where patients were infused with Lipoplatin and the platinum levels were measured in surgical specimens from primary or metastatic tumors and the adjacent normal tissue. Regulon’s anticancer treatment minimizes the side effects of classic chemotherapy. In simple terms all primary tumors and metastases are being targeted regardless of the tumor type or size following intravenous administration of our drug. Their targeting depends primarily on the degree of tumor vascularization. Tumors of the stomach and breast for example have the highest degree of vascularization and are expected to accumulate more platinum drug after intravenous administration.
Figure 3. Delivery of cisplatin “payload” directly to tumor cells facilitated by DPPG fusion circumventing the need for Ctr1-receptor mediated transportation required by naked cisplatin. After concentrating in tumors and metastases DPPG promotes the fusion of Lipoplatin with the cell membrane. Once they reach the tumor target Lipoplatin nanoparticles have the advantage, unique to Regulon’s technology, to fuse with the cell membrane of the tumor cell and empty their toxic payload inside the cytoplasm. Liposomes developed by others (e..g. Doxil of SPI-77 of Alza/J&J) are unable to do the fusion process; thus the toxic drug is emptied outside the tumor cell and is less effective.
Lipoplatin nanoparticles leading to delivery of their toxic payload inside the cytoplasm of the tumor cell where it is needed for anticancer efficacy (Figure 3). This is a major advantage in the implementation of the treatment in the clinic to enhance efficacy and eliminate toxicity. Crossing of the cell membrane barrier by Lipoplatin was also demonstrated in cell cultures (Figure 4).
Figure 4. Demonstration of the fusion or uptake of Lipoplatin nanoparticles using cancer cell cultures. The green donut-like structures are single cancer cells; their periphery where the cell mebrane is located fluoresces because it has uptaken fluorescent Lipoplatin nanoparticles or Regulon’s fusogenic liposomes as a control. Lipoplatin or DPPG-liposomes with fluorescent lipids enter rapidly MCF-7 human breast cancer cells thus providing proof of concept of membrane fusion or endocytosis to deliver the toxic cisplatin inside the tumor cell.
Lipoplatin is the only nanoparticle drug available that contains a heavy metal inside a liposome. Platinum can uptake high energy from external sources such as laser or gamma rays that can burst the nanoparticle to release the toxic drug or to heat up the surrounding cytoplasm. These exciting properties are under current investigation to explore the full potential of this exciting nanoparticle.
Figure 5. Encapsulation of the beta-galactosidase gene into a liposome of the same composition as the Lipoplatin and systemic delivery to SCID mice with human tumors stained preferentially the vasculature that was developed by the tumors under the skin of the animals to supply the tumor with nutrients. From Boulikas T Molecular mechanisms of cisplatin and its liposomally encapsulated form, Lipoplatin: Lipoplatin™ as a chemotherapy and antiangiogenesis drug. Cancer Therapy Vol 5, 349-376, 2007
The antiangiogenesis property of Lipoplatin has been suggested from the encapsulation of the beta-galactosidase gene into a liposome of the same composition as the Lipoplatin liposome; after systemic delivery to SCID mice with human tumors (Figure 5) the foreign “blue” gene stained preferentially the vasculature that the tumors under the skin of the animals developed to supply the tumor with nutrients. This shows that Regulon’s liposomes can target preferentially the vascular endothelial cells; in case of Lipoplatin, targeting of these cells with toxic cisplatin instead of the “blue” gene would cause their destruction. Thus, Lipoplatin limits tumor vascularization by attacking their endothelial cells in addition to the known property of cisplatin to attack the epithelial cell of the tumor.
The antimetastasis potential of Lipoplatin was shown in a study from the “Reference Oncology Center, Italian National Cancer Institute” in Aviano. Lipoplatin inhibited both migration and invasion of cervical cancer cells supporting its antimetastasis potential. This is a very important feature of Lipoplatin because migration and invasion are essential steps used by cancers to mediate their metastases.
In a paper that appeared in September 2013 in “Gynecologic Oncology” (http://www.ncbi.nlm.nih.gov/pubmed/24029417), the investigators, led by Dr. Donatella Aldinucci have examined the effectiveness of Lipoplatin in cisplatin-resistant cervical cancer cells. In the Aldinucci study, Lipoplatin was effective in both cervical cancer and cisplatin-resistant cervical cancer cells thus opening the possibility to apply Lipoplatin successfully against cervical cancer both as first and second-line. Furthermore, the same study has elucidated novel mechanisms on how Lipoplatin kills cervical cancer cells:
Regulon’s liposome encapsulation technology can be applied to most of the 1,000 FDA approved drugs, thus increasing the Company’s fundamental value.
The clinical development of Lipoplatin™ started in 2001. Lipoplatin™ is the liposome encapsulated form of Cisplatin. Cisplatin is one of the most widely used and most effective cytotoxic agents in the treatment of epithelial malignancies such as lung, head & neck, ovarian, bladder and testicular cancers. The continued clinical use is impeded by its severe adverse reactions including renal toxicity, gastrointestinal toxicity, peripheral neuropathy, asthenia, ototoxicity and optic neuropathy. However, Human studies have shown a different pharmacokinetic and biodistribution profile to that of cisplatin with serum longevity, a prelude to the extravasation and tumor invasion process by Lipoplatin™ nanoparticles.
Regulon has achieved a significant improvement (>90%), in the encapsulation of Cisplatin and has developed a reproducible manufacturing procedure.
Total platinum levels in plasma were dose-dependent and a half-life of 40-120 h was estimated for total platinum in sera of patients compared to 6h for cisplatin. Its urine excretion was also much slower and about 40% of the dose was excreted in the urine in 3 days compared again to ~8h for 50% excretion for cisplatin.
Additional studies on patients who underwent Lipoplatin infusion followed by prescheduled surgery have demonstrated a 10- to 200-fold higher accumulation in primary tumors and in metastases compared to the adjacent normal tissue. These are exciting results establishing Lipoplatin and Regulon’s liposome technology as a means to achieve high targeting. Even micrometastases, invisible in chest x-rays or CT scans, were proposed to be targeted by Lipoplatin because of the microvasculature sprouting in progress. The nanoparticle drug was inferred to target endothelial cells in tumor vasculature and its antioangiogenesis potential was proposed in addition to the classical chemotherapy activity.
Overall, Lipoplatin has proven to be a safe drug, the main toxicity being myelotoxicity. A Phase I study, where Lipoplatin was used as monotherapy, failed to reach the MTD. When used in combination with Gemcitabine, the dose of 120 mg/m2 was defined as MTD; higher doses were associated with increased myelotoxicity. A subsequent Phase I study determined the DLT for Lipoplatin monotherapy at 350 mg/m2 and the MTD at 300 mg/m2. For Lipoplatin-paclitaxel combination therapy, the DLT was 250 mg/m2for Lipoplatin and 175 mg/m2 for paclitaxel whereas the MTD was 200 mg/m2 for Lipoplatin and 175 mg/m2 for paclitaxel: Stathopoulos et al, 2010, Anticancer Res 30, 1317-1322
In a Phase II randomized comparative study on the efficacy and toxicity of 120 mg/m2 D1,8,15 Lipoplatin in combination with 1,000 mg/m2 gemcitabine D1,8 compared to 100 mg/m2 cisplatin D1 with same schedule of gemcitabine as first-line against NSCLC there were statistically significant less toxicities in the Lipoplatin arm and a better efficacy profile, especially in the non-squamous histological subtype. This schedule was promoted into a randomized Phase III study and an interim report showed statistically significant reduction in nephrotoxicity, asthenia, and neurotoxicity and enhanced efficacy in NSCLC adenocarcinoma.
The results of a different randomized comparative Phase III study on the efficacy and toxicity of Lipoplatin 200 mg/m2 D1 in combination with paclitaxel 135 mg/m2 D1 in a 14-day schedule compared to cisplatin 75 mg/m2 D1 with paclitaxel 135 mg/m2 D1 as first-line against NSCLC were reported with 114 and 115 patients in each arm. The study (see 9.12) demonstrated a statistically significant reduction in nephrotoxicity (6.1% vs 40%), and also a reduction of most other adverse effects including anemia, neutropenia and asthenia. The efficacy results established the noninferiority of Lipoplatin compared to cisplatin.
In a Phase II trial the toxicity and efficacy of Lipoplatin 120 mg/m2 D1,8,15 in combination with vinorelbine 30 mg/m2 D1,8 against breast cancer was studied. Of the 35 patients, 15 had previously received neoadjuvant treatment based on anthracyclines, 11 treatment with taxanes and 6 patients with both. The objective response rate was 53.1% and the median survival time was 22 months. Grade 3/4 neutropenia was observed in 44% of cycles, and febrile neutropenia was seen in 4 patients (11.4%). No grade 3/4 nephrotoxicity or neuropathy was noted. This combination was effective and well tolerated in patients with MBC. The authors proposed this scheme as first-line treatment.
In a Phase II study on the efficacy and toxicity of Lipoplatin in combination with 5-fluorodeoxyuridine and radiation therapy against advanced gastric tumors 4 out of 5 patients (80%) receiving five weekly cycles of treatment achieved complete response and in a follow up of nine months (Koukourakis et al, 2009). The high response rate observed in this Phase II study is suggested to arise from the high vascularization of gastric tumors compared to other solid tumors thus resulting in high Lipoplatin concentration (similar data were also observed in the patient tumor targeting study) and from rupture of the nanoparticles by the process of radiation therapy (see Mechanism of Action). Thus, radiation therapy might be proven the most efficacious combination for Lipoplatin.
A registrational Phase II/III study against pancreatic cancer is in progress under the orphan drug status granted to Lipoplatin by the European Medicines Agency. The Company has received scientific advice by EMEA for a pivotal randomized Phase III study using Lipoplatin 200 mg/m2 D1,8 in combination with pemetrexed as first line in non-squamous NSCLC compared to cisplatin with pemetrexed.
Lipoplatin has successfully completed a Phase III trial for non-small cell lung cancer (NSCLC) Stathopoulos et al, 2010
Nephrotoxicity in arm A patients treated with lipoplatin–paclitaxel was 6.1%, while for arm B patients treated with cisplatin–paclitaxel, it was 40.0%, P value < 0.001. Some arm A patients had increased blood urea and serum creatinine but this was temporary and these patients eventually received the full nine cycles. Other side-effects with a statistically significant difference occurred in arm B where GI tract nausea, vomiting and fatigue were worse than in arm A. Myelotoxicity was higher in arm B patients and the difference was statistically significant for grade 3–4 neutropenia. Six patients in arm A and 10 in arm B were hospitalized due to febrile neutropenia. Anemia was common: 43.9% in arm A and 54.9% in arm B. Grades 1–4 leucopenia were 33.3% and 45.2% in arm A and B patients, respectively; grades 3 and 4 leucopenia were 12.3% and 2.6%, respectively, in arm A and 18.3% and 8.7%, respectively, in arm B (Table 2; statistically significant difference P value 0.017). Asthenia was more common in arm B patients (71.3% versus 57% in arm A, P value 0.019). The side- effect comparison was carried out for 229 patients in total. Stathopoulos et al, 2010
In a publication in Cancer Chemother Pharmacol, (Stathopoulos et al, 2011), exciting data were announced from a randomized Phase III study on Lipoplatin™ in the treatment of non-squamous non-small cell lung cancer (NSCLC). This study used Lipoplatin in combination with paclitaxel as first line treatment against non-squamous NSCLC and compared response rates and toxicities to a similar group of patients treated with cisplatin plus paclitaxel. This study has demonstrated statistically significant (p value = 0.036) increase in tumor response rate in the Lipoplatin arm (59.22% of patients) versus the cisplatin arm (42.42%, of patients) while also reducing most major toxicities of cisplatin, especially nephrotoxicity.
Attempts to develop platinum compounds to reduce the side effects of cisplatin have resulted in the introduction of carboplatin and oxaliplatin. However, both of these drugs have proven to have inferior response rates to cisplatin especially in lung cancer. Other cytotoxic agents such as taxanes (paclitaxel, docetaxel), gemcitabine, vinorelbine, pemetrexed, and irinotecan have also been used as substitutes of cisplatin; however, none of these agents has demonstrated superior efficacy to cisplatin in lung cancer. This Lipoplatin study represents the first time a drug has improved on cisplatin’s response rate in non-squamous NSCLC.
Median survival times were 10 months for the Lipoplatin arm and 8 months for the cisplatin arm, with a p-value of 0.155. The median duration of response was 7 months for the Lipoplatin arm and 6 months for the cisplatin arm. Although not statistically significant, these results suggest the potential for superior overall survival (OS) for Lipoplatin compared to cisplatin, a hypothesis that will be tested in a larger trial. Furthermore, among the responders to Lipoplatin a subgroup of patients demonstrated a substantially higher overall survival than a comparable subgroup of cisplatin responders. After 10 months, 30% of patients in the Lipoplatin arm, as compared with just 16% of patients in the cisplatin arm, were without disease progression. By the end of the trial, there were 32 patients alive, 21 from the Lipoplatin arm (20.39%) and 11 from the cisplatin arm (11.11%). Thus, after 18 months, the number of surviving patients was approximately double for Lipoplatin versus cisplatin.
The clinical development of Lipoplatin in adenocarcinomas establishes this drug as the most active platinum drug with significantly lower side effects.
In conclusion, the clinical data support replacement of cisplatin by Lipoplatin. Lipoplatin has substantially reduced the renal toxicity, peripheral neuropathy, ototoxicity, myelotoxicity as well as nausea/vomiting and asthenia of cisplatin in Phase I, II and III clinical studies with enhanced or similar efficacy to cisplatin.