Description:1
FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE
SPECIFICATION
(See section 10 and rule 13)
Integrin (avß3) receptor-targeted PLGA nanoparticles containing cisplatin and upconversion nanoparticles for lung cancer
G D Goenka University, an Indian university of Sohna Gurugram Road, Sohna, Haryana, India, 122103
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
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FIELD OF INVENTION:
The present invention relates to pharmaceutical science field, which aims at design and development of Integrin (avß3) receptor-targeted PLGA nanoparticles containing cisplatin and upconversion nanoparticles improved theranostic effect in lung cancer therapy
BACK GROUND:
Lung cancer is a severe disease that arises from a malignant tumor developed in the respiratory epithelium of bronchi, bronchioles, and alveoli and is the leading cause of cancer deaths (approximately 1.76 million deaths annually). Lung cancer also has a 5-year survival rate of 18.6%, lower than other cancers such as colon (64.5%), breast (89.6%) and prostate (98.2%). Although, conventional modalities for treating lung cancer include surgery (complex operation), radiotherapy, and chemotherapy (most commonly used), depending on cancer stage. Chemotherapeutic drugs also affects normal cells, and interfere with their normal growth, resulting in extreme side effects and intolerability for patients. To address these challenges, medical science is continuously revolutionizing, focusing on the development of biodegradable multifunctional drug delivery systems (i.e., nanoparticles, liposomes, micelles and dendrimers etc.) at the nanometer scale. Over the decades, these nanocarriers have gained much attention in cancer research, being tested in small animals and in clinical trials to prove their effiacy and safety in cancer therapy.The novelty of our work to alter the physicochemical properties of cisplatin (CDDP) and upconversion nanoparticles (UCNP) which may be useful to the effective and safe theranostic applications in lung cancer therapy.
Danhier et al., have demonstrated the clinical applications of RGD grafted PLGA nanoparticles containing paclitaxel and JNJ-7706621. The study concluded that the prepared PLGA nanoparticles decorated with RGD peptide have shown
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signifiant effect in cytotoxicty study in HeLa cells and achieved lower IC50 vales at 5.5 µg/ ml in compared to free paclixatel. The observed result was further matched with the observation of cellular uptake study which revealed that after RGD peptide decorated PLGA nanoparticles were more effective to penetrate in cancer cells in compared without peptide modifid nanoparticles. Furthermore, the tumor inhibition study have also proved the effectiveness of RGD peptide decorated PLGA nanoparticles which achieved higher tumor growth inhibition in compared to other drug counterparts
Rios De La Rosa et al., have developed RGD conjugated nanoparticles containing rhodamine B (a florescent agent) for brain cancer imaging. The study have shown that the prepared nanoparticles were more capable for delivering doses of rhodamine B to U87MG (glioblastoma) cells and compared to A2780 (ovarian cancer) cells. This study also concluded that RGD conjugated PLGA nanoparticles could be used for the delivery of anticancer agents for the benefi of cancer patients.
Babu et al., 2017 fabricated chitosan-RGD decorated PLGA nanoparticles to improve therapeutic effiacy of paclitaxel in lung cancer therapy. The study showed that chitosan-RGD decorated PLGA nanoparticles have greatly improved cellular uptake via integrin receptor mediated endocytosis and induced G2/M cell cycle arrest and more cytotoxicity of paclitaxel than other counterparts in cancer cells.
Therefore, the present invention overcome the said problems and developed Pluronic F127-modifid PLGA nanoparticles with targeting RGD receptor ability for controlled and precise delivery of CDDP and UCNP for lung cancer therapy.
OBJECTIVE OF THE INVENTION:
1. It is an object of the invention to provide a Integrin (avß3) receptor-targeted PLGA nanoparticles containing cisplatin and upconversion nanoparticles improved theranostic effect in lung cancer therapy.
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2. It is another object of the invention to provide developed RGD receptor-targeted PLGA nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) in lung cancer therapy.
3. It is another object of the invention to alter the physicochemical properties of cisplatin (CDDP) and upconversion nanoparticles (UCNP) which may be useful to the effective and safe theranostic applications in lung cancer therapy.
4. It is another object of the invention to develop Pluronic F127-modifid PLGA nanoparticles with targeting RGD receptor ability for controlled and precise delivery of CDDP and UCNP for lung cancer therapy.
SUMMARY
Upon extensive pharmaceutical and biomedical research to treat lung cancer indicates that lung cancer remains one of the deadliest diseases and the leading cause of death in men and women worldwide. Lung cancer remains untreated and has a high mortality rate due to the limited potential for effective treatment with existing therapies. This highlights the urgent need to develop an effective, precise and sustainable solutions to treat lung cancer. In this study, we developed RGD receptor-targeted PLGA nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) in lung cancer therapy. Pluronic F127-RGD conjugate was synthesized by carbodiimide chemistry method and the conjugation was confimed by FTIR and 1HNMR spectroscopy techniques. PLGA (Poly(lactic-co-glycolic acid)) nanoparticles were developed by the double emulsifiation method, then the surface of the prepared nanoparticles was decorated with Pluronic F127-RGD conjugate. The prepared formulations were characterized for their particle size, polydispersity index, zeta potential, surface morphology, drug encapsulation effiiency, and in vitro drug release and haemolysis studies. Pharmacokinetic studies and safety parameters in BAL flid were assessed in rats. Histopathology of rat lung tissue was performed. The obtained results of particle sizes of the nanoparticle formulations were found 100–200 nm, indicating the homogeneity of dispersed colloidal nanoparticles
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formulations. Transmission Electron Microscopy (TEM) revealed the
spherical shape of the prepared nanoparticles. The drug encapsulation effiiency of PLGA nanoparticles was found to range from 60% to 80% with different nanoparticles counterparts. RGD receptor-targeted PLGA nanoparticles showed controlled drug release for up to 72 h. Further, RGD receptor-targeted PLGA nanoparticles achieved higher cytotoxicity in compared to CFT, CFT, and Ciszest-50 (marketed CDDP injection). The pharmacokinetic study revealed that RGD receptor-targeted PLGA nanoparticles were 4.6-fold more effective than Ciszest-50. Furthermore, RGD receptor-targeted PLGA nanoparticles exhibited negligible damage to lung tissue, low systemic toxicity, and high biocompatible and safety in lung tissue. The results of RGD receptor-targeted PLGA nanoparticles indicated that it is a promising anticancer system that could further exploited as a potent therapeutic approach for lung cancer.
BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:
Figure 1, illustrates a view of . (A) Schematic diagram for activation of Pluronic F-127 into Pluronic F-127-COOH (i), and sythesis of Pluronic F-127-RGD conjugate (ii), (B) preparations of CDDP and UCNP loaded PLGA nanoparticles (i) and RGD decorated CDDP (ii) and UCNP loaded PLGA nanoparticles (iii) for the present invention.
Fig. 2: illustrates a view of FTIR spectra of (A) Pluronic F-127, (B) activated Pluronic F-127-COOH and (C) Pluronic F-127-RGD conjugate for the present invention. Fig.3: illustrates a view of 1H NMR spectra of (A) Pluronic F127-COOH and (B) Pluronic F127-RGD conjugate for the present invention. Fig.4: illustrates a view of (A) Zeta potential of formulations i.e., Plain, CPF, CPFT and CPFT-RGD nanoparticles, (B) TEM image of PLGA nanoparticles; (i) plain nanoparticles with 200 nm scale (i), CPF nanoparticles showing a particle with 200
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nm scale (ii) CPFT nanoparticles showing a particle with 400 nm scale (iii), CPFT-RGD nanoparticles showing with 400 nm scale, and (C) % encapsulation efficiency of formulations i.e., CPF, CPFT and CPFT-RGD. Bars represent ± S.D (P<0.05, n = 3) for the present invention.
Fig: 5: illustrates a view of In-vitro drug release from CDDP loaded polymeric nanoparticles in PBS (pH 6.8). CDDP loaded PLGA nanoparticles (CPF, CPFT and CPFT-RGD), and Ciszest-50 injection. Bars represent ± S.D (P<0.05, n = 3) for the present invention.
Fig: 6: illustrates a view of In-vitro hemolysis of positive control (Triton X100), negative control (normal saline), Ciszest-50, CPF, CPFT and CPFT-RGD at 0.5, 2 and 4 h time point interval is represented as mean±SD (n = 3) for the present invention.
Fig: 7: illustrates a view of Cytotoxicity and antiproliferative profile of CDDP containing formulations such as Ciszest-50, CPF, CPFT and CPFT-RGDnanoparticles or Ciszest for A549 cells (n = 4). Bars represent ±S.D. (P<0.05) for the present invention.
Fig: 8: illustrates a view of Pharmacokinetic of CDDP formulations (CPF, CPFT and CPFT-RGD nanoparticles) and Ciszest-50 injection after i.v. administration at the dose of 5 mg/kg (n = 4) for the present invention.
Fig: 9: illustrates a view of Biochemical estimation in BAL fluids of rats after administration of normal saline, Ciszest-50, CPF, CPFT and CPFT-RGD nanaoparticles; (A) alkaline phosphatase activity (KA Units); (B) lactate dehydrogenase activity (U/L) and (C) total protein counts (g/dl) for the present invention.
Fig: 10: illustrates a view of Effect of CDDP formulations in the lungs after i.v. administration. Animals were sacrifice after 15 and 30 days of administration and the lungs were perfused with a normal saline solution by cannulating the both ventricles. The panels present macroscopic views of hematoxylin & eosin-stained lung sections from rats treated with normal saline solution (control), Ciszest-50, CPF, CPFT and RGD-CPFT nanoparticles at same CDDP dose of 5 mg/Kg for the present invention.
Further, skilled artisans will appreciate that elements in the figures are illustrated for
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simplicity and may not have been necessarily been drawn to scale. For example, the flowcharts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of
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steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.
The terms “having”, “comprising”, “including”, and variations thereof signify the presence of a component.
Now the present invention will be described below in detail with reference to the following embodiment.
Example 1
Lung cancer is a severe disease that arises from a malignant tumor developed in the respiratory epithelium of bronchi, bronchioles, and alveoli and is the leading cause of cancer deaths (approximately 1.76 million deaths annually). Lung cancer also has a 5-year survival rate of 18.6%, lower than other cancers such as colon (64.5%), breast (89.6%) and prostate (98.2%). Although, conventional modalities for treating lung cancer include surgery (complex operation), radiotherapy, and chemotherapy (most commonly used), depending on cancer stage. Chemotherapeutic drugs also affects normal cells, and interfere with their normal growth, resulting in extreme side effects and intolerability for patients. To address these challenges,
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medical science is continuously revolutionizing, focusing on the development of biodegradable multifunctional drug delivery systems (i.e., nanoparticles, liposomes, micelles and dendrimers etc.) at the nanometer scale. Over the decades, these nanocarriers have gained much attention in cancer research, being tested in small animals and in clinical trials to prove their effiacy and safety in cancer therapy.The novelty of our work to alter the physicochemical properties of cisplatin (CDDP) and upconversion nanoparticles (UCNP) which may be useful to the effective and safe theranostic applications in lung cancer therapy. We developed Pluronic F127-modifid PLGA nanoparticles with targeting RGD receptor ability for controlled and precise delivery of CDDP and UCNP for lung cancer therapy. The study was thoroughly conducted to prove biological applications of prepared formulation. The investigation includes, physicochemical characterization, hemolysis study, pharmacokinetic study, biochemical estimation of bronchoalveolar lavage (BAL) flids of rats, and histopathology of lung tissue and compared with Ciszest-50 (marketed cisplatin injection).
Example 2
Pluronic F127-RGD conjugate was successfully synthesized and confimed by FTIR and 1H NMR spectroscopy. This study confims that the double emulsifiation method as suitable for loading of both CDDP and UCNP in PLGA nanoparticles. PLGA nanoparticles have shown drug encapsulation effiiency upto 69.85%. Particles size and TEM analysis showed that the nanoparticles were uniformly distributed and spherical with a nanosize of 110–200 nm. After 72 h of in-vitro drug release study revealed a desired controlled drug release pattern of CDDP. Hemolysis studies showed that CPF, CPFT and CPFT-RGD nanoparticles were found blood compatible and safe. Further we performed pharmacokinetic evaluation as maximum of CPFT and CPFT-RGD nanoparticles showed 3.91 and 4.6-fold higher effectiveness when compared with Ciszest-50 injection in rats after 72 h i.v administration. CPFT-RGD nanoparticles have been proven to be effective, safer and biocompatible nanocarrier for the delivery of bioactive molecules for precise cancer therapy. Further, RGD receptor-targeted PLGA nanoparticles achieved higher cytotoxicity in compared to CPF, CPFT, and Ciszest-50 (CDDP injection). These results indicate that CPFTRGD nanoparticles may be further explored as novel nanodrug carrier for the delivery of anticancer drugs and other bioactive molecules in cancer therapy.
Example 3
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PLGA nanoparticles have shown drug encapsulation effiiency upto 69.85%. Particles size and TEM analysis showed that the nanoparticles were uniformly distributed and spherical with a nanosize of 110–200 nm. After 72 h of in-vitro drug release study revealed a desired controlled drug release pattern of CDDP. Hemolysis studies showed that CPF, CPFT and CPFT-RGD nanoparticles were found blood compatible and safe. Further we performed pharmacokinetic evaluation as maximum of CPFT and CPFT-RGD nanoparticles showed 3.91 and 4.6-fold higher effectiveness when compared with Ciszest-50 injection in rats after 72 h i.v administration. CPFT-RGD nanoparticles have been proven to be effective, safer and biocompatible nanocarrier for the delivery of bioactive molecules for precise cancer therapy. Further, RGD receptor-targeted PLGA nanoparticles achieved higher cytotoxicity in compared to CPF, CPFT, and Ciszest-50 (CDDP injection). These results indicate that CPFTRGD nanoparticles may be further explored as novel nanodrug carrier for the delivery of anticancer drugs and other bioactive molecules in cancer therapy.
Variations and modifications of the foregoing are within the scope of the present invention. Accordingly, many variations of these embodiments are envisaged within the scope of the present invention.
The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the present invention.
Acknowledgement
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Inventor Rahul Pratap Singh acknowledges the fincial support from Science and Engineeting Research Board (SERB), New Delhi, for providing fincial support (EEQ/2019/000218) under the scheme of Empowerment and Equity Opportunities for Excellence in Science.
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We claim,
1. A process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) in lung cancer therapy comprising the steps of:
(i) (ii) Succinic anhydride DMAP at 100 C RGD-NH2 NHS/EDC Pluronic F127-COOH Pluronic F127-RGD Pluronic F127 Pluronic F127-COOH A. Activation of Pluronic F127 by carbodiimide chemistry and its RGD conjugation B. Preparation of PLGA nanoparticles (i) Cisplatin Pluronic F127 PLGA Double Emulsification Method CPF: CDDP loaded PLGA nanoparticles Surface Decoration Pluronic F127-RGD (iii) Cisplatin Upconversion Pluronic F127 PLGA Double Emulsification Method (ii) CPFT: CDDP and UCNP loaded PLGA nanoparticles CPFT-RGD: RGD decorated CDDP and UCNP loaded PLGA nanoparticles CPFT: CDDP and UCNP loaded PLGA nanoparticles
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2. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the size of nanoparticle formulations were found 100–200 nm, indicating the homogeneity of dispersed colloidal nanoparticles formulations.
3. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the drug encapsulation efficiency of nanoparticles is 60% to 80%.
4. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the nanoparticles showed controlled drug release for up to 72 h.
5. A Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticle for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) synthesised by a process claimed in claim 1, wherein nanoparticles are 4.6-fold more effective than Ciszest-50.
Dated this 28/07/2023 G D Goenka University, Sohna Gurugram Road, Sohna, Haryana, India, 122103
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ABSTRACT
Integrin (avß3) receptor-targeted PLGA nanoparticles containing cisplatin and upconversion nanoparticles for lung cancer
The present invention relates to develop Integrin (avß3) receptor-targeted PLGA nanoparticles containing cisplatin and upconversion nanoparticles for lung cancer therapy. The obtained results of particle sizes of the nanoparticle formulations were found 100–200 nm, indicating the homogeneity of dispersed colloidal nanoparticles formulations. Transmission Electron Microscopy (TEM) revealed the spherical shape of the prepared nanoparticles. The drug encapsulation effiiency of PLGA nanoparticles was found to range from 60% to 80% with different nanoparticles counterparts. RGD receptor-targeted PLGA nanoparticles showed controlled drug release for up to 72 h. Further, RGD receptor-targeted PLGA nanoparticles achieved higher cytotoxicity in compared to CFT, CFT, and Ciszest-50 (marketed CDDP injection). The pharmacokinetic study revealed that RGD receptor-targeted PLGA nanoparticles were 4.6-fold more effective than Ciszest-50 , Claims:We claim,
1. A process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) in lung cancer therapy comprising the steps of:
(i) (ii) Succinic anhydride DMAP at 100 C RGD-NH2 NHS/EDC Pluronic F127-COOH Pluronic F127-RGD Pluronic F127 Pluronic F127-COOH A. Activation of Pluronic F127 by carbodiimide chemistry and its RGD conjugation B. Preparation of PLGA nanoparticles (i) Cisplatin Pluronic F127 PLGA Double Emulsification Method CPF: CDDP loaded PLGA nanoparticles Surface Decoration Pluronic F127-RGD (iii) Cisplatin Upconversion Pluronic F127 PLGA Double Emulsification Method (ii) CPFT: CDDP and UCNP loaded PLGA nanoparticles CPFT-RGD: RGD decorated CDDP and UCNP loaded PLGA nanoparticles CPFT: CDDP and UCNP loaded PLGA nanoparticles
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2. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the size of nanoparticle formulations were found 100–200 nm, indicating the homogeneity of dispersed colloidal nanoparticles formulations.
3. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the drug encapsulation efficiency of nanoparticles is 60% to 80%.
4. The process of synthesis of Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticles for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) as claimed in claim 1, wherein the nanoparticles showed controlled drug release for up to 72 h.
5. A Integrin (avß3) receptor -targeted PLGA (Poly(lactic-co-glycolic acid)) nanoparticle for the controlled and targeted codelivery of cisplatin (CDDP) and upconversion nanoparticles (UCNP) synthesised by a process claimed in claim 1, wherein nanoparticles are 4.6-fold more effective than Ciszest-50.