Application of array comparative genomic hybridization (aCGH) for identification of chromosomal aberrations in the recurrent pregnancy loss.

Spontaneous abortion occurs in 8-20% of recognized pregnancies and usually takes place in the first trimester (7-11 weeks). There are many causes of pregnancy loss, but the most important (about 75%) is the presence of chromosomal aberrations. We present the results of oligonucleotide array application in a cohort of 62 miscarriage cases. The inclusion criteria for the study were the loss after 8th week of pregnancy and the appearance of recurrent miscarriages. DNA was extracted from trophoblast or fetal skin fibroblasts. In the 62 tested materials from recurrent miscarriages, the detection rate was 56.5% (35/62). The most commonly found were aneuploidies (65%) (chromosomal trisomy 14, 16, 18, 21, and 22), Turner syndrome, and triploidy (17.1%). Other chromosomal abnormalities included pathogenic and likely pathogenic structural aberrations: 1) pathogenic: deletion 7p22.3p12.3 and duplication 9p24.3p13.2 inherited from the normal father, deletion 3q13.31q22.2 and deletion 3q22.3q23 of unknown inheritance and duplication of 17p12 inherited from father with foot malformation; 2) likely pathogenic variants: deletion 17p13.1 inherited from normal mother, deletion 5q14.3 of unknown inheritance and de novo deletion 1q21.1q21.2. Among these aberrations, six CNVs (copy number variants) were responsible for the miscarriage: deletion 7p22.3p12.3 and duplication 9p24.3p13.2, deletion 3q13.31q22.2 and deletion 3q22.3q23, and deletion 17p13.1 and deletion 1q21.1q21.2. Other two findings were classified as incidental findings (deletion 5q14.3 and 17p12 duplication). Our research shows that 17% of the aberrations (6/35 abnormal results) that cannot be identified by the routine kariotype analysis are structural aberrations containing genes important for fetal development, the mutations of which may cause spontaneous abortion.

Null variants in AGRN cause lethal fetal akinesia deformation sequence.

We present a case of lethal fetal akinesia deformation sequence (FADS) caused by a frameshift variant in trans with a 148 kbp deletion encompassing 3-36 exons of AGRN. Pathogenic variants in AGRN have been described in families with a form of congenital myasthenic syndrome (CMS), manifesting in the early childhood with variable fatigable muscle weakness. To the best of our knowledge, this is the first case of FADS caused by defects in AGRN gene. FADS has been reported to be caused by pathogenic variants in genes previously associated with CMS including these involved in endplate development and maintenance: MuSK, DOK7, and RAPSN. FADS seems to be the most severe form of CMS. None of the reported in the literature CMS cases associated with AGRN had two null variants, like the case presented herein. This indicates a strong genotype-phenotype correlation.

Effects of inclusion of cetirizine hydrochloride in ß-cyclodextrin.

Following the preparation of inclusion complex of cetirizine (CTZ) and ß-cyclodextrin (ß-CD), the compound was investigated to assess the possibility of modifying the physicochemical properties (solubility, release, stability, permeability) of CTZ after complexation that are vital for subsequent formulation studies involving the said complex. Changes in FT-IR/Raman spectra, DSC thermograms and XRD diffractograms confirmed the formation of a CTZ-ß-CD system. Hydrophilic interaction chromatography with a DAD detector was employed to determine alterations of the CTZ concentration during studies following complexation. An analysis of a phase-solubility diagram of cCTZ = fcß-CD indicated a linear rise in the solubility of CTZ as the concentration of ß-CD increased. The inclusion of CTZ in a system with ß-CD significantly reduced the instability of CTZ in the presence of oxidizing factors. It was also found that regardless of the pH of the acceptor fluids used in the release studies an increase was observed in the concentration of CTZ in CD system compared to its free form. The ability to permeate artificial biological membranes manifested by CTZ after complexation was enhanced as well. In summary, CD has significant potential to mask the bitter taste of CTZ and to counter the instability induced by oxidizing factors.

The Analysis of the Physicochemical Properties of Benzocaine Polymorphs.

The study was a pioneering attempt to assess the influence of the structural polymorphism (forms I, II, III) of benzocaine on its solubility, apparent solubility, and chemical stability, which are vital parameters for preformulation and formulation work. The impact of differences in the solubility of selected polymorphs of benzocaine on their permeability through artificial biological membranes (PAMPA system) was evaluated. The polymorphs of benzocaine were obtained by means of techniques commonly used for the preparation of various pharmaceutical dosage forms: ball milling, micro milling, and cryogenic grinding, which allowed for the appearance or preservation of form III, the initial conformation of benzocaine. Ball milling resulted in the conversion of form III to I, whereas micro milling yielded form II. As a result of cryogenic grinding, form III of benzocaine was preserved. The identification of all polymorphic forms of benzocaine was confirmed via X-ray powder diffraction (PXRD) supported by FT-IR spectroscopy coupled with density functional theory (DFT) calculations. The differences in solubility, dissolution, and permeability through artificial biological membranes resulting from the polymorphic forms of benzocaine were established by using chromatographic determinations. Accelerated stability tests indicated that all polymorphic forms were chemically stable at a required level.

Stability of cefozopran hydrochloride in aqueous solutions.

The influence of pH on the stability of cefozopran hydrochloride (CZH) was investigated in the pH range of 0.44-13.00. Six degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The degradation of CZH as a result of hydrolysis was a pseudo-first-order reaction. As general acid-base hydrolysis of CZH was not occurred in the solutions of hydrochloric acid, sodium hydroxide, acetate, borate and phosphate buffers, kobs = kpH because specific acid-base catalysis was observed. Specific acid-base catalysis of CZH consisted of the following reactions: hydrolysis of CZH catalyzed by hydrogen ions (kH+), hydrolysis of dications (k1H2O), monocations (k2H2O) and zwitter ions (k3H2O) and hydrolysis of zwitter ions (k1OH-) and monoanions (k2OH-) of CZH catalyzed by hydroxide ions. The total rate of the reaction was equal to the sum of partial reactions: [Formula: see text]. CZH similarly like other fourth generation cephalosporin was most stable at slightly acidic and neutral pH and less stable in alkaline pH. The cleavage of the ß-lactam ring resulting from a nucleophilic attack on the carbonyl carbon in the ß-lactam moiety is the preferred degradation pathway of ß-lactam antibiotics in aqueous solutions.

STABILITY OF CEFPIROME SULFATE IN AQUEOUS SOLUTIONS.

The influence of pH on the stability of cefpirome sulfate was investigated in the pH range of 0.44 - 13.00. The degradation of cefpirome sulfate as a result of hydrolysis was a pseudo-first-order reaction. General acid-base hydrolysis of cefpirome sulfate was not observed. In the solutions of hydrochloric acid, sodium hydroxide, acetate, borate and phosphate buffer, k(obs) = k(pH) because specific acid-base catalysis was observed. Specific acid-base catalysis of cefpirome sulfate consisted of the following reactions: hydrolysis of cefpirome sulfate catalyzed by hydrogen ions (kH+), hydrolysis of dications (k1H2O) monocations (k2 H2O), zwitter ions (k3H2O) and monoanions (k4 H2O) of cefpirome sulfate under the influence of water. The total rate of the reaction was equal to the sum of partial reactions k(pH) = kH+ x aH+ + kH2O x f1 + k2H2O x f2 + k3H2O x f3 + k4 H2O x f4. Based on the dependence k(pH) = f(pH) it was found that cefpirome sulfate was the most stable in aqueous solutions in the pH range of 4-6.

STUDIES OF THE CRYSTALLINE FORM OF CEFUROXIME AXETIL: IMPLICATIONS FOR ITS COMPATIBILITY WITH EXCIPIENTS.

Amorphous and crystalline forms of cefuroxime axetil were identified and characterized using DSC, XRPD, SEM, FT-IR and Raman spectroscopy. Based on the results of chromatographic studies, changes in the kinetic mechanism and rate of degradation of the crystalline form of cefuroxime axetil in binary systems with excipients were also evaluated. The findings suggest that the mechanism of degradation of cefuroxime axetil in such systems depends on two factors: the applied excipient and storage conditions. Cefuroxime axetil in combination with magnesium stearate, croscarmellose sodium and crospovidone, microcrystalline cellulose, aerosil is decomposed according to the first-order reaction model in dry air as well as at an increased relative air humidity, which may be associated with non-catalytic interactions between the active pharmaceutical ingredient and the excipients. However, in the presence of mannitol, under elevated humidity conditions (RH - 76%), the degradation of cefuroxime axetil follows the autocatalytic model. According to ESP maps, computed binding energies and HOMO - LUMO gaps, differences of degradation curves between cefuroxime axetil - mannitol and other investigated systems were explained. This study of the polymorphic transformation of the crystalline form of cefuroxime axetil and its binary systems with excipients after exposure to increased temperature and humidity indicated a conversion towards the amorphous form or the coexistence of both forms.

Solid-state stability and compatibility studies of clavulanate potassium.

The kinetic and thermodynamic parameters of degradation of clavulanate potassium in the solid state were studied by using a reversed phase high performance liquid chromatography (RP-HPLC) method. The degradation of clavulanate potassium was a first-order reaction depending on the substrate concentration at an increased relative air humidity (RH) and in dry air. The dependence ln k = f(1/T) became the ln k = (0.026 ± 166.35)-(2702.82 ± 1779.43)(1/T) in dry air and ln k = (1.65 ± 100.40) × 10(3)-(5748.81 ± 3659.67)(1/T) at 76.4% RH. The thermodynamic parameters Ea, ΔH(≠a), ΔS(≠a) of the degradation of clavulanate potassium in the solid state were calculated. The dependence ln k = f (RH%) assumed the form ln k = (8.78 ± 5.75) 10 (-2) (RH%) + (2.64 × 10(-8 )± 40.41). The compatibility of clavulanate potassium with commonly used excipients was studied at an increased temperature and in dry air. The geometric structure of molecule, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals were also determined in order to predict the structural changes and reactive sites in clavulanate potassium during degradation and compatibility studies in the solid state. The ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR) and Raman spectra of degraded samples of the compound were analyzed.

Assay of Diastereoisomers of Cefuroxime Axetil in Amorphous and Crystalline Forms Using UHPLC-DAD.

A sensitive UHPLC-DAD method was developed for determination of diastereoisomers of cefuroxime axetil in bulk substance in amorphous and crystalline forms as well as in pharmaceutical preparations. Chromatographic separation was achieved on Kinetex C-18 (100 mm × 2.1 mm, 1.7 µm) column with mobile phase consisting of 0.1 % formic acid:methanol (88:12, v/v), at the flow rate of 0.7 mL min-1 and total run time of 3 min. The wavelength of the DAD detector was set at 278 nm. Inter-day precision (RSD) was less than 3 % and accuracy level ranged between 98.31 and 104.99 %. Degradation products of cefuroxime axetil in aqueous solutions and in the solid state were identified with a EIS-Q-MS mass spectrometer. The solubility of above-mentioned polymorphic forms of cefuroxime axetil in suitable solvents is a crucial factor during preparation of samples and is essential for chromatographic separation of its diastereoisomers.

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form.

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile-0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min-1. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid-base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method's linearity was investigated in the ranges 0.40-1.40 µg mL-1 for isomer I and 0.40-2.40 µg mL-1 for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL-1 for isomer I and 0.0356 and 0.1078 µg mL-1 for isomer II, respectively.

[Meropenem--therapeutic recommendation after twenty years of presence on pharmaceutical market]. / Meropenem--rekomendacje terapeutyczne po 20 latach obecnosci na rynku farmaceutycznym.

Meropenem is the first representative of carbapenem analogues with methyl group, which has been applied in medicine. This drug has been approved by FDA (Food and Drug Administration) in 1996. Available results of clinical trials and scientific reports point surprising synergism of combination of meropenem with other chemotherapeutics, in the treatment of bacterial diseases. Present study based on available information presents indications for use the mentioned antibiotic in pharmacotherapy of infectious diseases. Meropenem is the first representative of carbapenem analogues, which contains methyl group. Introduction of a methyl group at the system of coupled rings: ß-lactam and pyrrolidine, solved the problem of degradation by the dehydropeptidase-I (DHP-I). In the consequence it is not necessary to use meropenem in the connection with specific DHP-I inhibitors. Meropenem, similarly to other carbapanem analogues, is intended for the treatment of severe inpatient and outpatient infections. Bacterial resistance to meropenem may be the result of: carbapenemases activity, decreased affinity to Penicillin Binding Proteins--PBP (mainly PBP 2 and PBP 3) and activation of efflux pump (antibiotic ejection outside the cell). Twenty-year period of application of meropenem in pharmacotherapy may cause the spread of methyl-ß-lactamases.

Development and validation of stability-indicating HPLC method for simultaneous determination of meropenem and potassium clavulanate.

A stability-indicating LC assay method was developed and validated for a simultaneous determination of meropenem and potassium clavulanate in the presence of degradation products formed during acid-base hydrolysis, oxidation and thermolysis. The isocratic RP-HPLC method was developed with a LiChrospher RP-18 (250 mm x 4.6 mm, 5 microm) column and gradient elution of 12 mmol/L ammonium acetate and acetonitrile. The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength 220 nm and the temperature 303 K. The method was validated with regard to linearity, accuracy, precision, selectivity and robustness, and was applied successfully for the determination of meropenem and potassium clavulanate separately as well as jointly in pharmaceutical formulations.

Stress Degradation Studies of Tebipenem and a Validated Stability-Indicating LC Method.

A inexpensive and rapid isocratic LC method has been developed for the quantitative determination of tebipenem-a new ß-lactam antibiotic. Stress degradation studies were performed on tebipenem in acidic (0.2 N hydrochloric acid) and basic (0.02 N sodium hydroxide) solutions, in a solution with oxidizing agent (3 % hydrogen peroxide), and in the solid state, during thermolysis and photolysis. For a chromatographic separation of tebipenem and its degradation products, a C-18 stationary phase and 12 mM ammonium acetate-acetonitrile (96:4 v/v) were used. A quantitative determination of tebipenem was carried out by using a PDA detector at 298 nm, with a flow rate of 1.2 mL min-1. The linear regression analysis for the calibration plots showed a good linear relationship (r = 0.999) in the concentration range 0.041-0.240 mg mL-1. The method demonstrated good precision (1.14-1.96 % RSD) and recovery (99.60-101.90 %). The limits of detection and quantitation were 9.69 and 29.36 µg mL-1, respectively. The analysis of tebipenem reactivity was supported by quantum chemical calculations based on the density functional theory (DFT). The analysis of the electron density of the HOMO and LUMO of tebipenem suggested the possibility of electron transport in the molecule during the degradation of bi-cyclic 4:5 fused penem rings.

Association between the Concentrations of Essential and Toxic Elements in Mid-Trimester Amniotic Fluid and Fetal Chromosomal Abnormalities in Pregnant Polish Women.

The present study aimed to investigate the relationship between the concentrations of essential and toxic elements present in the amniotic fluid (AF) and fetal chromosomal abnormalities in pregnant women. A total of 156 pregnant white Polish women aged between 20 and 43 years and screened to detect high risk for chromosomal defects in the first trimester were included in the study. AF samples were collected from these women during routine diagnostic and treatment procedures at mid-gestation (15-22 weeks of their pregnancies). The concentrations of various minerals in the AF were determined by inductively coupled plasma mass spectrometry. Genomic hybridization and cytogenetic karyotyping were performed to detect chromosomal aberrations in the fetuses. The genetic analysis revealed chromosomal aberrations in 19 fetuses (over 12% of all the evaluated women). The major abnormalities identified were trisomy 21 (N = 11), trisomy 18 (N = 2), and triploidy (N = 2). Fetuses with chromosomal abnormalities more frequently showed lower manganese concentration in the AF in the second trimester as compared to those with normal karyotype. A coincidence was observed between high iron levels in the AF and a higher risk of chromosomal abnormalities in the fetuses.

The Presence of Mycotoxins in Human Amniotic Fluid.

Mycotoxin exposure assessments through biomonitoring studies, based on the analysis of amniotic fluid, provides useful information about potential exposure of mothers and fetuses to ubiquitous toxic metabolites that are routinely found in food and the environment. In this study, amniotic fluid samples (n = 86) were collected via abdominal amniocentesis at 15-22 weeks of gestation from pregnant women with a high risk of chromosomal anomalies or genetic fetal defects detected during 1st trimester prenatal screening. These samples were analyzed for the presence of the most typical Aspergillus, Penicillium and Fusarium mycotoxins, with a focus on aflatoxins, ochratoxins and trichothecenes, using the LC-FLD/DAD method. The results showed that the toxin was present in over 75% of all the tested samples and in 73% of amniotic fluid samples from fetuses with genetic defects. The most frequently identified toxins were nivalenol (33.7%) ranging from

Prenatal diagnosis of glutaric acidemia type 2 with the use of exome sequencing - an up-to-date review and new case report.

INTRODUCTION: Inborn errors of metabolism (IEM) also called metabolic diseases constitute a large and heterogenous group of disorders characterized by a failure of essential cellular functions. Antenatal manifestation of IEM is absent or nonspecific, which makes prenatal diagnosis challenging. Glutaric acidemia type 2 (GA2) is a rare metabolic disease clinically manifested in three different ways: neonatal-onset with congenital anomalies, neonatal-onset without congenital anomalies and late-onset. Neonatal forms are usually lethal. Congenital anomalies present on prenatal ultrasound as large, hyperechoic or cystic kidneys with reduced amniotic fluid volume. MATERIAL AND METHODS: We present a systematic literature review describing prenatal diagnosis of GA2 and a new prenatal case. RESULTS: Ten prenatally diagnosed cases of GA2 have been published to date, mainly based on biochemical methods. New case of GA2 was diagnosed using exome sequencing method. DISCUSSION: All prenatal cases from literature review had positive history of GA2 running in the family. In our study trio exome sequencing was performed in case of fetal hyperechoic kidneys without a history of GA2. Consequently, we were able to identify two novel pathogenic variants of the ETFDH gene and to indicate their parental origin. SUMMARY: Exome sequencing approach used in case of fetal hyperechoic kidneys allows to identify pathogenic variants without earlier knowledge of the precise genetic background of the disease. Hyperechoic, enlarged kidneys could be one of the clinical features of metabolic diseases. After exclusion of chromosomal abnormalities, urinary tract obstruction and intrauterine infections, glutaric acidemia type 2 and number of monogenic disorders should be consider.

Wide Fontanels, Delayed Speech Development and Hoarse Voice as Useful Signs in the Diagnosis of KBG Syndrome: A Clinical Description of 23 Cases with Pathogenic Variants Involving the ANKRD11 Gene or Submicroscopic Chromosomal Rearrangements of 16q24.3.

KBG syndrome is a neurodevelopmental autosomal dominant disorder characterized by short stature, macrodontia, developmental delay, behavioral problems, speech delay and delayed closing of fontanels. Most patients with KBG syndrome are found to have a mutation in the ANKRD11 gene or a chromosomal rearrangement involving this gene. We hereby present clinical evaluations of 23 patients aged 4 months to 26 years manifesting clinical features of KBG syndrome. Mutation analysis in the patients was performed using panel or exome sequencing and array CGH. Besides possessing dysmorphic features typical of the KBG syndrome, nearly all patients had psychomotor hyperactivity (86%), 81% had delayed speech, 61% had poor weight gain, 56% had delayed closure of fontanel and 56% had a hoarse voice. Macrodontia and a height range of -1 SDs to -2 SDs were noted in about half of the patients; only two patients presented with short stature below -3 SDs. The fact that wide, delayed closing fontanels were observed in more than half of our patients with KBG syndrome confirms the role of the ANKRD11 gene in skull formation and suture fusion. This clinical feature could be key to the diagnosis of KBG syndrome, especially in young children. Hoarse voice is a previously undescribed phenotype of KBG syndrome and could further reinforce clinical diagnosis.

Mechanochemical activation with cyclodextrins followed by compaction as an effective approach to improving dissolution of rutin.

Rutin is one of the most important flavonoids with poor bioavailability. This work aimed at addressing the issue of poor biopharmaceutical performance of rutin by applying a combination of complexation with secondary processing into tablets. Mechanical activation was the most suitable method of rutin complex formation with (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD), while the ß-cyclodextrin (ß-CD) complex successfully formed by kneading with an ethanol/water mixture. Complexation was confirmed by thermal analysis, powder X-ray diffraction and vibrational spectroscopy. Dynamic vapour sorption showed that stability of powders at high humidity conditions was satisfactory, however, the ß-CD complex retained around 8% of moisture. The complexes were compacted with or without tricalcium phosphate (TRI-CAFOS) filler at a range of compression pressures (19-113 MPa). The best tabletability was determined for rutin/HP-ß-CD, compressibility for the TRI-CAFOS blends with complexes and compactibility for the rutin/HP-ß-CD + TRI-CAFOS mix. Dissolution studies showed quicker and more complete dissolution (pH 1.2) of rutin/HP-ß-CD tablets, however the compacts comprising the filler were superior than pure complexes. The tablets manufactured in this study appear to be promising delivery systems of rutin and it is recommended to combine rutin/HP-ß-CD with TRI-CAFOS and compact at 38-76 MPa.

Mucoadhesive Chitosan Delivery System with Chelidonii Herba Lyophilized Extract as a Promising Strategy for Vaginitis Treatment.

Chelidonium majus (also known as celandine) contains pharmacologically active compounds such as isoquinoline alkaloids (e.g., chelidonine, sanguinarine), flavonoids, saponins, carotenoids, and organic acids. Due to the presence of isoquinoline alkaloids, Chelidonii herba extracts are widely used as an antibacterial, antifungal, antiviral (including HSV-1 and HIV-1), and anti-inflammatory agent in the treatment of various diseases, while chitosan is a biocompatible and biodegradable carrier with valuable properties for mucoadhesive formulations preparation. Our work aimed to prepare mucoadhesive vaginal drug delivery systems composed of Chelidonii herba lyophilized extract and chitosan as an effective way to treat vaginitis. The pharmacological safety of usage of isoquinoline alkaloids, based on MTT test, were evaluated for the maximum doses 36.34 ± 0.29 µg/mL and 0.89 ± 1.16 µg/mL for chelidonine and sanguinarine, respectively. Dissolution rate profiles and permeability through artificial membranes for chelidonine and sanguinarine after their introduction into the chitosan system were studied. The low permeability for used save doses of isoquinoline alkaloids and results of microbiological studies allow confirmation that system Chelidonii herba lyophilized extract chitosan 80/500 1:1 (w/w) is a promising strategy for vaginal use. Ex vivo studies of mucoadhesive properties and evaluation of tableting features demonstrated that the formulation containing Chelidonii herba lyophilized extract (120.0 mg) with chitosan (80/500-100.0 mg) and polymer content (HPMC-100.0 mg, microcrystalline cellulose-50.0 mg, lactose monohydrate-30.0 mg and magnesium stearate-4.0 mg) is a vaginal dosage form with prolonging dissolution profile and high mucoadhesion properties (up to 4 h).

Hydrogel Delivery System Containing Calendulae flos Lyophilized Extract with Chitosan as a Supporting Strategy for Wound Healing Applications.

Calendulae flos is a valued plant material with known anti-inflammatory and antimicrobiological properties. The limitation for its use in the treatment of chronic wounds is the lack of adhesion to the required site of action. Obtaining the Calendulae flos lyophilized extract from water-ethanolic extract allowed to prepare valuable material whose biological activity in the wound healing process was confirmed by a positive result of the scratch test. The Calendulae flos lyophilized extract was standardized for the contents of the chlorogenic acid and the narcissin. The significant potency of the Calendulae flos pharmacological activity has become the reason for studies on its novel applications in combination with the multifunctional chitosan carrier, to create a new, valuable solution in the treatment of chronic wounds. The use of chitosan as a carrier allowed for the controlled release of the chlorogenic acid and the narcissin. These substances, characterized by prolonged release from the chitosan delivery system, were identified as well permeable, based on the results of the studies of the parallel artificial membrane permeability assay (PAMPA Skin) a model simulating permeability through membrane skin. The combination of the Calendulae flos lyophilized extract and the chitosan allowed for synergy of action towards hyaluronidase inhibition and effective microbiological activity. Optimization of the hypromellose hydrogel preparation ensuring the required rheological properties necessary for the release of the chlorogenic acid and the narcissin from the chitosan delivery system, as well as demonstrated antimicrobial activity allows indicating formulations of 3% Calendulae flos lyophilized extract with chitosan 80/500 in weight ratio 1:1 and 2% or 3% hypromellose as an important support with high compliance of response and effectiveness for patients suffering from chronic wounds.