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Case Report: Microfragmented Adipose Tissue Drug Delivery in Canine Mesothelioma: A Case Report on Safety, Feasibility, and Clinical Findings

  • San Michele Veterinary Hospital
  • Ospedale San Veterinario San Michele

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Mesothelioma is a rare lethal tumor of dogs and humans involving cavities of the body. Dogs are considered a model for new drugs and therapeutic methods since they present spontaneous diseases similar to humans. Microfragmented adipose tissue (MFAT) uploaded by paclitaxel (PTX) is a drug delivery medium providing slow release of chemotherapic drugs. A dog affected by pleural, pericardial, and peritoneal mesothelioma was treated by 17 intracavitary ultrasound-guided injections of MFAT-PTX over 22 months. A long-lasting improvement of general conditions was observed, treatment was well-tolerated, and no toxicity or hypersensitivity was reported. Pharmacokinetic (PK) data indicated low drug localization in the circulatory system and a tendency to enter or remain in the extravascular compartments of the body. Indeed, low levels of free-circulating drugs for a short time produced low toxicity, whereas, a higher intracavitary PTX concentration can have major pharmacological efficacy. To our knowledge, this is the first time that mesothelioma has been treated using such a procedure, and this should be considered as a novel therapeutic approach. The low systemic absorption suggests the possible role of MFAT-PTX for loco-regional/intratumoral therapy also useful in other types of tumors, and further investigation is warranted.
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Case Report: Microfragmented Adipose Tissue Drug Delivery in Canine
Mesothelioma: A Case Report on Safety, Feasibility, and Clinical Findings
ArticleinFrontiers in Veterinary Science · January 2021
DOI: 10.3389/fvets.2020.585427
14 authors, including:
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Offer Zeira
San Michele Veterinary Hospital
Francesco Petrella
University of Milan
Rita Paroni
University of Milan
Augusto Pessina
University of Milan
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published: 14 January 2021
doi: 10.3389/fvets.2020.585427
Frontiers in Veterinary Science | 1January 2021 | Volume 7 | Article 585427
Edited by:
David Bruyette,
Anivive Lifesciences, United States
Reviewed by:
Elena De Felice,
University of Camerino, Italy
Chad M. Johannes,
Iowa State University, United States
Francesco Petrella;
Specialty section:
This article was submitted to
Comparative and Clinical Medicine,
a section of the journal
Frontiers in Veterinary Science
Received: 20 July 2020
Accepted: 04 December 2020
Published: 14 January 2021
Zeira O, Ghezzi E, Pettinari L, Re V,
Lupi DM, Benali SL, Borgonovo S,
Alessandri G, Petrella F, Paroni R,
Dei Cas M, Tremolada C, Coccè V
and Pessina A (2021) Case Report:
Microfragmented Adipose Tissue
Drug Delivery in Canine
Mesothelioma: A Case Report on
Safety, Feasibility, and Clinical
Findings. Front. Vet. Sci. 7:585427.
doi: 10.3389/fvets.2020.585427
Case Report: Microfragmented
Adipose Tissue Drug Delivery in
Canine Mesothelioma: A Case Report
on Safety, Feasibility, and Clinical
Offer Zeira 1, Erika Ghezzi 1, Letizia Pettinari 1, Valentina Re 1, Davide M. Lupi 1,
Silvia L. Benali 2, Simone Borgonovo 3, Giulio Alessandri 4,5 , Francesco Petrella 6,7
Rita Paroni 5,8 , Michele Dei Cas 8, Carlo Tremolada 9, Valentina Coccè 5and
Augusto Pessina 5
1Department of Stem Cells and Regenerative Medicine, San Michele Veterinary Hospital, Tavazzano con Villavesco, Italy,
2Laboratorio La Vallonea, Milan, Italy, 3Clinica Veterinaria Crema, Crema, Italy, 4Department of Cerebrovascular Diseases,
Istituto di Ricovero e Cura a Carattere Scientifico, Besta Neurological Institute, Milan, Italy, 5Centro di Ricerca Coordinato
StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy, 6Department of Stem
Cells and Regenerative Medicine, Istituto di Ricovero e Cura a Carattere Scientifico, European Institute of Oncology, Milan,
Italy, 7Centro di Ricerca Coordinato StaMeTec, Department of Oncology and Emato-Oncology, University of Milan, Milan,
Italy, 8Department of Health Sciences, Università Degli Studi di Milano, Milan, Italy, 9Department of Stem Cells and
Regenerative Medicine, Istituto Image, Milan, Italy
Mesothelioma is a rare lethal tumor of dogs and humans involving cavities of the
body. Dogs are considered a model for new drugs and therapeutic methods since
they present spontaneous diseases similar to humans. Microfragmented adipose
tissue (MFAT) uploaded by paclitaxel (PTX) is a drug delivery medium providing
slow release of chemotherapic drugs. A dog affected by pleural, pericardial, and
peritoneal mesothelioma was treated by 17 intracavitary ultrasound-guided injections
of MFAT-PTX over 22 months. A long-lasting improvement of general conditions was
observed, treatment was well-tolerated, and no toxicity or hypersensitivity was reported.
Pharmacokinetic (PK) data indicated low drug localization in the circulatory system
and a tendency to enter or remain in the extravascular compartments of the body.
Indeed, low levels of free-circulating drugs for a short time produced low toxicity,
whereas, a higher intracavitary PTX concentration can have major pharmacological
efficacy. To our knowledge, this is the first time that mesothelioma has been treated
using such a procedure, and this should be considered as a novel therapeutic
approach. The low systemic absorption suggests the possible role of MFAT-PTX for
loco-regional/intratumoral therapy also useful in other types of tumors, and further
investigation is warranted.
Keywords: mesothelioma, paclitaxel, adipose tissue, dog, drug delivery
Mesothelioma is a rare neoplasm of dogs and humans affecting the lining epithelial cells of
the coelomic cavities of the body. It is a chemoresistant tumor and actually has no effective
therapeutic strategies. In human medicine, around 80% of this tumor occurs in men, median
survival is 8–18 months, and the survival rate is <12% in the 5 years after diagnosis.
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
In veterinary medicine, no sex predilection is reported. Median
survival for untreated dogs with mesothelioma is difficult to
assess since they are often euthanatized following diagnosis.
Various studies of treated dogs (surgery, intracavitary, and
intravenous chemotherapy) report survival time from 2 to 13
months (1). In both humans and dogs, the tumor may involve
cavities such as thorax, abdomen, pericardial sac, and the vaginal
tunics of the scrotum.
The domestic dog is considered to be an important animal
model for the evaluation of new drugs and therapeutic
methods since it presents spontaneous diseases very much
similar to human oncology (2,3), including mesothelioma.
Microfragmented adipose tissue (MFAT) is a potential
drug delivery medium that may provide a slow release of
chemotherapic drugs contiguous to the tumor (4). MFAT
consists of stromal vascular fraction, pericytes, and adipose
stromal stem cells, and it is known to have trophic, mitogenic,
anti-scarring, anti-apoptotic, immunomodulatory (5), and
antimicrobial actions produced by a large number of bioactive
elements, growth factors, and cytokines (57).
In order to obtain MFAT, we employed a commercially
available, enzyme-free technology (Lipogems) able to harvest
micro-fragmented fat preparation. This technology reduces the
size of the adipose tissue clusters by means of mild mechanical
forces while eliminating pro-inflammatory oil and blood residue.
The technique is gentle and provides micro-fragmented fat
in a short time (15–20) without expansion and/or enzymatic
treatment (8,9). Paclitaxel (PTX, Taxol R
) is an anticancer
chemotherapy drug that isclassified as a “plant alkaloid” and
is used in various types of solid tumors in human medicine.
Although the hypersensitivity and side effect related to the
toxicity of paclitaxel and cosolvents are well-known (10),
numerous studies have suggested the efficacy of paclitaxel in
different types of tumors using both systemic and subcutaneous
injection (1114). Whereas, intracavitary chemotherapy in
veterinary mesothelioma was described using platinum-based
drugs (15), paclitaxel is not considered the therapy of choice for
mesothelioma. In agreement with new approaches oriented to
verify and validate new paclitaxel formulations, the aim of our
study is to verify the safety, feasibility, and efficacy of intracavitary
administration of paclitaxel-loaded microfragmented adipose
tissue in mesothelioma in dogs.
A 6-year-old, mixed-breed, 24-kg, neutered dog, with a 2-
month history of a progressive weakness, loss of appetite,
productive cough, abdominal distension, and difficulty in
breathing, was selected for the treatment. Complete blood
count (CBC) and biochemical tests were assessed. Thoracic
radiographs detected severe pleural effusion and bilateral middle
and caudal lung lobe collapse. Abdominal radiographs and
ultrasound also evidenced severe effusion. Serosanguinous fluid
was removed from the pleural and abdominal spaces, underwent
cytological examination, and showed high malignant characters
(anisocytosis and anisokaryosis). Ultrasound-guided biopsies
were taken from the pleura, pericardium, and peritoneum,
and histological and immunohistochemical diagnoses were
performed; the final diagnosis was of mesothelioma.
Paclitaxel-Loaded Adipose Tissue
(MFAT-PTX) Preparation
Autologous adipose tissue, obtained by lipoaspirate from
the dog’s lumbar flanks, was microfragmented by using a
microfragmentation device (Lipogems, Italy) as previously
described (16). This procedure allows minimal manipulation
without the use of enzymatic procedures, and microfragmented
samples can be also cryopreserved or used as a scaffold
for paclitaxel (MFAT-PTX). For our study, fresh aliquots of
microfragmented adipose tissue were loaded with PTX by adding
the drug at a concentration of 1 mg/ml and stirring the mixture
for 30 min before use as previously reported (4).
Chemotherapeutic Protocol
Treatment protocol consisted of ultrasound-guided
administration into the abdominal and thoracic cavity of
7 ml of MFAT-PTX (1 mg/ml) corresponding to 0.29 mg/kg
(0.35 mg/m2) of free drug. First administration (T0) was done
15 days after presentation. Over the course of 22 months,
the dog underwent 17 treatments (both intrathoracic and
intra-abdominal), with an average of a treatment every 38 days
(shortest interval 14 days, longest 70 days).
The procedure is initiated by drainage of the abdomen and
chest from the exudate fluid followed by injection of 3 ml
of MFAT-PTX intraperitoneally +2 ml, respectively, into the
right and left pleural spaces (Figure 1). Clinical outcomes were
monitored at the time of each treatment and documented by
thorax radiographs, abdominal ultrasound, clinical examination,
and blood counts. Special attention was given to eventual
adverse effects.
Pharmacokinetics (PK) Study
The minimal parameters of PK [Cmax, Tmax , T1/2, volume of
distribution (Vd), and area under curve (AUC)] were evaluated
by checking the amount of drug in the blood 30 min and 2,
4, and 8 h after the first treatment of the MFAT-PTX. At the
10th treatment (T10), the residual amount of the drug was
also measured in the pleura and pericardium biopsies. The
amount of PTX was evaluated by liquid chromatography-coupled
mass spectrometry (LC-MS/MS) analysis as already reported (4).
Briefly, blood (100 µL) and tissues (about 50 mg) were added
in internal standard (25 µl of paclitaxel D5 10 µg/ml) and PTX
was extracted by 1 mL of a mixture methanol/isopropanol (6:4,
v:v). Dry extracts were redissolved with 200 µl of acetonitrile
and 10 µl injected in LC-MS/MS. The details for instrumental
conditions were already described elsewhere and kept essentially
unaltered (4).
Clinical Assessment
No major short- or long-term adverse effects were registered.
Complete blood counts and biochemistry performed each month
did not show abnormalities. In particular, during the whole
treatment leukocytes and platelet counts in the peripheral blood
Frontiers in Veterinary Science | 2January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 1 | Lipoaspirate collection and ultrasound-guided intrathoracic treatment with MFATPTX. (A) Lipoaspirate collection from the dog’s lumbar flanks. (B)
Procedure of treatment that was initiated by drainage of abdomen and chest from the exudate fluid followed by the injection of 3ml of MFAT-PTX intraperitoneally +
2 ml, respectively, into the right and the left pleural spaces. (C) Ultra sound view.
indicated constantly normal values, suggesting an absence of
systemic myelotoxicity. All phases of the treatments were feasible.
In order to avoid iatrogenic damage to the internal organs, major
attention was given to the intracavity administration when a low
amount of effusion was present.
The dog presented rapid improvement in its general
conditions after each exudate fluid drainage, followed by the
intracavitary administration. This effect lasts for an average
period of 30–40 days.
Notably, in the first 15 months of treatment the intervals
between treatments were longer (average 52 days) while in the last
7 months the intervals between treatments were shorter (average
24 days).
The patient presented good clinical conditions during
most of this period. During the final few days before each
treatment, exudate formation became visible, and the clinical
conditions worsened.
Evaluation of the clinical status was based on the periodical
veterinarian controls in which exudate fluid formation was
assessed by ultrasound, radiographs, and computed tomography
scan (CT) together with breathing quality. Both parameters
presented clear amelioration. Notably, the abdominal exudate
production completely ceased after the fourth treatment. In
addition, the owner’s evaluation was taken into consideration,
as they had knowledge of the dog’s “normal” vs. “abnormal”
behavior. The owner reported a good quality of life. The
dog was able to play with other dogs, go up the stairs, and
coughed rarely, breathed normally most of the time, and had
a good appetite.
Imaging Diagnostic Assessment
Post-treatment thoracic radiographs, performed once a month
in the first 6 months, showed progressive reduction of the
severe pleural effusion (Figure 2). The following radiographs,
every 3 months, evidenced only mild differences in the thoracic
effusion quantity while in the abdominal cavity no liquid could be
detected. The first thorax and abdominal CT scan, pre- and post-
contrast medium, performed 8 months after the initial treatment
evidenced mild to moderate pleural effusion, rare abdominal
effusion, pleura and peritoneum reactivity, and loco-regional
lymphadenopathy. A control CT after 12 months showed only
mild difference (Figure 2).
Pathological Assessment
The patient was euthanized after 22 months due to worsening of
his clinical conditions and underwent a complete post-mortem
exam. Macroscopically all the pleural surfaces presented isles
of yellowish fatty material, identified as MFAT-PTX (last
administration 2 weeks previously) (Figure 3). Cytological
smears of pleural and peritoneal effusion were evaluated,
presenting overlapping findings: hematic background with
scattered eosinophilic material, good cellularity, and mixed
cellular population; and presence of numerous cells in
clusters of small/medium size, having variable cytoplasm
nucleus/cytoplasm ratios, slightly basophilic cytoplasm with
occasional vacuolization, round/oval central or paracentral
nucleus with coarse chromatin, and prominent central nucleolus.
The cells present moderate to severe malignancy characters,
Frontiers in Veterinary Science | 3January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 2 | Radiography and computed tomography scan (CT) along the treatment. The figure reports the post-treatment thoracic radiographs (Rx) performed once
a month in the first 6 months. They showed progressive reduction of the severe pleural effusion. The computed tomography scan (CT) shows the first CT scan 8
months after first treatment. Transversal view of the soft tissue window, and post-contrast right side effusion. The control 12 months after the first CT scan shows a
bilateral pleural effusion mildly increased.
including anisocytosis and anisokaryosis and presence of
occasional leukocytes.
A biopsy of pericardium was fixed in 10% buffered formalin,
embedded in paraffin, and stained routinely with hematoxylin
and eosin. Immunohistochemistry was performed on paraffin
sections (30 µm) placed on Superfrost Plus slides (Superfrost R
Plus) with an automated immunostainer (Discovery Ultra-
Roche). The primary antibody was a monoclonal anti-mouse
mesothelial cell clone HBME-1 (Dako cod M3505) diluted 1:100
and incubated at room temperature for 20 min.
Histopathological sections of pericardium and pleura were
examined with focal evidence of a neoplastic cellular component
within the pleura engorging dilated vessels. Cells were round
to polyhedral and arranged in small groups or micropapillae.
The nucleus had a cytoplasmic ratio that was intermediate.
The cytoplasm moderate and eosinophilic, and the nucleus was
round to oval with 1–2 nucleoli. Anisokaryosis was marked and
anisocytosis moderate. Mitosis were 0–2 in 10 high-power fields
(400×). Immunohistochemistry for the HBME–mesothelioma
marker confirmed the mesothelial origin of the cells (Figure 3).
Pharmacokinetics (PK) Assessment
After treatment with MFAT-PTX, no drug was detected in the
blood at 30 min. At 2, 4, and 8h, the amounts detected in plasma
were 28.8, 19.25, and 4.89 ng/ml, respectively (Figure 4). Cmax
was 28.8 ng/ml, Tmax =2 h, and the half-time (T1/2)=5 h.
Based on the dose of PTX injected and the plasmatic Cmax , we
estimate an apparent volume of distribution at 2 h (Vd2h) of
243 liters. The calculation of plasmatic AUC 1–8 h demonstrated
that MFAT-PTX treatment makes available the drug in plasma
with a value of 117 ngh/ml. The residual amount in pleura
and pericardium measured at 30 days after the 10treatment by
HPLC suggested the presence of 3.6 and 13.3 ng/g, respectively,
which, by considering the weight of these tissues, can be evaluated
as a residual drug of about 2.16 µg in the pleura and 3.99 µg in
the pericardium.
Mesothelioma is a fatal disease in both dogs and humans,
and new effective therapeutic strategies are needed (17).
Our data suggest that localized delivery of microfragmented
adipose tissue (MFAT) uploaded by paclitaxel (MFAT-PTX)
directly into the peritoneal and thoracic cavity is feasible.
Administration in average intervals of 38 days was well-tolerated
in the dog, and no unique toxicity or hypersensitivity was
noted. This result is of particular interest. Due to the low
aqueous solubility of paclitaxel, Taxol R
formulations include
Frontiers in Veterinary Science | 4January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
FIGURE 3 | Macroscopic and histological analysis of pleura. (A) The macroscopic analysis shows the presence on the pleural surfaces isles of yellowish fatty material,
identified as MFAT-PTX (last administration 2 weeks previously). (B,C) Histological section of pleura (hematoxylin and eosin stain) shows neoplastic cells within the
lumen of dilated vessel. Immunohistochemistry for the HBME–mesothelioma marker shows moderate to intense membranous immunolabeling of the tumor cells with
apical membrane accentuation.
FIGURE 4 | Plasmatic concentration of paclitaxel (PTX). The figure shows the
plasmatic concentration of PTX after the first treatment with MFAT-PTX as
reported in the chemotherapeutic protocol. The amount of PTX was checked
in the blood after 30 min and 2, 4, and 8 h.
Cremophor R
(polyoxyethylated castor oil) and ethanol as an
excipient. Such formulations overcome poor solubilization
of paclitaxel for parenteral use. However, Cremophor R
induced complement activation is believed to be the cause
of common hypersensitivity reactions related to Taxol R
in humans and other species (10,18,19). The absence of
hypersensitivity in our case may be explained by various
ways. MFAT is known to have strong immunomodulation
activity (9). Another possible explanation is the long
intervals between treatments together with the slow release of
low doses.
The MFAT-PTX scheme of treatment seems to be able to
produce a local, rather long-term, antineoplastic effect without
any systemic myelotoxicity. A CT control scan after 12 months
showed no major difference in the effusion quantity, pleural and
peritoneal reactivity, and lymphadenopathy, which indicates a
reduction in tumor progression. The long-lasting improvement
in the dog clinical conditions was regularly reported from both
veterinarian and owner and is probably due to the slow release
of paclitaxel from the microfragmented adipose tissue in the
thorax and abdomen, as already reported (4). As far as the
thoracic cavity is concerned, from our data in the first 15
months of treatment, the intervals between local administrations
were longer and became progressively shorter in the following
months (from average of 52–24 days). The need to shorten the
intervals in the last months of treatment was due to a slow and
progressive increase in the thoracic effusion. On the other hand,
in the peritoneal space, effusion quantity was very much reduced
within 6 months after the initial treatment and remained such
until the last treatment. This observation is important since the
dose of paclitaxel and MFAT volume remained the same in all
treatments. It may find an explanation by assuming that the
tumor developed some grade of resistance, but it does not explain
why the peritoneal compartment behaved in a different way,
Frontiers in Veterinary Science | 5January 2021 | Volume 7 | Article 585427
Zeira et al. Case Report: Loco-Regional Treatment of Dog’s Mesothelioma
maintaining a very low level of effusion during the whole period.
In order to answer this query, a much larger number of patients
are warranted.
The PK data showed a very high value of Vd indicating that,
after treatment, the drug localization in the circulatory system is
low and that the drug has a propensity to enter or remain in the
extravascular compartments of the body. This is compatible with
the chemical structure of PTX, which is a lipophilic molecule. It
is also a consequence of the loco-regional treatment with MFAT-
PTX resulting in a more significant distribution of PTX into the
areas with higher lipid density.
As known, this parameter together with the above reported
gives an index of the systemic drug exposure. The low value
is predictive of low systemic toxicity due to the loco-regional
treatment producing low levels of free-circulating drug for
a short time, whereas, locally, PTX concentration can have
pharmacological efficacy. This is also confirmed by the low
modulation of blood cell counts during the treatment and by the
presence of significant residual amounts of PTX found in both
pleura and pericardium 1 month after treatment.
Even though our experience with this therapeutic procedure
includes more than one patient, we have decided to limit our
report to this single case due to its very intense follow-up and
detailed investigation from diagnosis to post-mortem assessment.
To our knowledge, this is the first time that mesothelioma
is treated using such a procedure in a dog and should be
considered as a novel therapeutic approach for mesothelioma
treatment. Furthermore, the lack of systemic absorption after
intra-abdominal and intrathoracic administration suggests a
possible role of MFAT-PTX for intratumoral therapy. Such a
procedure may also be useful in other types of tumors, and
further investigation is warranted.
We have used this new procedure in 2 other cases of
mesothelioma. Unfortunately, both cases were presented to
our hospital in a rather advanced clinical status and died
within 1 month for cardiopulmonary failure. However, the same
procedure was applied to other 2 patients with tumors such as
spleen hemangiosarcoma and ovarian carcinoma with diffuse
abdominal involvement. At the present time, results are similar to
our mesothelioma case by means of no major short- or long-term
adverse effects and overall survival time.
The lack of complications in the dog should be taken
into account when considering this treatment in other species,
including man. The study of spontaneous, naturally occurring
tumors in dogs is a model that provides a valuable role in
developing potentially successful, innovative treatment regimens
for translational medicine, facilitating the transfer of knowledge
from the “bench” to the “bedside.”
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
The procedures involving MFAT-PTX were performed in
accordance with the guidelines defined by the Italian Presidency
of the Council of Ministers and following the guidelines
published by the General Directory of Animal Health and
Veterinary drugs of the Italian Ministry of Health. The
owners considered euthanasia for their dog but accepted our
proposed treatment as a last possibility. They were thoroughly
informed about the entire procedure and signed a formal
agreement with the San Michele Veterinary Hospital in
acceptance of both anesthesia and therapy. They also accepted
that their dog would undergo post-mortem examination. The
veterinary hospital followed guidelines established for Good
Clinical Practice.
OZ, LP, EG, VR, DL, SLB, and SB: medical diagnosis,
management of case, and collection of data. RP, MD, and CT:
laboratory analysis. AP, EG, VC, GA, and FP: collection of data,
writing, and editing of manuscript. AP, VC, and FP: review
of final submission. All authors contributed to the article and
approved the submitted version.
All the procedures were performed as part of the employment
of the authors OZ, EG, LP, VR, DL, SLB, and SB at San Michele
Veterinary Hospital, GA in Besta Institute, FP at European
Institute of Oncology, and RP, MD, VC, and AP at Milan
University, CT in Lipogems International.
MD was supported by the Ph.D. program in Molecular and
Translational Medicine, Università degli Studi di Milano,
Italy. The authors are grateful to Dr. Paolo Montuschi
for the illustration of Figure 1 and thank the Laboratory
of Histopathology, Istituto Zooprofilattico Sperimentale
delle Venezie (PD-Italy), for the information regarding
immunohistochemistry protocol.
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Conflict of Interest: CT is the scientific director of Lipogems International.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2021 Zeira, Ghezzi, Pettinari, Re, Lupi, Benali, Borgonovo, Alessandri,
Petrella, Paroni, Dei Cas, Tremolada, Coccè and Pessina. This is an open-access
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Frontiers in Veterinary Science | 7January 2021 | Volume 7 | Article 585427
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... Dog survival was significantly improved and, more importantly, the absence of toxicity and side effects due to DMFAT-PTX treatment. To note, under standard systemic therapy, PTX is particularly toxic in canine species [42]. Whether this approach could also work in humans remains to be determined. ...
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Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers.
Mesothelioma is an uncommon cancer in dogs for which there is no established standard of care. Chemotherapy is often suggested despite no definitive proof of efficacy. The aim of this study was to evaluate the impact of chemotherapy on survival of dogs with mesothelioma. A retrospective multicentric study was carried out. To be included, dogs needed to present an evocative clinical evolution and a morphological diagnosis of mesothelioma. Exclusion of other cause of effusion and complete clinical follow‐up were also required. Fourty dogs were included, 27 received chemotherapy (group 1) and 13 did not (group 2). Groups were heterogeneous regarding the proportion of animals undergoing surgery as part of their treatment (16 in group 1, 2 in group 2; p = 0.016) and homogeneous otherwise. Univariate analysis showed that dogs from group 1 survived significantly longer than dogs from group 2 (MST: 366 vs 74 days; p < 0.001). Complete resolution of effusion after the first chemotherapy administration positively correlated with survival in group 1 (MST: 415 vs 160 days; p < 0.01). All other variable tested had no significant impact on survival in univariate analysis, but dogs undergoing surgery and dogs having serous membranes' modification at medical imaging tended to survive longer. Multivariate analysis confirmed that chemotherapy was the sole variable independently associated with survival in our study (odds ratio 5.57–6.12; p < 0.01). This article is protected by copyright. All rights reserved.
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Similar to the disease affecting humans, osteoarthritis (OA) is a painful musculoskeletal condition affecting 20% of the adult canine population. Several solutions have been proposed, but the results achieved to date are far from being satisfactory. New approaches, such as intra-articular delivery of cells (including mesenchymal stromal cells), have been proposed. Among the many sources, the adipose tissue is considered very promising. We evaluated the safety, feasibility, and efficacy of a single intra-articular injection of autologous and micro-fragmented adipose tissue (MFAT) in 130 dogs with spontaneous OA. MFAT was obtained using a minimally invasive technique in a closed system and injected in the intra- and/or peri-articular space. Clinical outcomes were determined using orthopedic examination and owners' scores for up to 6 months. In 78% of the dogs, improvement in the orthopedic score was registered 1 month after treatment and continued gradually up to 6 months when 88% of the dogs improved, 11% did not change, and 1% worsened compared with baseline. Considering the owners' scores at 6 months, 92% of the dogs significantly improved, 6% improved only slightly, and 2% worsened compared with baseline. No local or systemic major adverse effects were recorded. The results of this study suggest that MFAT injection in dogs with OA is safe, feasible, and beneficial. The procedure is time sparing and cost-effective. Post injection cytological investigation, together with the clinical evidence, suggests a long-term pain control role of this treatment. The spontaneous OA dog model has a key role in developing successful treatments for translational medicine. Stem Cells Translational Medicine 2018;1-10.
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Background Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population. Methods Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1. ResultsOur results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1. Conclusions Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.
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Intravenous paclitaxel has been underused in dogs due to severe and acute hypersensitivity reactions. Subcutaneous (SC) administration of paclitaxel and its safety are unknown. In this preliminary study, SC administration of paclitaxel was evaluated for hypersensitivity reactions and toxicity in 21 dogs with advanced cancer. Dogs received 1 to 5 paclitaxel doses, ranging from 85 to 170 mg/m(2), SC every 14 or 21 days. A total of 40 paclitaxel doses were administered and none of the 21 dogs developed systemic or acute local hypersensitivity reactions. Severe skin lesions at the injection site developed in 2 dogs after the 4th injection at the same location. Grade 4 neutropenia was observed in 50% of the dogs 5 days after the first treatment at 115 mg/m(2) (n = 14). Two animals developed Grade 5 diarrhea and died likely due to hemodynamic failure or sepsis. Paclitaxel can be administered SC in dogs with no hypersensitivity reaction.
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Paclitaxel is a commonly used chemotherapeutic agent with a broad spectrum of activity against cancers in humans. In 1992, paclitaxel was approved by the U.S. Food and Drug Administration (FDA) as Taxol(®) for use in advanced ovarian cancer. Two years later, it was approved for the treatment of metastatic breast cancer. Paclitaxel was originally isolated from the bark of the Pacific yew tree, Taxus brevifolia in 1971. Taxanes are a family of microtubule inhibitors. As a member of this family, paclitaxel suppresses spindle microtubule dynamics. This activity results in the blockage of the metaphase-anaphase transitions, and ultimately in the inhibition of mitosis, and induction of apoptosis in a wide spectrum of cancer cells. Additional anticancer activities of paclitaxel have been defined that are independent of these effects on the microtubules and may include the suppression of cell proliferation as well as antiangiogenic effects. Based on its targeting of a fundamental feature of the cancer phenotype, the mitotic complex, it is not surprising that paclitaxel has been found to be active in a wide variety of cancers in humans. This review summarizes the evidence in support of paclitaxel's broad anticancer activity and introduces the rationale for, and the progress in development of novel formulations of paclitaxel that may preferentially target cancers and that are not associated with the risks for hypersensitivity in dogs. Of note, a novel nanoparticle formulation of paclitaxel that substantially limits hypersensitivity was recently given conditional approval by the FDA Center for Veterinary Medicine for use in dogs with resectable and nonresectable squamous cell carcinoma and nonresectable stage III, IV and V mammary carcinoma. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.
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Taxanes (a class of chemotherapeutic agents) are an important cause of hypersensitivity reactions (HSRs) in cancer patients. During the last decade, the development of rapid drug desensitization has been key to allow patients with HSRs to taxanes to be safely re-treated although the mechanisms of these HSRs are not fully understood. Earlier studies suggested that solvents, such as Cremophor EL used to solubilize paclitaxel, were responsible for HSRs through complement activation, but recent findings have raised the possibility that some of these HSRs are IgE-mediated. Taxane skin testing, which identifies patients with an IgE-mediated sensitivity, appears as a promising diagnostic and risk stratification tool in the management of patients with HSRs to taxanes. The management of patients following a HSR involves risk stratification and re-exposure could be performed either through rapid drug desensitization or graded challenge based on the severity of the initial HSR and the skin test result. Rapid drug desensitization has been shown to be an effective and safe method to re-introduce taxanes in hundreds of patients, including those with life-threatening HSRs. Patients with non-severe delayed skin HSRs may benefit from rapid drug desensitization since they may be at increased risk for an immediate HSR upon re-exposure. This review focuses on the clinical presentation, diagnosis, and novel mechanisms of immediate HSRs to taxanes. A new management strategy for HSRs to taxanes based on skin testing and rapid drug desensitization is proposed.
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Mesenchymal stem cells (MSCs) are currently being widely investigated both in the lab and in clinical trials for multiple disease states. The differentiation, trophic, and immunomodulatory characteristics of MSCs contribute to their therapeutic effects. Another often overlooked factor related to efficacy is the degree of engraftment. When reported, engraftment is generally low and transient in nature. MSC delivery methods should be tailored to the lesion being treated, which may be local or systemic, and customized to the mechanism of action of the MSCs, which can also be local or systemic. Engraftment efficiency is enhanced by using intra-arterial delivery instead of intravenous delivery, thus avoiding the "first-pass" accumulation of MSCs in the lung. Several methodologies to target MSCs to specific organs are being developed. These cell targeting methodologies focus on the modification of cell surface molecules through chemical, genetic, and coating techniques to promote selective adherence to particular organs or tissues. Future improvements in targeting and delivery methodologies to improve engraftment are expected to improve therapeutic results, extend the duration of efficacy, and reduce the effective (MSC) therapeutic dose.
Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or adipose tissue can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural scaffold of MSCs, to deliver PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with a long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, DMFAT loaded with PTX prevents or delays NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may represent very innovative natural biomaterials able to localize and release anti-cancer molecules at the tumor site, helping to fight cancer in human.
The purpose of this study was to develop paclitaxel (PTX) formulations with solid dispersion (SD) and micelles (M) in order to improve solubility and oral absorption in rats. In addition, the enhanced anti-cancer effects of PTX formulations were compared in various breast cancer cell lines. The SD formulations with various copolymers were prepared using a solvent evaporation method, and micelles with Soluplus® mixed with D-α-tocopheryl polyethylene glycol-1000-succinate (TPGS) were prepared using a film hydration method. The physical properties of SD and M formulations were evaluated. The dissolution (%) of SD4 and SD9 formulations, and the solubility of M2 were significantly higher than those of PTX. The SD formulations and micelles were also stable for 3 and 1 month, respectively. The anti-cancer effects of SD4, SD9, and M2 significantly increased in breast cancer cells, whereas the blank formulations were not toxic to normal cells. The SD4, SD9 and M formulations improved the permeability of Cou-6 compared to Cou-6 solution in Madin-Darby canine kidney cells (MDCK line). The SD formulations and micelles had enhanced bioavailability (BA) compared to that of PTX, showing relative BA values of 667.3% (SD4), 359.6% (SD9), and 365.4% (M2). This study demonstrates the technologies to increase the anti-cancer effects and BA of PTX, via SD and micelle formulations, and, to our knowledge, is the first comparison of the two formulations.
Carriers used to solubilize taxane chemotherapy drugs cause severe hypersensitivity. Nanoparticle formulations can provide improved dissolution and bioavailability of taxanes. Thus, a nanoparticulate, excipient‐free formulation of paclitaxel (CTI52010) was evaluated in tumour‐bearing dogs with intravenous and subcutaneous delivery. Tumour‐bearing dogs were treated with intravenous CTI52010 using a modified rapid dose escalation scheme. Subcutaneous administration was then planned for a small cohort of dogs for comparison. For both groups, serial blood samples were collected after first dosing for pharmacokinetic analysis by LCMSMS. Tumour response was measured using RECIST criteria. Toxicity was recorded using VCOG‐CTCAEv1.1. Fifteen dogs were treated with intravenous delivery at increasing dosages (80‐136 mg/m2), with one objective response in the urethral component of a prostatic carcinoma (probable transitional cell carcinoma) and four dogs with durable stable disease (two carcinomas, two sarcomas). Pharmacokinetic data indicate a rapid initial clearing of the drug from serum followed by an extended elimination half‐life, similar to normal dogs and suggesting reticuloendothelial clearance. Parameters and toxicity were highly variable and a maximally tolerated dosage could not be reliably confirmed. Three dogs were treated with subcutaneous delivery and no drug was detected in circulation, resulting in termination of the study. This novel formulation of paclitaxel is well tolerated in dogs and no unique toxicity or hypersensitivity was noted. The preliminary responses suggest biologic activity. The lack of systemic absorption after subcutaneous administration suggests a possible role for intratumoural anticancer therapy.
Paclitaxel (PTX), a taxane plant product, is one of the most effective broad-spectrum anti-cancer agents and approved for the treatment of a variety of cancers including ovarian, breast, lung, head and neck as well as Kaposi's sarcoma. Poor aqueous solubility and serious side effects associated with commercial preparation of PTX (Taxol®) triggered the development of alternative PTX formulations. Over past three decades plethora of research work have been done in order to develop cremophor free formulations. Various nanocarriers systems including nanoparticles, liposomes, micelles, bioconjugates, dendrimers have been employed in order to improve its solubility and eliminate undesired side effects. These nanocarriers offers advantage of high degree of encapsulation and cellular uptake, escape from elimination by P-glycoprotein (P-gp) mediated efflux and can be explored for targeted drug delivery. The potential of these nanocarriers can be reflected by the fact that various nanocarriers of PTX are in different stages of clinical trials and a few have already been commercialized including Abraxane®, Lipusu and Genexol PM®. This review focuses on the various challenges associated with PTX formulation development, limitations of existing formulation and novel approaches for the development of alternative formulations for PTX and also highlights the development of novel formulations in clinical settings.